Comparative biological properties of the four
stereoisomers of difethialone, a second-generation
anticoagulant rodenticide, in rats: development of a
model allowing to choose the appropriate stereoisomeric
ratio

Sébastien Lefebvre, Isabelle Fourel, Nolan Chatron, Hervé Caruel, Etienne

Benoit, Virginie Lattard

► To cite this version:

Sébastien Lefebvre, Isabelle Fourel, Nolan Chatron, Hervé Caruel, Etienne Benoit, et al. Comparative biological properties of the four stereoisomers of difethialone, a second-generation anticoagulant rodenticide, in rats: development of a model allowing to choose the appropriate stereoisomeric ratio. Archives of Toxicology, 2020, 94 (3), pp.795-801. 10.1007/s00204-020-02662-0. hal-02476692

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# Pharmacokinetic and inhibition potency of the Four stereoisomers of difethialone in rats—Development of modeling allowing the choice of appropriate stereoisomers ratio

Sébastien Lefebvre<fnref n="1" /> Isabelle Fourel<fnref n="1" />, Nolan Chatron<fnref n="1" />, Hervé Caruel², Etienne Benoit<fnref n="1" />, Virginie Lattard<fnref n="1" />

## Abstract

The current management of rodent pest populations is based on second-generation anticoagulant rodenticides (SGAR). These molecules, of which difethialone is part, are much more efficient than the first generation. Nevertheless, this efficiency comes with a major drawback, SGARs are tissue-persistent that increases the exposure of rodent predators to them. According to its chemical structure, difethialone has four stereoisomers, whose specific inhibition potency and pharmacokinetic have never been described and might be useful to design new eco-friendly rodenticides. The study aimed to investigate the ability to inhibit anticoagulant target enzyme (VKORC1) and the pharmacokinetics in rats of the four difethialone stereoisomers in rats. We show that stereoisomers are all highly efficient to inhibit VKORC1 activity, but they have distinct initial half-life with 6.0h, 25.4h, 69.3h and 82.3h for respectively E4-trans, E2-cis, E1-trans and E3-cis stereoisomer. These results open the way of the development of eco-friendly and efficient rodenticide by mixing some of these stereoisomers. Preferential incorporation of the E4-trans stereoisomer (high inhibitory VKORC1 potency, relatively shorter liver half-life) into difethialone rodenticides baits might produce a more eco-friendly product than current commercially-available difethialone formulations. In addition, we put forward modeling to help design bait according to the circumstance of use (presence of non-target species, food competition, etc) by modulating the theoretical AUC and the theoretical concentration of the product at the death of the rodent pest. Thus, this modeling might allow to diminish the use of laboratory animal in assay.

## Keywords

Modeling — Difethialone — Rodents

## Contents

<table><tr><td>Introduction</td><td>1</td></tr><tr><td>1 Material and methods</td><td>2</td></tr><tr><td>1.1 Chemicals</td><td>2</td></tr><tr><td>1.2 Animals</td><td>2</td></tr><tr><td>1.3 Pharmacokinetic study</td><td>2</td></tr><tr><td>1.4 VKOR activity essay and kinetics</td><td>2</td></tr><tr><td>1.5 Dosages</td><td>3</td></tr><tr><td>1.6 Data analysis</td><td>3</td></tr><tr><td>2 Results</td><td>3</td></tr><tr><td>2.1 Inhibiting activity of the four individual stereoisomers of difethialone</td><td>3</td></tr><tr><td>2.2 Determination of the pharmacokinetics of the four individual stereoisomers of difethialone</td><td>3</td></tr></table>

2.3 Development of a pharmacokinetic model able to predict the half-life of a mixture 3

3 Discussion 4

References 6

## Introduction

The use of second generation anticoagulant rodenticides (SGAR) is the cornerstone of the management of the rodent pest population. Indeed, as the first-generation anticoagulant rodenticides (FGARs), SGARs have a delayed action avoiding food aversion due to the association by rodents of bait consumption and death of other rodents, if this occurs quickly after bait consumption. In addition, SGARs like FGARs have an efficient and safe antidote, vitamin K1. A considerable advantage over FGAR, SGAR molecules are also efficient on rodents carrying Vkorc1 mutations leading to resistance to

<footnote n="1">¹USC 1233 RS2GP, INRA, VetAgro Sup, University of Lyon, F-69280 Marcy l'Etoile, France ²Liphatech, Bonnel, 47480 Pont du Casse, France</footnote>

<footnote>*Corresponding author: virginie.lattard@vetagro-sup.fr</footnote>
vitamin K anticoagulant, this is the main reason for the devel-
opment of these molecules. However, this advantage comes
with a dark side, SGARs are tissue-persistent. This last point
is a major concern today, considering the ecotoxicology of the
SGAR. Indeed, many studies have pinpointed an exposure of
rodent predators to SGAR [1–6]

In the two last years, significant progress has been made
in the development of new AR. It is based on the stereochem-
istry of SGAR and more specifically on the modification of
cis/trans diastereoisomers proportions. Indeed, for the same
AR molecule, both diastereoisomers exhibit always different
half-life compared to the other one while keeping their in-
hibitory potency [7, 8]. Moreover, a recent ecological study
found that only trans-bromadiolone is involved in the poi-
soning of non-targeted species whereas the rodent bait con-
tained both diastereoisomers [9, 10] However, considering
difethialone, the diastereoisomers approach failed. The initial
half-lives of both diastereoisomers are not sufficiently differ-
ent. To go through the diastereomers concept and to improve
again the efficiency and the eco-compatibility of rodenticides,
we have separately studied properties of the two enantiomers
of each diastereomer of difethialone.

This study opens an innovative way to improve
difethialone by producing a new mix of the four stereoisomers
instead of the mix currently used in the different formulations
containing difethialone, the latter being composed of more
than 90% of cis-isomers. This new approach implies to study
the pharmacokinetics of the four molecules of difethialone to
describe their respective elimination kinetic and inquire about
the possibility for improved ecotoxicology. Then, their ability
to inhibit VKORC1 activity has to be confirmed as equivalent
to actual formulation to validate their efficacy.

The context of rodent pest management can widely dif-
fer according to the prevalence of resistant phenotype among
rodent populations, the availability of palatable foods that
can reduce the interest for the baits or even the presence of
non-target species or predators. Hence, it may be interesting
to increase or decrease the persistence of formulated products
to achieve the best compromise between safety and efficiency
appropriate to each ecosystem taking into account resistance,
non-target species presence, a food competition, and the en-
vironment. Nevertheless, how the choice the mixes to test
for each situation among the wide range of mixes that can be
produced with four stereoisomers? It is fundamental to ratio-
nalize the choice of mix composition by mathematic modeling
in order to reduce laboratory animal use in new rodenticide
research and homologation tests. Thus, the end of this study
proposes a modeling approach to optimize rodenticide formu-
lation before animal testing.

# 1. Material and methods

**1.1 Chemicals**

Difethialone with the diastereoisomers ratio of 45% of cis-
isomers (corresponding to the racemic mixture of 1S,3R and
1R,3S isomers) and 55% of trans-isomers (corresponding to

the racemic mixture of 1S,3S and 1R,3R isomers) and pu-
rity above 98% was provided by Liphatech (Pont de Casse,
France). The ratio was confirmed by reversed-phase LC-
MS/MS [11]. Individual stereoisomers of difethialone were
provided by Liphatech by separation of the stereoisomers con-
tained in difethialone mixture by the chiral chromatography
method described below.

HPLC-grade acetonitrile and methanol, acetone, dichloromethane and hexane for analysis were supplied by Merck (Darmstadt, Germany). The cartridges used for solid-phase extraction were Oasis® HLB (Hydrophilic-Lipophilic-Balanced) (1mL) purchased from Waters (Milford, Massachusetts, USA). Isoflurane® and vitamin K1 were from Alcyon, (Miribel, France). Vitamin K1 was converted to vitamin K epoxide according to the method described by Tishler et al. [12]. Purity was estimated by LC/MS using an analytical standard (Cayman Chemical, MI, USA) and was higher than 99%. HPLC grade water was prepared using a Milli-Q plus system, (Millipore, Saint-Quentin en Yvelines, France) and used for preparation of HPLC eluents.

**1.2 Animals**

Eight-week-old male OFA-Sprague Dawley rats (each weigh-
ing 175-200 g) were obtained from a commercial breeder
(Charles Rivers, l'Arbresle, France) and were acclimated for
a minimum period of 7 days. The rats were housed three per
cage under a constant photoperiod (12h of light per day)and
ambient temperature (20°C ±2). Animals were kept in stan-
dard cages (Eurostandard, Type IV, Tecniplast, Limonest,
France), and received standard feed (Scientific Animal Food
and Engineering, reference A04) and water ad libitum.

**1.3 Pharmacokinetic study**

Experimental research on the rats was performed according to
an experimental protocol following international guidelines
and with approval from the ethics committee of the Veterinary
School of Lyon (authorization n°201704190941578).

Male OFA-Sprague Dawley rats received through per os
administration of 3.4 mg/kg of difethialone composed of 45%
of cis- and 55% of trans-isomers dissolved in 10% DMSO
and 90% vegetable oil. Rats were maintained in life by daily
subcutaneous administration of vitamin K1 (5 mg.kg-1). 4,
9, 24, 48, 120, 168 and 216 hours after difethialone admin-
istration, 3 rats were anesthetized with isoflurane and blood
was taken by cardiac puncture into citrated tubes. Finally,
rats were euthanized with CO2 and the liver of each rat was
immediately collected and stored at -20°C until analysis.

**1.4 VKOR activity essay and kinetics**

Liver microsomes were prepared from fresh livers of Sprague
Dawley rats by differential centrifugation according to the pro-
tocol described by Hodroge et al.[13]. Then, the microsomes
are pooled from five individuals. Microsomal vitamin K epox-
ide reductase (VKOR) activity was assayed according to the
protocol described by Hodroge et al. [13, 14]. The inhibiting
effect of the silica gel column-purified cis- or trans-isomers
of difethialone was evaluated by the determination of Ki after
the addition of various concentrations of the anticoagulant to
the standard reaction in the presence of increasing amounts of
vitamin K epoxide (from 0.001 to 0.2 mM) using anticoagu-
lant concentrations from approximately 0.05 to 20 Ki. Three
replications were performed for each stereoisomer.

**1.5 Dosages**

Difethialone stereoisomers are dosed as described in Foure1
et al. [15]

**1.6 Data analysis**

The VKA elimination pharmacokinetics are composed of two
distinct phases, with two different half-lives, an initial phase
during a few days with a half-life of the order of some hours
[8] and a terminal-phase mays last several months with a
half-life of the order of some days considering molecule[16].
Currently, the underlying mechanism is not understood. Con-
sidering ecotoxicological and efficacy studies, only the initial
half-life is relevant as rodents die between five to ten days.
Thus, the initial half-life determines part of the molecule's
effectiveness in killing rodents and the remnant concentration
that can be consumed by a predator. The terminal half-life is
only observed when animals survive (often experimentally by
co-administration of vitamin K). Thus, in this study, we only
consider the initial half-life of the difethialone stereoisomers.
Half-lives are calculated for each stereoisomer with all values
from the time to peak to 216h.

Statistical analyses were performed with R v. 3.1.2. Re-
sults were expressed as mean values ±SD or fitted value and
confidence interval. Constant of inhibitions or regression
curve parameters were compared with Fisher's test. Areas
under the curves are compared with a Student's test corrected
by Bonferroni correction.

**2. Results**

**2.1 Inhibiting activity of the four individual**
**stereoisomers of difethialone**

Prior to our study, the constant of inhibition (Ki) of each
stereoisomer was evaluated in vitro on rat liver microsomes
expressing wild type VKORC1 (Sprague Dawley rats), in
order to confirm their action on vitamin K regeneration cycle.
Values are represented in figure 1. Even though, the Ki of
E2-cis stereoisomer was significantly greater than the three
others (P<0.05) with a Ki about twice as high as the others, all
Ki were in range between 22.37 and 51.98 nM comparable to
those of effective molecules used in the field. As an example,
the Ki value of warfarin, which is ineffective in the field, is
500 nM.

**2.2 Determination of the pharmacokinetics of the**
**four individual stereoisomers of difethialone**

The first step of our study of the four stereoisomers of
difethialone was to compare their respective pharmacokinetic
parameters in rats. At t=0h, 3.4 mg/kg of difethialone has been

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Constant of inhibition of each difethialone stereoisomer for wild Vkorc1 from liver microsomes of laboratory rats. Values are presented with 95% confidence interval. E2-cis Ki is significantly higher than those of three other difethialone stereoisomer (P<0.05).</figcaption></figure>

administered to each rat, then at 4, 9, 24, 48, 120, 168 and 216 hours a group of three rats was euthanized and their liver was collected. The hepatic difethialone concentrations of each stereoisomer versus time are shown in Figure 2 A, B, C and D for respectively the trans isomers (E1-trans and E4-trans) and the cis-isomers (E2-cis and E3-cis). The pharmacokinetic parameters of each stereoisomer were reported in Table 1. To calculate the initial half-life of each stereoisomer concentration we used only values after the peak concentration time in an exponential decay model. The peak concentration time occurred at 9h for E1-trans, E3-cis and E2-cis stereoisomers and at 4h for E4-trans stereoisomer.

Half-life of the E4-trans stereoisomer (6.0 h) is signif-
icantly lower than the one of the three others (P<0.0001).
Likewise, E2-cis half-life is significantly lower compared to
E1-trans and E3-cis stereoisomers (P<0.05). Considering
the area under the curve, stereoisomer AUCs are significantly
different between them (P<0.001).

**2.3 Development of a pharmacokinetic model able
to predict the half-life of a mixture**

Finally, we tested the ability of a simple model made with
elimination constants previously estimated to predict the
difethialone liver concentration versus time after adminis-
tration of a stereoisomers mix. In order to build this model,
we assume that (i) the absorption constant of each of the four
stereoisomers is common (ii) after 24h post oral administra-
tion, the variation of liver difethialone concentration only
depends on the elimination rate (iii) the elimination rate of
each stereoisomer is not influenced by the presence of the
other stereoisomer. Thus, the equation of the difethialone
concentration versus time can be defined as in equation 1. In
this equation, f1 to f4 are the fractions of each stereoisomer
<table><caption>**Table 1.** Pharmacokinetic parameters of each difethialone stereoisomer liver concentration after a single per os administration of 3.4 mg/kg of difethialone composed of 45% of cis- and 55% of trans-isomer. Values are presented with 95% confidence interval.</caption>
   <thead>
    <tr>
     <td>
      Stereoisomer
     </td>
     <td>
      Elimination coefficient (h-1)
     </td>
     <td>
      Initial Half-life (h)
     </td>
     <td>
      R
      <sup>
       2
      </sup>
     </td>
     <td>
      Area under the curve (h x μg/g of liver)
     </td>
    </tr>
   </thead>
   <tbody>
    <tr>
     <td>
      E1-trans
     </td>
     <td>
      0.009 [0.006-0.014]
     </td>
     <td>
      69.3 [48.2-123.4]
     </td>
     <td>
      0.793
     </td>
     <td>
      458.1 [381.1-535.2]
     </td>
    </tr>
    <tr>
     <td>
      E4-trans
     </td>
     <td>
      0.114 [0.083-0.146]
     </td>
     <td>
      6.0 [4.8-8.3]
     </td>
     <td>
      0.959
     </td>
     <td>
      54.5 [43.5-65.6]
     </td>
    </tr>
    <tr>
     <td>
      E2-cis
     </td>
     <td>
      0.027 [0.015-0.039]
     </td>
     <td>
      25.4[17.5-46.1]
     </td>
     <td>
      0.871
     </td>
     <td>
      226.9 [166.1-287.6]
     </td>
    </tr>
    <tr>
     <td>
      E3-cis
     </td>
     <td>
      0.008 [0.004-0.012]
     </td>
     <td>
      82.3 [57.7-143.8]
     </td>
     <td>
      0.777
     </td>
     <td>
      952.9 [806.2-1,099.8]
     </td>
    </tr>
   </tbody>
  </table>

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Difethialone stereoisomer liver concentration over time after a single per os administration of 3.4 mg/kg of difethialone composed of 45% of cis- and 55% of trans-isomers. Values are represented as mean of concentrations ± standard deviation, 100% is set as the max mean of each stereoisomer. Parameters of fitting curves are presented in table 1.</figcaption></figure>

in the product administered per os, and C0 is the theoretical
concentration if the mix would have been administered by
intravenous. To verify this model's ability for liver concen-
tration prediction of stereoisomers mix, we used two pharma-
cokinetic data published in [8]. One of these mixes mainly
contains trans stereoisomers: 47% of E4-trans and 47% of E1-
trans with 3% of each two other stereoisomers, and a cis mix
that is a racemic composition of E3-cis and E2-cis stereoiso-
mers. In this validation, only the 24h concentration is used to
fit the C0 value. Then, fitting indicators were calculating only
with other point concentrations (figure 3). Models have Rp2
of 0.9184 for trans mix and of 0.9100 for cis mix.

**3. Discussion**

The management of rodent populations is based on the use
of anticoagulant rodenticides. Indeed, physical methods as

$$
C(t) = C_0 * (f4 * e^{-0.1147*t} + f1 * e^{-0.009998*t} \\
\qquad + f3 * e^{-0.008419*t} + f2 * e^{-0.0273*t})
$$

1. Equation predicting the concentration of difethialone in rat liver as a function of the time. The constants f1, f2, f3 and f4 are respectively the fraction of E1-trans, E2-cis, E3-cis and E4-trans in the product.).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Liver concentration of difethialone over time after administration of two difethialone mixes: (A) trans mix with 47% of E4-trans, 47% of E1-trans, 3% of E3-cis and 3% of E2-cis and (B) cis mix with 50% of E3-cis and 50% of E2-cis. Points represent mean of experimental values ± standard deviation. Green, blue, yellow and red areas represent respectively the proportion of E4-Trans, E1-trans, E3-cis and E4-trans stereoisomers liver concentrations obtained with the model. The R² of the model are 0.9184 for trans mix and 0.9100 for cis mix.</figcaption></figure>

trapping are limited to small invasions of rodents [17]. In addition, there are many problems with the effectiveness of other chemical rodenticides. For example, due to their rapid action, their use results in an association between the bait and its toxicity, leading for the rodent to an aversion to the consumption of the bait [18]. Furthermore, in the case of human or animal intoxication, other chemical rodenticides have no antidote, unlike anticoagulant rodenticides [18]. The FGARs are few remnant, but also hardly efficient when many rodent populations with a resistant mutation of Vkorc1 have emerged in the pas [19]. Conversely, since they were discovered, the SGARs have been categorized as highly tissue persistent and effective in a single administration at lower concentrations compared to FGARs, including against rodents with resistant mutation [13]. These characteristics have established the renown of second-generation molecules in the management of rodent populations. Nevertheless, their reluctance is both an advantage for their efficacy and a drawback for their ecotoxicology. The new approach proposed recently by

Damin-Pernik et al. [7, 8], based on the diastereomers of existing SGARs to reduce their ecotoxicology, is here expanded to the four stereoisomers of difethialone.

The capacity of each difethialone stereoisomer to inhibit the wild rat Vkorc1 enzyme activity has been determined in vitro on a fresh rat liver microsome. Each stereoisomer inhibits the enzyme with a Ki within a range of 20 to 60 nM. These constant of inhibition are similar to those of the SGARs and chlorophacinone which are used in the field. Indeed, Ki of difenacoum, chlorophacinone and bromadiolone are respectively of 30, 40 and 70 nM [13].

Our results show that each stereoisomer has its own initial half-life, which is included between 6.0h and 82.3h. Thus, difethialone E4-trans stereoisomer has a comparable half-life to warfarin enantiomers. Indeed, E4-trans half-life is 6.0h compared to 7.6h and 11h half-life of both warfarin enantiomers [20]. However, the four difethialone stereoisomers reveal a lower Ki than warfarin (500 nM)[13]. Regarding E2-cis and E3-cis, which compose the commercial difethialone currently used, E3-cis half-life is three times greater than E2-cis half-life. This important difference may explain the biphasic elimination described when commercial difethialone liver elimination was studied [16] E2-cis would be early eliminated, constituting the first elimination stage, followed by E3-cis late efflux leading to the second elimination step. These results push us toward questioning the assessment of composed products half-life. Indeed, the half-life of the stereoisomers mix depends on the chosen range of measurement. Thus, for the first time, the global half-life of a mix is linked to the ratio and the half-life of the less remnant stereoisomer. Consequently, as the global half-life of a stereoisomer mix is increasing over time, the mix should not only be assessed by its final half-life. Indeed, with this unique indicator, it is not possible to estimate what is the amount of product remaining in the rodent when it dies and being at risk for the environment. Moreover, if the only aim is to reduce the persistence of the product, the choice of E4-trans stereoisomer is obvious since it is the less remnant difethialone stereoisomer (Figure 2 and Table 1). However, the area under the curve of such products would be so low that its efficiency would be marred.

In our opinion, two new pharmacokinetic indicators have to be designed in order to assess both the presumptive efficacy and the presumptive persistence of an anticoagulant. First, to assess the presumptive efficacy, we propose to consider the relative AUC between 24h and 120h. The goal of this indicator is to confirm that, if rodents eat the target concentration of bait, the AUC within 5 days after the ingestion is sufficient to conduct to its death. The second one is the percent of the 24 h concentration remaining at 120h after administration. Indeed, the death of rodents occurs most of the time from this fifth day after administration. Consequently, this indicator should be minimized and the AUC should be maximized.

We created a model based on the half-lives of each stereoisomer (equation 1). This model has been validated with experimental data of pharmacokinetic of difethialone
products with different stereoisomers ratios. Thus, we were able to simulate the elimination pharmacokinetics of different hypothetic difethialone mixes. These simulations allowed us to determine the best theoretical mix composition. While such simulations do not replace pharmacokinetic or efficacy studies, they can help to reduce the number of tests on animals, in accordance with the 3R principle. Once the constraints of efficacy and ecotoxicity have been defined, which is equivalent to defining a minimum AUC (efficacy) and a maximum anticoagulant concentration at the time the rodent pest begins to be symptomatic and die (ecotoxicity). The model makes it possible to target the best stereoisomer mixtures to be tested, which allows sparing laboratory animals by avoiding to test inefficient bait or bait with an ecotoxicity not adapted to the intended use.

To preselect some mixes with our model, we determined the best stereoisomers composition to maximize the AUC, according to a fixed persistence of difethialone at 120h. The results of these simulations are presented in Figure 4. According to this diagram, it is possible to design rodenticides with a wide range of AUC and persistence. The diagram pinpoints the importance of E2-cis stereoisomer in the design of new products. Indeed, with combinations of E2-cis and one of the trans stereoisomers (E1-trans or E4-trans), it is possible to design a large range of products adapted to different situations. In a vulnerable environment with few food competition (rodents eat the bait several times), a rodent population with moderate or no resistance might be controlled with a moderate persistence bait (E2-cis and E1-trans). Conversely, to control resistant populations or in environments where a one-shot bait is mandatory (important food competition), the use of more persistent baits would be more relevant to reach or maintain sufficient hepatic concentrations (E4-trans fraction and E2-cis fraction, but still without E3-cis which is really too persistent and even almost not eliminable). Obviously, the level of actions to prevent non-target poisoning has to be in accordance with the persistence of the used product.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Evolution of difethialone AUC between 24 and 120h (curve and left axis) simulated with the best bait mix of the four difethialone stereoisomers (color area and right axis) for a fixed residual concentration of difethialone at 120h (in percent of the 24h concentration).</figcaption></figure>

However, these kinds of simulation have some limitations and can only be lifted by confirming the efficacy and per-

sistence of the product directly on target species. The first limitation is the conditions of the pharmacokinetics studies, especially the administered dose, limited information is available on the self-induction of AR elimination. If such modulation exists, the pharmacokinetics of AR might be different according to the administered dose. The second main limitation is the behavior of wild rodent regarding to the poisoned bait. All new baits have to be tested in vivo in at least a baits choice feeding efficacy test, to check that the bait palatability is sufficient to ensure an adequate intake of active products in one or more times.

In conclusion, this study shows that four difethialone stereoisomers are able to inhibit the wild Vkorc1 rat enzyme with the same efficiency. Moreover, the pharmacokinetics of these stereoisomers are widely different which allows the creation of new baits that might assure the efficiency of difethialone and modulate a persistence adapted to the treated environment. Finally, this article presents a model to select better difethialone formulation. The developed model brings a new powerful method to design more efficient ARs and could be applied to other ARs stereoisomers formulation studies.

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