Design of a structure-activity relationship model of vitamin K epoxide reductase (VKORC1) inhibitors combining chemical synthesis of new compounds, enzymatic assays and molecular modelling.

Vitamin K antagonists (VKAs) anticoagulants have been used since the 1950s as medicines and rodenticides. These molecules are mainly 4-hydroxycoumarin derivatives and act by inhibiting the vitamin K epoxide reductase (VKORC1), an endoplasmic reticulum membrane resident enzyme. However, many VKORC1 mutations have been reported over the last decade, inducing VKAs resistances and thus treatments failures. Although studies have reported experimental and computational investigations of VKAs based on VKORC1 structural homology models, the development of new effective anticoagulants has been quite complex due to the lack of structural data and reliable structure-activity relationships. However, the recent publication of VKORC1 crystal structure provides new information for further studies. Based on these findings, we combined chemical synthesis, enzymatic assays and molecular modelling methods to design a structure-activity relationship (SAR) model. Our results proved that the lipophilicity, the membrane permeability of inhibitors and their affinity towards human VKORC1 enzyme are the main characteristics for potent anticoagulants. Our SAR model managed to rank compounds according to their ability to inhibit the human VKORC1. Such a tool might constitute an alternative to evaluate new molecules potency before their chemical synthesis and biological assessment and might assist the development of new VKAs.

Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra- and inter-species model to predict the suitable formulation mix.

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

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.

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 6.0 h, 25.4 h, 69.3 h, and 82.3 h 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 result in a more eco-friendly product than current commercially available difethialone formulations. In addition, we put forward modelling to help design bait according to the circumstance of use (presence of non-target species, food competition, etc.) by modulating the theorical AUC and and the theorical 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.

Management of Rodent Populations by Anticoagulant Rodenticides: Toward Third-Generation Anticoagulant Rodenticides.

Second-generation anticoagulant rodenticides (SGARs) have been used since the 1980s for pest management. They are highly efficient even in warfarin-resistant rodents. Nevertheless, because of their tissue persistence, nontarget poisoning by SGARs is commonly described in wildlife. Due to this major problem, a new generation of anticoagulants must be developed to limit this risk. This study proposes a method of developing a new generation of anticoagulant rodenticides by revisiting the old SGARs based on the concept of stereochemistry. Each current SGAR is a mixture of diastereomers. Diastereomers of each compound were purified, and their biologic properties were compared by determining their ability to inhibit vitamin K epoxide reductase (VKOR) activity involved in the activation of vitamin K-dependent clotting factors and their toxicokinetic properties. Systematically, for each SGAR, both diastereomers are as effective in inhibiting VKOR activity. However, their toxicokinetic properties are very different, with one of the two diastereomers always more rapidly cleared than the other one. For all SGARs except flocoumafen, the less persistent diastereomer is always the less predominant isomer present in the current mixture. Therefore, the development of baits containing only the less persistent diastereomer would avoid the ecotoxicological risk associated with their use without decreasing their efficacy.

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.

Interest of the faecal and plasma matrix for monitoring the exposure of wildlife or domestic animals to anticoagulant rodenticides.

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGAR), are known to be a potential threat to unintended species due to their tissue persistence. The liver is the storage tissue of ARs and is a matrix of choice in diagnosing exposure and intoxication of non-target fauna. However, it is only available on dead animals. Blood and faeces can be used on living animals. These two biological matrices were compared in terms of their relevance to exposure to ARs. In addressing this question, we compared the faecal, plasma and liver concentrations of bromadiolone, one of the SGAR frequently implicated in wildlife exposure. We studied this comparison at the individual level and at the population level, considering three influencing factors: dose, sex and time. Our findings demonstrate that faecal analyses are more valuable than plasma analyses for monitoring AR exposure of domestic and wild animals, even if faecal concentrations cannot be correlated with liver concentrations.

Oil removal from water by selective sorption on hydrophobic cotton fibers. 1. Study of sorption properties and comparison with other cotton fiber-based sorbents.

Hydrophobic cotton fibers, obtained by acylation of cellulose with fatty acid using microwaves radiations, have a high selective affinity for vegetable or mineral oil, fuel, and petroleum, in aqueous medium. Their sorption capacity (SC) (weight of liquid picked up by a given weight of sorbent) is about 20 g/g, after draining. They are reusable after simple squeezing, and their SC reaches a constant value, ca. 12 g/g. Moreover, this product is stable in water, whereas raw cotton can develop molds, after oil sorption. Besides, it is also biodegradable.

Oil removal from water by sorption on hydrophobic cotton fibers. 2. Study of sorption properties in dynamic mode.

The recovery of oil from an oil-in-water emulsion, during a flow through a bed of cotton rendered hydrophobic by acylation of cellulose was defined by sorption and coalescence phenomena. During percolation, the column "hold-up" (difference between injected and rejected oil) became constant at the equilibrium volume, i.e., as soon as the instant oil concentration in the effluent (C) was equal to the oil concentration in the initial emulsion (C0). This equilibrium permitted the measurement of the cotton sorption capacity (SC), which increased with C0 up to the cotton saturation. The oil-water separation improved at a lower temperature, lower flow, a deeper medium, and larger oil drops. The system was modeled as a piston flow-through in order to generalize the results.