Stéphane Besse

Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum.

Difenacoum, an antivitamin K anticoagulant, has been widely used as rodenticide to manage populations of rodents. Difenacoum belongs to the second generation of anticoagulant, and, as all the molecules belonging to the second generation of anticoagulant, difenacoum is often involved in primary poisonings of domestic animals and secondary poisonings of wildlife by feeding contaminated rodents. To develop a new and ecofriendly difenacoum, we explored in this study the differences in properties between diastereomers of difenacoum. Indeed, the currently commercial difenacoum 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 their efficiency to inhibit the coagulation of rodents were explored. Tissue persistence of trans-isomers was shown to be shorter than that of cis-isomers with a half-life fivefold shorter. Efficiency to inhibit the vitamin K epoxide reductase activity involved in the coagulation process was shown to be similar between cis- and trans-isomers. The use of trans-isomers of difenacoum allowed to drastically reduce difenacoum residues in liver and other tissues of rodents when the rodent is moribund. Therefore, secondary poisonings of wildlife should be decreased by the use of difenacoum largely enriched in trans-isomers.

New insights into the catalytic mechanism of vitamin K epoxide reductase (VKORC1) - The catalytic properties of the major mutations of rVKORC1 explain the biological cost associated to mutations

The systematic use of antivitamin K anticoagulants (AVK) as rodenticides caused the selection of rats resistant to AVKs. The resistance is mainly associated to genetic polymorphisms in the Vkorc1 gene encoding the VKORC1 enzyme responsible for the reduction of vitamin K 2,3‐epoxide to vitamin K. Five major mutations, which are responsible for AVK resistance, have been described. Possible explanations for the biological cost of these mutations have been suggested. This biological cost might be linked to an increase in the vitamin K requirements. To analyze the possible involvement of VKORC1 in this biological cost, rVKORC1 and its major mutants were expressed in Pichia pastoris as membrane‐bound proteins and their catalytic properties were determined for vitamin K and 3‐OH‐vitamin K production. In this report, we showed that mutations at Leu‐120 and Tyr‐139 dramatically affect the vitamin K epoxide reductase activity. Moreover, this study allowed the detection of an additional production of 3‐hydroxyvitamin K for all the mutants in position 139. This result suggests the involvement of Tyr‐139 residue in the second half‐step of the catalytic mechanism corresponding to the dehydration of vitamin K epoxide. As a consequence, the biological cost observed in Y139C and Y139S resistant rat strains is at least partially explained by the catalytic properties of the mutated VKORC1 involving a loss of vitamin K from the vitamin K cycle through the formation of 3‐hydroxyvitamin K and a very low catalytic efficiency of the VKOR activity.

Origin of the gender differences of the natural resistance to antivitamin K anticoagulants in rats.

Vitamin K antagonists (VKA) are used either in human medicine to prevent thromboembolic disorders or as rodenticides for pest control management. In rodents, female rats are described to be more tolerant to the action of vitamin K antagonists than males. Nevertheless, the mechanism of this greatest tolerance is still unknown and this study aims to identify the origin of this greatest tolerance after VKA administration. Therefore, difethialone, one of the most powerful VKA was used in this study. A possible difference in the pharmacokinetics of difethialone between males and females was first investigated. The determination of the pharmacokinetic parameters allowed to exclude a pharmacokinetic origin of the greatest tolerance of females to VKA. Thus, a natural resistance to difethialone of the liver VKOR activity, which is the target of VKA, was thus explored in females. The determination of Ki towards difethialone in liver microsomes allowed to also exclude this hypothesis. Therefore, equipment in vitamin K-dependent clotting factors and properties of vitamin K-dependent clotting factors were explored. Basal activity of clotting factors VII and X were found significantly higher in females of respectively 43% and 21%. Moreover, after VKA administration, half-lives of clotting factors II and X were found significantly longer in females of respectively 27% and 10% and a lag time of 4h before the beginning of the decay of factor VII was observed only in females after difethialone administration. The greater tolerance of female rats to VKA is thus due a stronger basal pool of vitamin K-dependent clotting factors VII and X and to a slower decline of vitamin K-dependent clotting factors II, VII and X after VKA administration.

Study of the efficiency of anticoagulant rodenticides to control Mus musculus domesticus introgressed with Mus spretus Vkorc1.

BACKGROUND Antivitamin K anticoagulant (AVK) rodenticides are commonly used to control rodent pests worldwide. They specifically inhibit the VKORC1 enzyme essential for the recycling of vitamin K, and thus prevent blood clotting and cause death by haemorrhage. Numerous mutations or polymorphisms of the Vkorc1 gene were reported in rodents, and some led to resistance to rodenticides. In house mice (Mus musculus domesticus), adaptive introgression of the Vkorc1 gene from the Algerian mouse (Mus spretus) was reported. This adaptive introgression causes the substitution of four amino acids in M. musculus domesticus. RESULTS The consequences of introgression were assessed by (i) the characterisation of the in vivo resistant phenotype of adaptive Vkorc1spr -introgressed mice, (ii) the characterisation of the ex vivo resistance phenotype of the liver VKOR activity and (iii) the comparison of these results with the properties of recombinant VKORC1spr protein expressed in yeast. The resistance factor (from 1 to 120) induced by the four introgressed polymorphisms obtained using these three approaches was dependent on the AVKs used but were highly correlated among the three approaches. CONCLUSION The four introgressed polymorphisms were clearly the cause of the strong resistant phenotype observed in the field. In the context of strong selection pressure due to the extensive use of AVKs, this resistant phenotype may explain the widespread distribution of this genotype from Spain to Germany.

Synthesis and biological evaluation of C-3 aliphatic coumarins as vitamin K antagonists

Since the discovery of Warfarin in the 1940s, the design of new warfarin-derived anticoagulants for rodent management has been challenging, with mainly structural modifications performed on the C3 position of the coumarin skeleton. In order to better understand the pharmacomodulation of such derivatives, we have synthesized a family of C3 (linear and branched) alkyl-4-hydroxycoumarins, which led to the identification of compounds 5e and 5f as potential short-term active anticoagulants.