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The conjugative transfer of the plasmid carrying the bla(CTX-M-9) gene from Salmonella enterica serovar Virchow isolated from a chicken farm to a recipient Escherichia coli strain was evaluated in vitro and in axenic rats inoculated with both strains, with or without selective pressure due to therapeutic doses of cefixime. The transfer of the bla(CTX-M-9) gene of S. enterica serovar Virchow to E. coli was confirmed in vitro, at a low frequency of 5.9 x 10(-8) transconjugants/donors. This transfer rate was higher in gnotobiotic rats and reached approximately 10(-5) transconjugants/donors without selective pressure. This frequency was not affected by the addition of therapeutic doses of cefixime. Thus, estimates of in vitro transfer underestimated potential transfer in the digestive tract, and therapeutic doses of cefixime did not increase the selection for transconjugants.

A semi-quantitative immunological method (BIACORE method) for detecting sulfamethazine in bovine milk was developed and validated using the total error approach. The acceptance limits were set at +/-40% and the risk of procedure of (1-beta) proportion measurements falling outside the acceptance limits was chosen at 5%. Different response functions were tested on the basis of the accuracy index (I(A)). The best model was a weighted (1/X(2)) quadratic regression and the simplest one was an unweighted quadratic regression. This approach identified the weak point of the method, which was precision. Finally this BIACORE method was able to detect positive samples containing sulfamethazine in the dosing range between 50 and 150 ng/ml.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for use in pharmacokinetic studies in order to determine the concentrations of monensin in plasma and edible tissues of chicken. Two sample preparations were performed, one for determining monensin concentrations in plasma using acetonitrile for protein precipitation and another one for determining monensin concentrations in muscle, liver, and fat using methanol-water followed by a clean up on a solid-phase extraction cartridge. Sample extracts were injected into the LC-MS/MS system, and a gradient elution was performed on a C18 column. Narasin was used as internal standard. The LC-MS/MS method was validated using an approach based on accuracy profiles, and applicability of the method was demonstrated for the determination of monensin in chicken plasma, muscle, liver, and fat in a pharmacokinetic study.

The ecological impact of different doses of ciprofloxacin was investigated in an experimental germ-free rat model into which human fecal flora was inoculated. Animals received oral doses (gavage) of 0, 0.25, 2.5, and 25 mg/kg body weight (bw) of ciprofloxacin once daily for 5 weeks. All doses of ciprofloxacin significantly reduced aerobic populations. Elimination of Enterobacteriaceae and reduction of bifodibacteria were noticed in the group treated with 25 mg/kg of the antibiotic. The rest of the intestinal flora was not affected. These effects were reversible after the treatment ended. The percentage of resistant enterococci increased in rats treated with 2.5 and 25 mg/kg; however, this increase was not statistically significant. There was a significant (P < 0.05) emergence of ciprofloxacin-resistant Bacteroides fragilis group with 25 mg/kg bw, which is equivalent to a human therapeutic dosage of the antibiotic. The MIC values and the percentage of resistance remained elevated 2 weeks after the end of treatment in this anaerobic population. Although sub-populations of enterococci and Enterobacteriaceae showed decreased susceptibility after ciprofloxacin administration, resistance was not evident. The ability of an exogenous strain of Salmonella to colonize the intestine of animals treated with 25 mg/kg of ciprofloxacin confirmed that the drug disrupted the colonization barrier effect of the indigenous flora at the high dose level tested. No changes in the metabolic parameters occurred during the antibiotic treatment. The results obtained in the HFA rat model were similar to those obtained in our previous study using the HFA mice model where ciprofloxacin at 0.125, 1.25, and 12.5 mg/kg bw induced a decrease of enterococci and Enterobacteriaceae populations. The high dose of ciprofloxacin also induced a decrease in bifidobacteria counts, an increase in levels of resistant B. fragilis group and a significant (P < 0.05) disruption of the colonization resistance of the barrier flora in HFA mice.

In order to achieve the harmonization of validation strategies, the interpretation of several validation criteria is proposed. Furthermore, a simple and visual decision tool to assess the validity of an analytical procedure is described: the accuracy profile based on the estimation of the total error of the measurements. This profile build with beta-expectation tolerance intervals can also compute with efficiency the uncertainty related to the results of a laboratory, which is an essential parameter for the accreditation of laboratories under ISO 17025.

Two interlaboratory studies were organized in 2002-2003 in order to check the proficiency of laboratories in confirming the presence of sulfonamide residues in muscle and milk. These studies involved 25 EU National Reference Laboratories (NRLs) from 21 different European Countries in charge of statutory monitoring of antimicrobial residues in food of animal origin at a national level. The study was conducted according to international and national guidelines by the Community Reference Laboratory (CRL) in charge of antimicrobial substances. Four different test matrices of sheep muscle and four different test matrices of bovine milk containing different sulfonamide substances were prepared and sent to the participants. Each participant was asked to use his own routine confirmatory method and to analyse each sample in triplicate within a period of about six weeks during which the stability of the materials was checked by the organizer. The sulfonamide content of each material was determined by calculating the robust means of all the results and the deviation of the results from the assigned values was assessed by calculating Z-scores. Overall, results were satisfactory, particularly considering that it was the first proficiency test dealing with sulfonamides organised by the Community Reference Laboratory.

Acrylamide is present as a contaminant in the human diet in heated food products. It has been found to be carcinogenic in laboratory rats and has been classified as probably carcinogenic in humans. In order to clarify the possible involvement of a primary genotoxic mechanism in acrylamide-induced carcinogenicity, both the presence of DNA damage, measured by the comet assay, and the formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade), derived from reaction of the active metabolite glycidamide (GA) with the DNA, analyzed by LC/MS/MS, were assessed in selected rat tissues. Rats were administered with single oral doses of acrylamide (18, 36 or 54 mg/kg body weight (b.w.) and the organs (blood leukocytes, brain, bone marrow, liver, testes and adrenals) were sampled at different times after treatment. Results from GA-induced DNA adduct measurements indicated a relatively even organ distribution of the adducts in brain, testes and liver. Organ-specificity in acrylamide carcinogenesis can therefore not be explained by a selective accumulation of GA-DNA adducts in the target organs, at least not after a single dose exposure. The DNA adduct profiles and half-lives were similar in the different organs; except that the N3-GA-Ade adduct was more rapidly removed from tissues than the N7-GA-Gua adduct. Increased extent of DNA migration, as measured by the in vivo rat comet assay, was found in brain and testes, and these specific results seem to be in accordance with the known organ-specificity in acrylamide carcinogenesis in rat. Only weak and transient DNA damage was recorded in the liver, bone marrow and adrenals. The DNA-damaging effect of the compound observed in the blood leukocytes could be a simple biomarker of acrylamide exposure and genotoxicity.

An animal model was designed to study the chronopharmacokinetics of intravenous drugs and applied to anticancer agents vinorelbine (VNB) and methotrexate (MTX). Each experiment was performed on four pigs housed in a standardized light-dark cycle (12:12). Four pigs received a 0.16-mg bolus of VNB, followed by a 60-h continuous infusion at 0.48 mg/h. After hydration and urine alkalinization, four other pigs received a 2 mg/kg bolus of MTX, followed by two concomitant 60-h continuous infusions, one with MTX (8 mg/kg/h) and the other for hydration and folinic acid rescue (1.5 mg/kg/24 h). Serum cortisol was determined in each blood sample collected in these pigs. Blood samples were collected each hour for 60 h. The infusion flow rates and drug solution concentrations were controlled throughout the experiments. Analysis of VNB serum concentrations did not show any circadian rhythm of VNB serum concentrations. One pig administered MTX exhibited severe toxicity. Interestingly, no circadian rhythm of serum cortisol concentration was observed in this pig, whereas the three others exhibited a statistically significant cortisol circadian rhythm with a peak secretion in the morning. Two of these three pigs showed a significant 24-h rhythm of MTX with acrophase occurring at approximately 1:00 PM in both. The maximal concentration was found at 12:00 AM in the third pig. After the data were pooled, a highly significant (P < 0.01) circadian rhythm in MTX serum concentrations (57%) was found, with acrophase at midday. The pig represents a useful model for the study of chronopharmacokinetics of drugs given intravenously in human. The MTX chronokinetic variation found herein may be of interest for the improvement of chemotherapy in cancer patients.

A collaborative study involving 14 laboratories was conducted to determine residues of ampicillin in porcine muscle tissue by using a liquid chromatographic method developed for multipenicillin analysis that can quantitate 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer, pH 9, followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50 degrees C and with 1,2,4-triazole and mercury(II) chloride solution, pH 9.0, at 65 degrees C. The derivatized compounds are eluted isocratically on a C8 column with a mobile phase of acetonitrile and phosphate buffer (pH 6; 0.1 M) containing sodium thiosulfate and the ion-pair reagent tetrabutylammonium hydrogen sulfate. The penicillins are detected by UV absorption at 325 nm. The limit of detection and the limit of determination (quantitation) of the method were calculated to be approximately 3-5 and 25 microg/kg, respectively, in accordance with the criteria of European Union (EU) Decision No. 93/256/EEC. In this first interlaboratory study, collaborators were instructed to monitor 4 different penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, and amoxicillin) by analyzing 8 blind samples of muscle tissue in triplicate. These samples were prepared from 2 materials containing different concentrations of incurred ampicillin (63.5 microg/kg for material No. 1 and 358.1 microg/kg for material No. 2) and 1 blank material. The repeatability relative standard deviation and the reproducibility relative standard deviation were 10.2 and 17.4%, respectively, for material No. 1 and 7.0 and 16.0%, respectively, for material No. 2. These results demonstrate that the method is suitable for the determination of ampicillin residues in muscle tissue at the EU maximum residue limit (50 microg/kg) and above. However, the identification of positives by this procedure may need additional confirmation by techniques with greater specificity, such as liquid chromatography combined with mass spectrometry, or tandem mass spectrometry. Investigations regarding the basis of interlaboratory testing studies will further demonstrate the suitability of multiresidue methodology for detecting and quantitating other compounds in the family of penicillin antibiotics.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.

Pharmacokinetic variables of skeletal muscle creatine kinase were determined in sheep after intravenous and intramuscular administration of the semipurified enzyme. Catheters implanted in the jugular vein were used for both intravenous injections and blood withdrawals. Blood sample collection by vacutainer and hemolysis may in fact have considerable effects on the measurement of creatine kinase activity in plasma. The change in the enzyme activity versus time in the plasma, after intravenous administration (123 +/- 38 U/kg) of creatine kinase, was fitted by a biexponential model. The mean volume of the central compartment (45 +/- 5 mL/kg) was approximately equal to the plasma volume. Plasma half-life and plasma clearance of creatine kinase were 3.7 +/- 1.7 h and 23 +/- 8 mL.kg-1.h-1, respectively. Mean plasma bioavailability of creatine kinase after intramuscular administration (357 +/- 36 U/kg) in both the loins and the gluteal mass was 42%. Maximal plasma activity was observed 4 and 5 h after injection and the half-life of the terminal phase was 7.3 or 8.6 h according to the muscle. The extent of muscle damage after intramuscular administrations of 21 veterinary drug formulations (one product per animal) was estimated from the total creatine kinase activity released in plasma during the 72 h following the injection. Equivalent weights of damaged muscle ranged from 1.4 to 83.3 g according to the irritant potency of the test formulation. Results differed only moderately between the injection sites (right and left gluteal mass) in the same animal. It can be concluded from this study that, in sheep: i) the bioavailability of creatine kinase from different injection sites (gluteal mass and loins) is comparable; and ii) the intra-individual variability in the estimation of muscle damage is moderate. Once validated, this non-invasive approach for local tolerance studies could be of value in assessing and comparing the irritant potency of veterinary drugs and in reducing the number of animals required.

Pharmacodynamics studies consider three main parameters: the size of the bacterial population, the concentration of the antibiotic and the duration of its action. The pharmacodynamic characteristics of colistin were studied in vitro with Escherichia coli. The bacterial kinetics were fitted using differential equations. The mathematical model gave qualitative and quantitative information about the characteristics of the antibiotic-bacteria association. Above all, when linked to a pharmacokinetic model, the model permitted the prediction of the drug's efficiency. Simulations of various dosage levels in which the administration route, dose size, or interval between doses varied, permitted a more rational optimization than a prediction of efficacy based on the time taken to achieve antibiotic plasmatic concentrations above the minimal inhibitory concentration. Pharmacokinetic/pharmacodynamic modeling seems to be an interesting possibility for determining antibiotic dosing levels during the preclinical phase.

The modelling of in vitro growth curves of Staphylococcus aureus ATCC 6538P at different pHs, of spiramycin killing curves and of the dose-effect relationship was performed. The growth curve was characterised by a Volterra-Kostitzin differential model, the main parameters of which were the bacterial growth rate constant of 0.95 +/- 0.13 h-1 and a lag-time of 0.74 +/- 0.36 h. The Zhi model used for bactericidal kinetics showed the same bacterial killing rate constant from a minimum concentration characteristic of "time-dependent" antibiotics. This differential model incorporated with pharmacokinetic models could lead to a rationale for proper dosage regimens.

The disposition of radiotocopherol after intraperitoneal administration of 14C-labelled-DL-alpha-tocopherol (1 microCi/kg bw, dose 1) and 3H-labelled-DL-alpha-tocopherol (4 microCi/kg bw dose 4) and of D-alpha-tocopherol, after intraperitoneal administration of DL-alpha-tocopherol acetate (100 mg/kg bw) was investigated in sheep. Plasma samples were taken at regular intervals after dosing and assayed for radioactivity and D-alpha-tocopherol. Plasma profiles were modelized using a compartmental approach and the input entry rate (for radioactivity) was identified using a numerical deconvolution method. Based on plasma specific activity (dose 1 and dose 4) and D-alpha-tocopherol plasma concentration (unlabelled alpha-tocopherol acetate), half-lives were not significantly different (99.6 +/- 28.3 h, 121.3 +/- 38.5 h and 75.0 +/- 49.75 h after dose 1, dose 4 and unlabelled alpha-tocopherol respectively). The times to reach maximal plasma concentrations were similar for the 3 test articles (mean values ranging from 21.5 to 26.7 h). The only significant difference was observed for the apparent volume of distribution (with respect to bioavailability) which was much larger for the unlabelled test article. Deconvolution study of plasma specific activity showed: i), that the maximal input rate was reached only after a short delay (3 to 12 h); ii), that half of the activity was absorbed after a delay of 38.13 +/- 16.76 h (dose 1) and 44.3 +/- 6.50 h (dose 4); and iii), that 90% of the activity was absorbed after 151.2 +/- 27.3 h (dose 1) and 162.3 +/- 11.2 (dose 4). It is concluded that the intraperitoneal route is of interest for alpha-tocopherol administration, but that more information is required to determine the exact process of absorption.

Pharmacokinetic characteristics of an extemporaneous form of colistin sulfate in young calves were studied for a dosage of 25,000 IU.kg-1. The intravenous route (IV) is characterized by a 3-compartment model whose main parameters are: volume of distribution (1.02 l.kg), body clearance (0.15 l.h-1 kg-1) and mean residence time (3.87 h). By intramuscular route (IM), a mean serum peak of 37 IU.ml-1 was reached at a mean time of 0.5 h. The mean half-time of terminal phase (6.47 h) does not differ significantly from that of the intramuscular route (4.52 h). Absolute bioavailability calculated based on 4 calves was 109 +/- 28%. Repeated IM administrations seem to be adapted to maintain a bactericidal activity and to reduce risks of toxicity and neurological disorders (25,000 IU.kg-1) every 12 h over 3d.

Absorption rate and plasma and fat disposition of lindane after various lindane percutaneous treatments in shorn and unshorn sheep were investigated. To analyze data with a deconvolution method, IV administration was performed to determine the basic pharmacokinetic values of lindane in sheep. After IV administration, the steady state volume of distribution was very high (8.07 +/- 3.60 L/kg of body weight), and the mean residence time was long (28.1 +/- 11.7 hours). Deconvolution analysis indicated that lindane absorption was continuous until 33 to 41 days after spraying with a 0.025% lindane solution. Total amount of absorbed lindane in shorn (15,171 +/- 4,463 micrograms/kg) sheep was about twice that in unshorn (7,615 +/- 3,128 micrograms/kg) sheep; from deconvolution analysis, it was calculated that the time required for 50% of the available dose to be absorbed was between 115 and 179 hours. After percutaneous lindane administration, the fat concentration was compared with the available lindane dose. The apparent half-life of lindane elimination in fat was 225 +/- 47.4 hours, which is similar to the value calculated for the absorption rate constant. By comparing fat and plasma concentrations, it was calculated that for a mean plasma concentration of 5 ng/ml, the fat lindane concentration was 1.65 +/- 0.87 micrograms/g (ie, lower than the generally accepted tolerance level of 2 micrograms/g).

The oxytocic effect of a prostaglandin F2 alpha analogue, fenprostalene, was assessed in 4 ovariectomized ewes fitted with electrodes in both uterine horns and in the cervix. In the absence of estradiol priming, significant motility changes were not elicited by fenprostalene. Conversely, when ewes were primed with 17-beta-estradiol, fenprostalene markedly increased the electrical activity in the uterus and cervix. After a single subcutaneous fenprostalene administration (5 micrograms/kg), activity values remained about twice that of the control values during 8.52 +/- 3.31 hours. When the same dosage was administered IM, similar post-injection activity values were obtained, but only during 5.88 +/- 0.72 hours. Oxytocic effects of fenprostalene were far longer than those elicited by a single IM administration of 50 micrograms of prostaglandin F2 alpha (tham salt)/kg (0.91 +/- 0.32 hours) or by a single IM administration of 1 microgram of cloprostenol/kg (1.88 +/- 0.81 hours). Using the dose-effect relationship curve obtained from the same ewes by continuous IV infusions of oxytocin (OXT), the postinjection activity values reached after a single subcutaneous administration of fenprostalene were equivalent to those of an IV infusion of OXT at an average dose of 4.09 ImU of OXT/kg/hr for 6 to 13 hours, according to the values of the particular ewe concerned. These long-lasting oxytocic properties, in addition to its luteolytic capabilities, would make fenprostalene a suitable drug for promoting effective evacuation of the uterus when required in daily veterinary practice.

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.

Four ovariectomized cows were used to compare the uterotonic (oxytocic) properties of the prostaglandins F2alpha analogue fenprostalene to cloprostenol and PGF2alpha-tromethamine salt (dinoprost). Uterine activity was measured by electromyography with the duration and magnitude of activity quantified by microcomputer. The administration of 1 mg of fenprostalene to estradiol primed animals significantly increased uterine motility for approximately 19 h. This was significantly longer than the duration observed for either cloprostenol (500 mug, i.m., 8.9 h) or dinoprost (25 mg, i.m., 7.7 h). However, the level of activity was similar for the 3 compounds tested, with postinjection levels of oxytocic effect averaging 369 % for treated animals compared to 100 % for controls. Therefore, the difference in effects for the three prostaglandins may be due more to pharmacokinetic properties rather than to different potencies of the three compounds. In addition, a pregnant cow (100 d gestation) was treated with fenprostalene (1 mg, s.c.). Fenprostalene treatment resulted in unchanged uterine activity for a 6-h period, followed by a four-fold increase in genital tract activity which lasted for 12 h. Thereafter, activity was inhibited for one day, followed by a sharp increase in uterine activity leading to abortion within 66 to 72 h after fenprostalene injection. The placenta was expelled 7 days after treatment.