Pharmacokinetic-pharmacodynamic cutoff values for benzylpenicillin in horses to support the establishment of clinical breakpoints for benzylpenicillin antimicrobial susceptibility testing in horses.

Introduction: The aim of this international project was to establish a species-specific Clinical Breakpoint for interpretation of Antimicrobial Susceptibility Testing of benzylpenicillin (BP) in horses. Methods: A population pharmacokinetic model of BP disposition was developed to compute PK/PD cutoff values of BP for different formulations that are commonly used in equine medicine around the world (France, Sweden, USA and Japan). Investigated substances were potassium BP, sodium BP, procaine BP, a combination of procaine BP and benzathine BP and penethamate, a prodrug of BP. Data were collected from 40 horses that provided 63 rich profiles of BP corresponding to a total of 1022 individual BP plasma concentrations. Results: A 3-compartment disposition model was selected. For each of these formulations, the PK/PD cutoff was estimated for different dosage regimens using Monte Carlo simulations. The fAUC/MIC or fT>MIC were calculated with a free BP fraction set at 0.4. For fAUC/MIC, a target value of 72 h (for a 72h treatment) was considered. For fT>MIC, efficacy was assumed when free plasma concentrations were above the explored MIC (0.0625-2 mg/L) for 30 or 40 % of the dosing interval. For continuous infusion, a fT>MIC of 90 % was considered. It was shown that a PK/PD cutoff of 0.25 mg/L can be achieved in 90 % of horses with routine regimen (typically 22,000 IU/kg or 12.4 mg/kg per day) with IM procaine BP once a day (France, Japan, Sweden but not USA1) and with IM sodium BP at 14.07 mg/kg, twice a day or IV sodium BP infusion of 12.4 mg/kg per day. In contrast, penethamate and the combination of procaine BP and benzathine BP were unable to achieve this PK/PD cutoff not even an MIC of 0.125 mg/L. Discussion: The PK/PD cutoff of 0.25 mg/L is one dilution lower than the clinical breakpoint released by the CLSI (0.5 mg/ L). From our simulations, the CLSI clinical breakpoint can be achieved with IM procaine BP twice a day at 22,000 IU i.e. 12.4 mg/kg.

Doxycycline serum protein binding in pigs reveals a relatively high free fraction.

Doxycycline is an antibiotic widely used in pig farming. As with all antibiotics, only the free concentrations are considered to be bacteriologically active. Historically, the free fraction (fu) in pig plasma has been estimated at 7%, which, given the effective dosage regime used in pigs, leads to free plasma concentrations of doxycycline largely lower than the minimum inhibitory concentrations of the target pathogens. This apparent inconsistency led us to reassess plasma protein binding of doxycycline in pigs. Using an equilibrium dialysis method, the extent of doxycycline binding was measured individually in 26 pigs for total doxycycline concentration ranging from 10 to 1000 µmol/L. Analysis of the data using a non-linear mixed-effects model demonstrated linearity of plasma protein binding with a mean fu value of 31% and a relatively low inter-subject variability of approximately 10%. This new data showing that the free fraction is four times greater than what could have been anticipated from historical data is discussed in particular for the calculation of the PK/PD cut-offs, which are used to establish the clinical breakpoints for antimicrobial susceptibility testing.

Comparison of toxicokinetic properties of eleven analogues of Bisphenol A in pig after intravenous and oral administrations.

Due to the restrictions of its use, Bisphenol A (BPA) has been replaced by many structurally related bisphenols (BPs) in consumer products. The endocrine disrupting potential similar to that of BPA has been described for several bisphenols, there is therefore an urgent need of toxicokinetic (TK) data for these emerging BPs in order to evaluate if their internal exposure could increase the risk of endocrine disruption. We investigated TK behaviors of eleven BPA substitutes (BPS, BPAF, BPB, BPF, BPM, BPZ, 3-3BPA, BP4-4, BPAP, BPP, and BPFL) by intravenous and oral administrations of mixtures of them to piglets and serial collection of blood over 72 h and urine over 24 h, to evaluate their disposition. Data were analyzed using nonlinear mixed-effects modeling and a comparison was made with TK predicted by the generic model HTTK package. The low urinary excretion of some BPs, in particular BPM, BPP and BPFL, is an important aspect to consider in predicting human exposure based on urine biomonitoring. Despite their structural similarities, for the same oral dose, all BPA analogues investigated showed a higher systemic exposure (area under the plasma concentration-time curve (AUC) of the unconjugated Bisphenol) than BPA (2 to 4 fold for 3-3BPA, BPAF, BPB and BPZ, 7-20 fold for BP4-4, BPAP, BPP, BPFL, BPF and BPM and 150 fold for BPS) due mainly to a considerable variation of oral bioavailability (proportion of BP administered by oral route that attains the systemic circulation unchanged). Given similarities in the digestive tract between pigs and humans, our TK data suggest that replacing BPA with some of its alternatives, particularly BPS, will likely lead to higher internal exposure to potential endocrine disruptive compounds. These findings are crucial for evaluating the risk of human exposure to these emerging BPs.

Population Pharmacokinetics of Intravenous Amoxicillin Combined With Clavulanic Acid in Healthy and Critically Ill Dogs.

Background: Data regarding antimicrobial pharmacokinetics (PK) in critically ill dogs are lacking and likely differ from those of healthy dogs. The aim of this work is to describe a population PK model for intravenous (IV) amoxicillin-clavulanic acid (AMC) in both healthy and sick dogs and to simulate a range of clinical dosing scenarios to compute PK/PD cutoffs for both populations. Methods: This study used a prospective clinical trial in normal and critically ill dogs. Twelve client-owned dogs hospitalized in the intensive care unit (ICU) received IV AMC 20 mg/kg every 8 h (0.5-h infusion) during at least 48 h. Eight blood samples were collected at predetermined times, including four trough samples before the next administration. Clinical covariates and outcome were recorded, including survival to discharge and bacteriologic clinical failure. Satellite PK data were obtained de novo from a group of 12 healthy research dogs that were dosed with a single AMC 20 mg/kg IV. Non-linear mixed-effects model was used to estimate the PK parameters (and the effect of health upon them) together with variability within and between subjects. Monte Carlo simulations were performed with seven dosage regimens (standard and increased doses). The correlation between model-derived drug exposure and clinical covariates was tested with Spearman's non-parametric correlation analysis. Outcome was recorded including survival to discharge and bacteriologic clinical failure. Results: A total of 218 amoxicillin concentrations in plasma were available for healthy and sick dogs. A tricompartmental model best described the data. Amoxicillin clearance was reduced by 56% in sick dogs (0.147 L/kg/h) compared with healthy dogs (0.336 L/kg/h); intercompartmental clearance was also decreased (p <0.01). None of the clinical data covariates were significantly correlated with individual exposure. Monte Carlo simulations showed that higher PK/PD cutoff values of 8 mg/L could be reached in sick dogs by extending the infusion to 3 h or doubling the dose. Conclusions: The PK of AMC is profoundly different in critically ill dogs compared with normal dogs, with much higher interindividual variability and a lower systemic clearance. Our study allows to generate hypotheses with regard to higher AMC exposure in clinical dogs and provides supporting data to revise current AMC clinical breakpoint for IV administration.

Prediction of Minocycline Activity in the Gut From a Pig Preclinical Model Using a Pharmacokinetic -Pharmacodynamic Approach.

The increase of multidrug-resistant (MDR) bacteria has renewed interest in old antibiotics, such as minocycline, that can be active against various MDR Gram-negative pathogens. The elimination of minocycline by both kidneys and liver makes it suitable for impaired renal function patients. However, the drawback is the possible elimination of a high amount of drug in the intestines, with potential impact on the digestive microbiota during treatment. This study aimed to predict the potential activity of minocycline against Enterobacterales in the gut after parenteral administration, by combining in vivo and in vitro studies. Total minocycline concentrations were determined by UPLC-UV in the plasma and intestinal content of piglets following intravenous administration. In parallel, the in vitro activity of minocycline was assessed against two Escherichia coli strains in sterilized intestinal contents, and compared to activity in a standard broth. We found that minocycline concentrations were 6-39 times higher in intestinal contents than plasma. Furthermore, minocycline was 5- to 245-fold less active in large intestine content than in a standard broth. Using this PK-PD approach, we propose a preclinical pig model describing the link between systemic and gut exposure to minocycline, and exploring its activity against intestinal Enterobacterales by taking into account the impact of intestinal contents.

Kinetic disposition of diazepam and its metabolites after intravenous administration of diazepam in the horse: Relevance for doping control.

In horses, the benzodiazepine diazepam (DIA) is used as sedative for pre-medication or as an anxiolytic to facilitate horse examinations. As the sedative effects can also be abused for doping purposes, DIA is prohibited in equine sports. DIA is extensively metabolized to several active metabolites such as nordazepam, temazepam and oxazepam (OXA). For veterinarians, taking into account the detection times of DIA and its active metabolites is needed for minimizing the risk of an anti-doping rule violation. Therefore, a pharmacokinetic study on 6 horses was conducted using a single intravenous (IV) dose of 0.2 mg/kg DIA Plasma and urine samples were collected at specified intervals until 16 and 26 days post-administration, respectively. Samples were analysed by a sensitive liquid chromatography-electrospray ionization/tandem mass spectrometry method. DIA showed a triphasic elimination pattern in the horse. The mean plasma clearance of DIA was 5.9 ml/min/kg, and the plasma elimination half-life in the terminal phase was 19.9 h. Applying the Toutain model approach, an effective plasma concentration of DIA was estimated at 24 ng/ml, and irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were computed to 0.047 and 0.1 ng/ml, respectively. The detection time according to the European Horserace Scientific Liaison Committee (EHSLC), that is the time for which observed DIA plasma concentrations of all investigated horses were below the IPC was 10 days. Using Monte Carlo Simulations, it was estimated that concentrations of DIA in plasma would fall below the IPC 18 days after the DIA administration for 90% of horses. However, in the present study, a single administration of DIA could be detected for 24 days in urine via the presence of OXA, its dominant metabolite.

Levers to Improve Antibiotic Treatment of Lambs via Drinking Water in Sheep Fattening Houses: The Example of the Sulfadimethoxine/Trimethoprim Combination.

To limit the spread of bacterial diseases in sheep fattening houses, antibiotics are often administered collectively. Collective treatments can be delivered by drinking water but data on the drug's solubility in water or on plasma exposure of the animals are lacking. We first assessed the solubility of products containing sulfadimethoxine (SDM), associated or not with trimethoprim (TMP), in different waters. We then compared in lambs the SDM and TMP pharmacokinetic profiles after individual intravenous (IV) and oral administrations of SDM-TMP in experimental settings (n = 8) and after a collective treatment by drinking water with SDM-TMP or SDM alone in a sheep fattening house (n = 100 for each treatment). The individual water consumption during the collective treatments was also monitored to characterize the ingestion variability. We showed that TMP had a short terminal half-life and very low oral bioavailability, demonstrating that it would be unable to potentiate SDM by oral route. Conversely, SDM had a long terminal half-life of 18 h and excellent oral bioavailability. However, delivery by drinking water resulted in a very high interindividual variability of SDM plasma concentrations, meaning that although disease spread could be controlled at the group level, some individuals would inevitably be under- or over-exposed to the antibiotic.

Transcriptomic modifications of the thyroid gland upon exposure to phytosanitary-grade fipronil: Evidence for the activation of compensatory pathways.

Fipronil is a phenylpyrazole insecticide used for the control of a variety of pest for domestic, veterinary and agricultural uses. Fipronil exposure is associated to thyroid disruption in the rat. It increases thyroid hormone (TH) hepatic clearance. The effect on thyroxine (T4) clearance is about four fold higher than the effect on T4 plasma concentrations suggesting that the thyroid gland might develop compensatory mechanisms. The aim of this study was to document the potential effects of fipronil treatment on the thyroid transcriptome together with its effects on TSH and TH blood levels under well characterized internal exposure to fipronil and its main metabolite fipronil sulfone. Fipronil (3 mg/kg/d by gavage for 14 days) clearance increased while its half-life decreased (about 10 fold) throughout treatment. Fipronil treatment in adult female rats significantly decreased total T4 and free triiodothyronine (T3) concentrations. Key genes related to thyroid hormone synthesis and/or cellular dynamic were modulated by fipronil exposure. RT-PCR confirmed that thyroglobulin gene expression was upregulated. A trend toward higher Na/I symporter expression was also noted, while sulfotransferase 1a1 gene expression was down-regulated. The expression of genes potentially involved in thyroid cell dynamic were upregulated (e.g. prostaglandin synthase 1, amphiregulin and Rhoa). Our results indicate that both pathways of TH synthesis and thyroid cell dynamics are transcriptional targets of fipronil and/or its main sulfone metabolite. The underlying mechanisms remain to be elucidated.

The nuclear receptors pregnane X receptor and constitutive androstane receptor contribute to the impact of fipronil on hepatic gene expression linked to thyroid hormone metabolism.

Fipronil is described as a thyroid disruptor in rat. Based on the hypothesis that this results from a perturbation of hepatic thyroid hormone metabolism, our goal was to investigate the pathways involved in fipronil-induced liver gene expression regulations. First, we performed a microarray screening in the liver of rats treated with fipronil or vehicle. Fipronil treatment led to the upregulation of several genes involved in the metabolism of xenobiotics, including the cytochrome P450 Cyp2b1, Cyp2b2 and Cyp3a1, the carboxylesterases Ces2 and Ces6, the phase II enzymes Ugt1a1, Sult1b1 and Gsta2, and the membrane transporters Abcc2, Abcc3, Abcg5, Abcg8, Slco1a1 and Slco1a4. Based on a large overlap with the target genes of constitutive androstane receptor (CAR) and pregnane X receptor (PXR), we postulated that these two nuclear receptors are involved in mediating the effects of fipronil on liver gene expression in rodents. We controlled that liver gene expression changes induced by fipronil were generally reproduced in mice, and then studied the effects of fipronil in wild-type, CAR- and PXR-deficient mice. For most of the genes studied, the gene expression modulations were abolished in the liver of PXR-deficient mice and were reduced in the liver of CAR-deficient mice. However, CAR and PXR activation in mouse liver was not associated with a marked increase of thyroid hormone clearance, as observed in rat. Nevertheless, our data clearly indicate that PXR and CAR are key modulators of the hepatic gene expression profile following fipronil treatment which, in rats, may contribute to increase thyroid hormone clearance.

Maternal and fetal exposure to bisphenol a is associated with alterations of thyroid function in pregnant ewes and their newborn lambs.

The putative thyroid-disrupting properties of bisphenol A (BPA) highlight the need for an evaluation of fetal exposure and its consequence on the mother/newborn thyroid functions in models relevant to human. The goals of this study were to characterize in sheep a relevant model for human pregnancy and thyroid physiology, the internal exposures of the fetuses and their mothers to BPA and its main metabolite BPA-glucuronide (Gluc), and to determine to what extent it might be associated with thyroid disruption. Ewes were treated with BPA [5 mg/(kg · d) sc] or vehicle from d 28 until the end of pregnancy. Unconjugated BPA did not appear to accumulate in pregnant ewes, and its concentration was similar in the newborns and their mothers (0.13 ± 0.02 and 0.18 ± 0.03 nmol/ml in cord and maternal blood, respectively). In amniotic fluid and cord blood, BPA-Gluc concentrations were about 1300-fold higher than those of BPA. Total T(4) concentrations were decreased in BPA-treated pregnant ewes and in the cord and the jugular blood of their newborns (30% decrease). A similar difference was observed for free T(4) plasma concentrations in the jugular blood of the newborns. Our results show in a long-gestation species with a similar regulatory scheme of thyroid function as humans that BPA in utero exposure can be associated with hypothyroidism in the newborns. If such an effect were to be confirmed for a more relevant exposure scheme to BPA, this would constitute a major issue for BPA risk assessment.

CYP450-dependent biotransformation of the insecticide fipronil into fipronil sulfone can mediate fipronil-induced thyroid disruption in rats.

In rats, the widely used insecticide fipronil increases the clearance of thyroxine (T(4)). This effect is associated with a high plasma concentration of fipronil sulfone, the fipronil main metabolite in several species including rats and humans. In sheep, following fipronil treatment, fipronil sulfone plasma concentration and thyroid disruption are much lower than in rats. We postulated that fipronil biotransformation into fipronil sulfone by hepatic cytochromes P450 (CYP) could act as a potential thyroid disruptor. The aim of this study was to determine if fipronil sulfone treatment could reproduce the fipronil treatment effects on T(4) clearance and CYP induction in rats. Fipronil and fipronil sulfone treatments (3.4 µmol/kg/day per os, 14 days) increased total and free T(4) clearances to the same extent in THX + T(3), euthyroid-like rats. Both treatments induced a 2.5-fold increase in Ugt1a1 and Sult1b1 messenger RNA (mRNA) expressions and a twofold increase in UGT1A activity suggesting that T(4) elimination was mediated, at least in part, by hepatic uridine 5'-diphospho-glucuronosyltransferases (UGT) and/or sulfotransferases (SULT) induction. Both treatments induced a 10-fold increase in Cyp3a1 and Cyp2b2 mRNA expressions concomitant with a threefold increase in CYP3A immunoreactivity and a 1.7-fold increase in antipyrine clearance, a biomarker of CYP3A activity. All these results showed that fipronil sulfone treatment could reproduce the fipronil treatment effects on T(4) clearance and hepatic enzyme induction in rats. The potential of fipronil sulfone to act as a thyroid disruptor is all the more critical because it persists much longer in the organism than fipronil itself.