Jérôme Henri

Low or High Doses of Cefquinome Targeting Low or High Bacterial Inocula Cure Klebsiella pneumoniae Lung Infections but Differentially Impact the Levels of Antibiotic Resistance in Fecal Flora

ABSTRACT The combination of efficacious treatment against bacterial infections and mitigation of antibiotic resistance amplification in gut microbiota is a major challenge for antimicrobial therapy in food-producing animals. In rats, we evaluated the impact of cefquinome, a fourth-generation cephalosporin, on both Klebsiella pneumoniae lung infection and intestinal flora harboring CTX-M-producing Enterobacteriaceae. Germfree rats received a fecal flora specimen from specific-pathogen-free pigs, to which a CTX-M-producing Escherichia coli strain had been added. K. pneumoniae cells were inoculated in the lungs of these gnotobiotic rats by using either a low (105 CFU) or a high (109 CFU) inoculum. Without treatment, all animals infected with the low or high K. pneumoniae inoculum developed pneumonia and died before 120 h postchallenge. In the treated groups, the low-inoculum rats received a 4-day treatment of 5 mg/kg of body weight cefquinome beginning at 24 h postchallenge (prepatent phase of the disease), and the high-inoculum rats received a 4-day treatment of 50 mg/kg cefquinome beginning when the animals expressed clinical signs of infection (patent phase of the disease). The dose of 50 mg/kg targeting the high K. pneumoniae inoculum cured all the treated rats and resulted in a massive amplification of CTX-M-producing Enterobacteriaceae. A dose of 5 mg/kg targeting the low K. pneumoniae inoculum cured all the rats and averted an outbreak of clinical disease, all without any amplification of CTX-M-producing Enterobacteriaceae. These findings might have implications for the development of new antimicrobial treatment strategies that ensure a cure for bacterial infections while avoiding the amplification of resistance genes of human concern in the gut microbiota of food-producing animals.

Comparative Cytotoxicity, Oxidative Stress, and Cytokine Secretion Induced by Two Cyanotoxin Variants, Microcystin LR and RR, in Human Intestinal Caco-2 Cells

While MC‐LR and MC‐RR share significant structural similarity, MC‐RR is less cytotoxic than MC‐LR. In the current study, we have compared the effects of MC‐LR and MC‐RR in Caco‐2 cells by evaluating cytotoxicity, oxidative stress (reactive oxygen species production), and the cellular proinflammatory response (IL‐6 and IL‐8 production). Following treatment with 100 µM microcystins (MC), cytotoxicity was two‐fold greater with MC‐LR as compared to MC‐RR after 24 h exposure. Whereas the reactive oxygen species production and IL‐6 secretion were similar following a 24‐h treatment with either MC, 100 µM MC‐LR induced a five‐fold greater IL‐8 secretion when compared to MC‐RR. Our study has demonstrated that, although both MC‐LR and MC‐RR induced some cytotoxicity in human intestinal cells, a major difference in IL‐8 production was observed between the two variants.

Bioavailability, distribution and depletion of monensin in chickens.

The pharmacokinetics of monensin including apparent volume of distribution, total body clearance, systemic bioavailability, partition coefficients and tissue residues were determined in chickens. The drug was given by intravenous injection in the left wing vein at the dose of 0.46 mg/kg and by intracrop administration at the dose of 4 mg/kg according to a destructive sampling. The pharmacokinetic variables were compared after noncompartmental, naïve averaged, naïve pooled and nonlinear mixed-effects modelling analyses. Partition coefficients and tissue residues were determined after a treatment with feed additives (125 mg/kg of feed) of 33 days. The clearance, volume of distribution and bioavailabilty were approximately 2.2 L/h/kg, approximately 9 L/kg and approximately 30% respectively except with nonlinear mixed effects models that presented values of 1.77 L/h/kg, 14.05 L/kg and 11.36% respectively. Tissue/plasma partition coefficients were estimated to 0.83, 3.39 and 0.51 for liver, fat and thigh muscle respectively. Monensin residues after treatment were not detected 6 h after withdrawal except for fat where monensin was still quantifiable 12 h after. Pharmacokinetic variables seem to be inaccurate when assessed with non linear mixed-effects modelling associated to destructive sampling in chickens. Values varied slightly with noncompartmental, naïve averaged and naïve pooled analyses. The absorption, elimination and partition parameters will be incorporated into a physiologically based pharmacokinetic model and the depletion study will be used to test the ability of this model to describe monensin residues in edible tissues under different dosage regimens.

Comparison of the oral bioavailability and tissue disposition of monensin and salinomycin in chickens and turkeys

The current study describes the pharmacokinetic parameters of two carboxylic polyether ionophores: monensin in turkeys and salinomycin in chickens. These data can be used to understand and predict the occurrence of undesirable residues of coccidiostats in edible tissues of these animal species. Special attention is paid to the distribution of residues between the different edible tissues during and at the end of the treatment period. For the bioavailability studies, monensin was administered to turkeys intravenously, in the left wing vein, at a dose of 0.4 mg /kg and orally at a dose of 20 mg /kg. Salinomycin was administered to chickens intravenously, in the left wing vein, at a dose of 0.25 mg /kg and orally at a dose of 2.5 mg /kg. Residue studies were carried out with supplemented feed at the rate of 100 mg /kg of feed for monensin in turkeys and 70 mg /kg for salinomycin in chickens, respectively. Coccidiostats had a low bioavailability in poultry (around 30% for monensin in chickens, around 1% for monensin in turkeys and around 15% for salinomycin in chickens). Monensin in chickens had a longer terminal half-life (between 3.07 and 5.55 h) than both monensin in turkeys (between 1.36 and 1.55 h) and salinomycin in chickens (between 1.33 and 1.79 h). The tissue /plasma partition coefficients showed a higher affinity of both monensin and salinomycin for fat, followed by liver and muscle tissue. The depletion data showed a fairly rapid elimination of coccidiostats in all the tissues after cessation of treatment. According to the results of depletion studies, a withdrawal period of 1 day seems sufficient to avoid undesirable exposure of consumers.

A physiologically based pharmacokinetic model for chickens exposed to feed supplemented with monensin during their lifetime

We developed a flow-limited physiologically based pharmacokinetic model for residues of monensin in chickens and evaluated its predictive ability by comparing it with an external data set describing concentration decays after the end of treatment. One advantage of this model is that the values for most parameters (34 of 38) were taken directly from the literature or from field data (for growth and feed intake). Our model included growth (changes in body weight) to describe exposure throughout the life of the chicken. We carried out a local sensitivity analysis to evaluate the relative importance of model parameters on model outputs and revealed the predominant influence of 19 parameters (including three estimated ones): seven pharmacokinetic parameters, five physiological parameters and seven animal performance parameters. Our model estimated the relative bioavailability of monensin as feed additive at 3.9%, which is even lower than the absolute bioavailability in solution (29.91%). Our model can be used for extrapolations of farming conditions, such as monensin supplementation or building lighting programme (which may have a significant impact for short half-life molecules such as monensin). This validated PBPK model may also be useful for interspecies extrapolations or withdrawal period calculations for modified dosage regimens.

Cytochrome P450-dependent metabolism of monensin in hepatic microsomes from chickens and turkeys.

Monensin is a polyether ionophore used as feed additive to prevent coccidiosis in poultry. Monensin is safe and effective for the target species when used at recommended dosages but the therapeutic index of this ionophore is rather narrow and several accidental poisonings are quoted in the literature involving horses, pigs, cattle, turkeys, sheep, goats and rabbits (Langston et al., 1985). These recommended dosages, in addition to the safety and efficiency of use, are established to prevent the presence of unwanted residues (with the respect of a withdrawal time) because of the cardiovascular system (Pressman, 1980) perturbations provoked by coccidiostats even at low dose. To predict residual concentrations of monensin in the organs of birds and then to assess the scenarios of contamination of food as a risk for the consumer, a physiologically based pharmacokinetic (PBPK) model describing the disposition of monensin in chickens and turkeys is currently under investigation. The aim of the present study is to quantitate the hepatic metabolism of the parent compound, using hepatic microsomes. The description of the metabolic clearance is essential in the model to evaluate its part in the total body clearance of monensin. Especially as its metabolites are not of toxicological interest, it is interesting to follow monensin disappearance. A metabolic balance (Davison, 1984) established in colostomized and bile-cannulated chickens indicated a fairly rapid excretion of the radioactivity mainly in the faeces (about 96%) and to a limited extent in the urine (about 1%). Extensive biotransformation studies of monensin in the rat, chicken and turkey have been conducted in order to identify the metabolites (Donoho et al., 1978). Recently (Anonymous, 2004), metabolites in chicken have been quantified using HPLC ⁄ESI-MS. These data indicate that monensin gives rise to nine metabolites. It has been decided to measure monensin depletion rather than formation of metabolites as usually presented in comparable studies because of the large number of metabolites. Furthermore, according to the parsimony principle, it simplifies the PBPK model and it is sufficient to follow the behaviour of the parent compound, which is of particular interest as a marker of total residues. All these reasons explain the choice of the substrate depletion approach (Obach & Reed-Hagen, 2002). The metabolites result from demethylation and a subsequent oxidation, decarboxylation, and from mono or dihydroxylation at different positions of the rings, which correspond essentially to the O-demethylation and simultaneous hydroxylation. These data also indicate that the metabolic pathways are very similar in chicken, turkey and rat. From a quantitative point of view, all metabolites identified in the chicken liver represented each less than 10% of the labelling and unchanged monensin contributed to about 5% only (Anonymous, 2004). Livers were removed from Ross chickens of 7 weeks of age and BUT9 turkeys of 12 weeks old. Six birds of each species were used to obtain three samples of two pooled livers per species. Each sample was obtained with two livers, one from a bird of each sex.

The present and future of withdrawal period calculations for milk in the European Union: Focus on heterogeneous, nonmonotonic data

Harmonization of the method for calculating the withdrawal period for milk dates from the 1990s. European harmonization has led to guidance with three accepted methods for determining the withdrawal period for milk that are currently applicable. These three methods can be used by marketing authorization holders, but, in some cases, their diversity can lead to very different withdrawal periods. This is particularly the case when concentrations in milk are nonmonotonic and heterogeneous, meaning that concentrations strictly increase and then strictly decrease with significant interindividual variability in the time to reach the maximal concentration. Here, we first describe the concepts associated with the different methods used in the harmonized approach currently applicable for the determination of milk withdrawal periods, and then, we propose the application of a modern pharmacometric tool. Finally, with a nonmonotonic heterogeneous dataset, we illustrate the usefulness of this tool in comparison with the three currently applicable methods and discuss the limitations and advantages of each method.

Tools to evaluate pharmacokinetics data for establishing maximum residue limits for approved veterinary drugs: examples from JECFA's work

Maximum residue limits (MRLs) for residues of veterinary drugs are the maximum concentrations of residues permitted in or on a food by national or regional legislation. In the process of MRLs recommendations by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), analysis of pharmacokinetic data describing the ADME process (absorption, distribution, metabolism and excretion) is a crucial step and requires the use of different pharmacokinetic tools. The results of animal metabolism studies are the prime determinants of the residue definition in food commodities. Substances labelled with radioactive isotopes are used so that the disposition of the residue can be followed as total residue and main metabolites concentrations. Residue depletion studies with radiolabelled parent drug will lead to the estimate of the time course of the total residue and to determine a marker residue. Depletion studies with an unlabelled drug provide more information on the time course of the marker residue in raw commodities after administration under approved practical conditions of use. By use of this information and after conversion with the total/residue marker ratio, MRLs are derived by comparison of the acceptable daily intake with the daily intakes calculated with different scenarios of dietary exposure. Progress in pharmacokinetic model such as physiologically based pharmacokinetics and population pharmacokinetics will drive the future research in this field to improved veterinary drug development. Copyright

A Population WB-PBPK Model of Colistin and its Prodrug CMS in Pigs: Focus on the Renal Distribution and Excretion

PurposeThe objective was the development of a whole-body physiologically-based pharmacokinetic (WB-PBPK) model for colistin, and its prodrug colistimethate sodium (CMS), in pigs to explore their tissue distribution, especially in kidneys.MethodsPlasma and tissue concentrations of CMS and colistin were measured after systemic administrations of different dosing regimens of CMS in pigs. The WB-PBPK model was developed based on these data according to a non-linear mixed effect approach and using NONMEM software. A detailed sub-model was implemented for kidneys to handle the complex disposition of CMS and colistin within this organ.ResultsThe WB-PBPK model well captured the kinetic profiles of CMS and colistin in plasma. In kidneys, an accumulation and slow elimination of colistin were observed and well described by the model. Kidneys seemed to have a major role in the elimination processes, through tubular secretion of CMS and intracellular degradation of colistin. Lastly, to illustrate the usefulness of the PBPK model, an estimation of the withdrawal periods after veterinary use of CMS in pigs was made.ConclusionsThe WB-PBPK model gives an insight into the renal distribution and elimination of CMS and colistin in pigs; it may be further developed to explore the colistin induced-nephrotoxicity in humans.

Lack of experimental evidence to support mcr-1-positive escherichia coli strain selection during oral administration of colistin at recommended and higher dose given by gavage in weaned piglets

In this study, we assessed the selective effect of colistin administered orally to healthy weaned piglets harbouring an intestinal mcr-1-positive Escherichia coli strain. Maximum recommended dose and a higher dose often used in European pig farms were given by gavage. No selection of the mcr-1-positive strain was observed in our controlled conditions, irrespective of the dose. Further investigations in real farming conditions seem necessary.