The present and future of withdrawal period calculations for milk in the European Union: dealing with data below the limit of quantification.

The assessment of withdrawal periods for milk is affected by the occurrence of data below the lower analytical quantification limit (BLQ data) and the resulting uncertainty. The current regulatory approach for dealing with BLQ residues is simple and easy: BLQ data (and missing data) are arbitrarily reassigned a value of one-half the LOQ before any calculation on the data with one of the three currently applicable methods. Here, we reconsider the determination of the withdrawal period of milk with data below the limit of quantification. Theoretical background on analytical limits and pharmacometric considerations will be established. Then, we analyze the uncertainty problems caused by the current approach and propose a calculation solution (maximum-likelihood estimation handling left-censored data) included in nonlinear mixed-effects modeling. Finally, we illustrate this issue using a case example.

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.

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.

Antimicrobial residues in foods of animal origin in Africa: public health risks.

The authors report on the current status of work on residues of veterinary medicinal products and, in particular, antimicrobial residues in foods of animal origin. This review focuses on residues of veterinary antimicrobials, antimicrobials used in livestock production, the concept of residues, and antimicrobial residues in foods of animal origin. Only one antimicrobial substance has been approved in the West African Economic and Monetary Union, compared with 16 substances in Benin and 56 in the European Union. The issue of antimicrobial residues in foods of animal origin has rarely been a serious concern in developing countries, in contrast to the situation in Europe. However, while the prevalence of veterinary drug residues in foods of animal origin is less than 1% in Europe, in some African countries it can be as high as 94%. Antimicrobial residues in foods of animal origin can cause allergies, cancer, alterations in the intestinal flora, bacterial resistance and the inhibition of fermentation in the dairy industry. The harmonisation of regulations in Africa could reduce the circulation of prohibited antimicrobials and lead to the implementation of a plan for the control and surveillance of residues from veterinary medicinal products in foods of animal origin.

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.

Pharmacodynamic modeling of in vitro activity of marbofloxacin against Escherichia coli strains.

A mathematical pharmacodynamic model was developed to describe the bactericidal activity of marbofloxacin against Escherichia coli strains with reduced susceptibility levels (determined using MICs) under optimal and intestinal growth conditions. Model parameters were estimated using nonlinear least-square curve-fitting procedures for each E. coli strain. Parameters related to bactericidal activity were subsequently analyzed using a maximum-effect (E(max)) model adapted to account for a direct and a delayed effect. While net growth rates did not vary significantly with strain susceptibility, culture medium had a major effect. The bactericidal activity of marbofloxacin was closely associated with the concentration and the duration of exposure of the bacteria to the antimicrobial agent. The value of the concentration inducing a half-maximum effect (C(50)) was highly correlated with MIC values (R(2) = 0.87 and R(2) = 0.94 under intestinal and optimal conditions, respectively). Our model reproduced the time-kill kinetics with good accuracy (R(2) of >0.90) and helped explain observed regrowth.

Transfer of plasmid-mediated CTX-M-9 from Salmonella enterica serotype Virchow to Enterobacteriaceae in human flora-associated rats treated with cefixime.

Food animals are a potential source of CTX-M resistance genes for humans. We evaluated the transfer of the bla(CTX-M-9) gene from an animal strain of Salmonella enterica serotype Virchow to Enterobacteriaceae of the human intestinal flora by using human flora-associated (HFA) rats with and without cefixime treatment. In the absence of antibiotic, no transconjugant enterobacteria were found in the feces of HFA rats. However, the transfer rate was high if Escherichia coli J5 recipient strains were coinoculated orally with Salmonella. S. enterica serotype Virchow persisted in the rat fecal flora both during and after treatment with therapeutic doses of cefixime. The drug did not increase the transfer rate, and E. coli J5 transconjugants were eliminated from the flora before the end of cefixime treatment. No cefixime was recovered in the rat feces. In the presence of recipient strains, the bla(CTX-M-9) resistance gene was transferred from a strain of animal origin to the human intestinal flora, although transconjugant colonization was transient. Antibiotic use enhanced the persistence of donor strains, increasing the resistance gene pool and the risk of its spread.

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.

Harmonization of strategies for the validation of quantitative analytical procedures: a SFSTP proposal part IV. Examples of application.

A harmonized approach for the validation of analytical methods based on accuracy profile was introduced by a SFSTP commission on the validation of analytical procedure. This fourth and last document aims at illustrating this methodology and the statistics used. Therefore the validation of real case methods are proposed such as methods for the quality control of drugs, for the quantitation of impurities in drug substances, for bioanalysis or for the determination of nutriments. Furthermore, different types of analytical methods are used in order to demonstrate the applicability of the proposed approach to a wide range of methods such as liquid chromatography (LC-UV, LC-MS), spectrophotometry or ELISA.

Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal--part III.

In the first two documents [Ph. Hubert, J.J. Nguyen-Huu, B. Boulanger, E. Chapuzet, P. Chiap, N. Cohen, P.A. Compagnon, W. Dewé, M. Feinberg, M. Lallier, M. Laurentie, N. Mercier, G. Muzard, C. Nivet, L. Valat, J. Pharm. Biomed. Anal. 36 (2004) 579-586; Ph. Hubert, J.J. Nguyen-Huu, B. Boulanger, E. Chapuzet, P. Chiap, N. Cohen, P.A. Compagnon, W. Dewé, M. Feinberg, M. Lallier, M. Laurentie, N. Mercier, G. Muzard, C. Nivet, L. Valat, E. Rozet, J. Pharm. Biomed. Anal., in press], a recent SFSTP Commission on the validation of analytical procedure has introduced a harmonized approach for the validation of analytical procedures. In order to complete this guide, the statistical methodology allowing to correctly conclude about the validity of a procedure is proposed in this third part of the guide. Indeed all the steps to obtain the decision tool namely the accuracy profile are described and illustrated step by step by a numerical example. This tool, based on the concept of total error (bias+standard deviation) build with a beta-expectation tolerance interval, allows to easily take the right decision and simultaneously minimizing the risk of the future use of the analytical procedure.

Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal--part II.

As reported in a previous paper, the main objective of the new commission of the Société Française des Sciences et Techniques Pharmaceutiques (SFSTP) was the harmonisation of approaches for the validation of quantitative analytical procedures. In a series of meetings, members of this Commission have first tried to review the objectives of analytical methods and the objectives of validation methods and to recommend the use of two-sided beta-expectation tolerance intervals for total error of validation samples (accuracy profile) in the acceptance/rejection of analytical method in validation phase. In the context of the harmonization, the other objectives were: (i) to propose a consensus on the norms usually recognized, while widely incorporating the ISO terminology; (ii) to recommend to validate the analytical procedure accordingly to the way it will be used in routine; (iii) to elaborate a rational, practical and statistically reliable strategy to assure the quality of the analytical results generated. This strategy has been formalised in a guide and the three latter objectives made by the Commission are summarised in the present paper which is the second part of summary report of the SFSTP commission. The SFSTP guide has been produced to help analysts to validate their analytical methods. It is the result of a consensus between professionals having expertise in analytical and/or statistical fields. The suggestions presented in this paper should therefore help the analyst to design and perform the minimum number validation experiments needed to obtain all the required information to establish and demonstrate the reliability of its analytical procedure.

Comparison of faecal and optimal growth conditions on in vitro pharmacodynamic activity of marbofloxacin against Escherichia coli.

The objective of the study was to compare the in vitro activity of marbofloxacin against Escherichia coli (E. coli) strains with differing marbofloxacin susceptibility levels under optimal growth conditions and under condition mimicking faecal environment in time-kill kinetic studies. Under optimal growth conditions, marbofloxacin exerted a bactericidal concentration-dependent activity against all E. coli strains with bactericidal concentrations equal to 1 or 4 times MIC. Under faecal growth conditions, marbofloxacin maintained a bactericidal concentration-dependent activity but a 4- to 16-fold increase in bactericidal concentration was required to produce a similar magnitude of effect at 8 h. The bactericidal activity decreased between 8 and 24 h and allowed a residual bacterial population to subsist with a significant regrowth for some of them. Under no-growth conditions, marbofloxacin produced a very low decrease of non-dividing bacteria during a short time. No concentration produced a reduction > or = 3log10 in viable count excepted for two susceptible strains at concentration > or = 64 x MIC after 4 h exposure. The pharmacodynamic parameters from time-kill kinetic studies provide a useful means of studying antimicrobial activity. The importance of using different growth conditions is indicated by the difference in the killing of E. coli in the absence of active dividing cells and in the presence of autoclaved faecal content, both of which have a detrimental effect on the activity of marbofloxacin.

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 © 2016 John Wiley & Sons, Ltd.

Stability of butyrylcholinesterase: thermal inactivation in water and deuterium oxide.

Irreversible thermal inactivation of the tetrameric form of human plasma butyrylcholinesterase (cholinesterase; EC 3.1.1.8) was studied in water and in deuterium oxide at pH 7 in the temperature range 53-65 degrees C. The enzyme inactivation follows a complex kinetics that may be described by the sum of two apparent first-order processes. The Eyring plot for enzyme inactivation exhibits a wavelike discontinuity over a span of 2 C degrees around 58 degrees C. This transition was interpreted in terms of equilibrium between two temperature-dependent conformational states. Though 2H2O does not alter the overall multistep inactivation process, a slight solvent isotope effect was observed: a stabilizing effect and a shift in the transition temperature. A comparison between several enzyme preparations revealed differences in thermodynamic activation parameters of inactivation suggesting microheterogeneity in enzyme structures. Kinetics of inactivation of usual (E1uE1u) and atypical (E1aEa1a++) enzymes were compared. The atypical enzyme was found to be more stable than the usual phenotype.

Instantaneous secretion rate of growth hormone in lambs: relationships with sleep, food intake, and posture.

Relationships among sleep, feeding behavior, posture, and GH secretion were investigated in two groups of ruminant lambs in January (n = 6) and May (n = 3). Lambs were placed in individual cages and fed ad libitum. Behavioral features were obtained from continuous polygraphic recording. Blood was collected from undisturbed sheep every 3 min for 24 h via an indwelling catheter connected to a peristaltic pump. One month after the sampling session, ovine GH (oGH) was iv administered to evaluate oGH kinetic parameters. From GH plasma concentrations and oGH kinetic parameters, the instantaneous secretion rate of GH was reconstituted using a numerical deconvolution method. All lambs exhibited normal behavioral patterns. The clearance of oGH was similar for the two groups, and the daily production rates of GH were estimated at 14.60 +/- 7.99 micrograms/kg.24 h in January and 10.57 +/- 5.21 micrograms/kg.24 h in May. Analysis of concentration profiles indicate an episodic pattern of GH secretion into plasma. The mean number of peaks was 16.22 +/- 4.47/24 h, and the mean duration was 47.2 +/- 12.8 min for the nine sheep. When instantaneous secretion rates were taken into account, the number of identified peaks was similar, but the mean duration was reduced (32.9 +/- 9.8 min for the nine sheep). Significant relationships were not found between GH plasma concentration profiles and the state of vigilance, food behavior, or posture. Conversely, when the instantaneous secretion rates were taken into account, the highest GH production rate was detected during rest, i.e. slow wave sleep and rapid eye movement sleep, absence of food intake or rumination, and lying down. It is emphasized that the use of GH instantaneous secretion rate instead of GH concentration is of importance when evaluating the relationships between GH dynamics and short duration events. It is concluded that the influence of vigilance on GH secretion, which has already been demonstrated in humans, is likely to exist in other species.

Pulsatile secretion of LH in the ram: a re-evaluation using a discrete deconvolution analysis.

The purpose of the present experiment was to characterize LH secretion pulsatility in rams by analysing the instantaneous secretion rate profile obtained by deconvoluting the plasma concentration profile. Plasma LH concentration profiles were obtained by collecting blood samples every 6 min for 24 h during two different sessions separated by an interval of 15 days. Individual kinetic parameters of ovine LH (oLH) were determined following i.v. injection of oLH. By deconvoluting the plasma concentration profile, it was shown that a pulse has an effective duration of only 20.41 +/- 7.69 (S.D.) min whereas the mean duration estimated from measurement of plasma concentrations was 61.00 +/- 15.16 min. The number of pulses was similar before and after deconvolution (7.80 +/- 1.99 vs 9.70 +/- 3.44 pulses/24 h respectively). Using deconvolution the calculated production rate was 2.26 +/- 0.94 micrograms/kg per 24 h, about 50% of this production being located in the pulses. Statistical analysis of pulsatility revealed that pulse occurrence was a nonperiodic event and that the amplitude of LH pulses and the associated amount of LH released were correlated with the duration of the preceding quiescence period, but had no statistically significant influence on the duration of the following quiescence period.

Activity of the genital tract and plasma levels of oxytocin and cortisol at the time of mating in the ewe.

Experiments were conducted in the ewe to determine the effects of mating on the activity of the genital tract and on blood levels of oxytocin and cortisol. The activity of the uterus and cervix was recorded by electromyography, oxytocin was measured by radioimmunoassay, and cortisol by high performance liquid chromatography. Mating itself did not increase circulating oxytocin or cortisol; uterine motility remained unchanged during and after copulation but the cervix was significantly stimulated during teasing and after copulation. It is suggested that increased cervical activity resulting from adrenergic mechanisms may facilitate the generation of a cervical reserve of spermatozoa.

Determination of four nitroimidazole residues in poultry meat by liquid chromatography-mass spectrometry.

A multi-residue liquid chromatography-mass spectrometry (LC-MS) assay method is described for the determination of four nitroimidazoles in poultry muscle. The extraction procedure is based on liquid-liquid extraction with ethyl acetate followed by an evaporation step. A deuterated internal standard is used. The LC separation was made on a C18 bonded silica column with an aqueous formic acid (0.2%) solution-methanol-acetonitrile (81:13:6) mobile phase. Following electrospray ionization, the protonated molecular ion [M+H]+ is obtained for each compound. Monitoring several ions for each nitroimidazole provides the specificity required for confirmatory assay. Validation of the method was performed to estimate linearity, intra-day and inter-day repeatability, accuracy and detection limit. The present method is capable of identifying nitroimidazole residues in muscle at levels below 5 microg/kg.

Stability of penicillin antibiotic residues in meat during storage. Ampicillin.

Incurred samples from a pig treated with ampicillin, one of the most important penicillin antibiotic drugs used in food-producing animal treatments, were analyzed at the residue level of the drug in muscle tissue (approximately 100 microg kg(-1)) during their freezing storage and using three different techniques: quantitative microbiological assay, HPLC-UV and LC-MS. Two parameters have been specifically monitored: storage temperature (-20 and -75 degrees C) and storage packaging (ground meat or bulk meat). No significant decrease was observed during the first 3 months of storage monitoring at -20 and -75 degrees C. On the contrary, the sample preparation significantly affected the drug concentration in muscle from the very beginning of the storage. Grinding the meat before storage allowed to keep the drug near the higher level of concentration (approximately 100 microg kg(-1)) when bulk meat stored frozen systematically led to a decreased value (approximately 75 microg kg(-1)). After 8 months of storage at -20 degrees C, a significant decrease arose and was never observed at -75 degrees C. All the results were similarly obtained with the three different techniques used simultaneously, which allows to indicate a good correlation between the techniques.

Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal--Part I.

This paper is the first part of a summary report of a new commission of the Société Française des Sciences et Techniques Pharmaceutiques (SFSTP). The main objective of this commission was the harmonization of approaches for the validation of quantitative analytical procedures. Indeed, the principle of the validation of theses procedures is today widely spread in all the domains of activities where measurements are made. Nevertheless, this simple question of acceptability or not of an analytical procedure for a given application, remains incompletely determined in several cases despite the various regulations relating to the good practices (GLP, GMP, ...) and other documents of normative character (ISO, ICH, FDA, ...). There are many official documents describing the criteria of validation to be tested, but they do not propose any experimental protocol and limit themselves most often to the general concepts. For those reasons, two previous SFSTP commissions elaborated validation guides to concretely help the industrial scientists in charge of drug development to apply those regulatory recommendations. If these two first guides widely contributed to the use and progress of analytical validations, they present, nevertheless, weaknesses regarding the conclusions of the performed statistical tests and the decisions to be made with respect to the acceptance limits defined by the use of an analytical procedure. The present paper proposes to review even the bases of the analytical validation for developing harmonized approach, by distinguishing notably the diagnosis rules and the decision rules. This latter rule is based on the use of the accuracy profile, uses the notion of total error and allows to simplify the approach of the validation of an analytical procedure while checking the associated risk to its usage. Thanks to this novel validation approach, it is possible to unambiguously demonstrate the fitness for purpose of a new method as stated in all regulatory documents.