Population Pharmacokinetics and Absolute Oral Bioavailability of Lasalocid after Single Intravenous and Intracrop Administration in Laying Hens.

Lasalocid sodium is a polyether carboxylic ionophore agent authorized by the EU for use as a coccidiostat in broilers, turkeys, and pullets up to 16 weeks of age, except for laying hens. However, laying hens are the most common nontarget species exposed to lasalocid sodium, mainly due to cross-contamination from feed mills. This exposure may result in potential drug residue deposition in eggs, which could potentially expose consumers to the drug. The breeds commonly used for commercial egg production in Poland are Isa Brown and Green-legged Partridge hens, which have been found to significantly differ in egg-laying performance. This variability may also affect the pharmacokinetics of lasalocid. Data on lasalocid plasma pharmacokinetics in laying hens are lacking. In this study, we aimed to determine typical population pharmacokinetic parameters, absolute oral bioavailability, and how breed may influence the pharmacokinetics of lasalocid. Twenty-layer hens of the two breeds were used in this study. Lasalocid was administered orally at a single dose of either 1 mg or 5 mg/kg body weight or intravenously at a dose of 1 mg/kg body weight, in a crossover design with a three-week washout period between study periods. Blood samples were collected for 72 h, and lasalocid concentrations were measured using high-performance liquid chromatography with fluorescence detection. A population pharmacokinetic analysis was conducted using nonlinear mixed effects modeling. Standard numerical and graphical criteria were used to select the best model, and a stepwise covariate modeling approach was used to determine any influencing factors. The best model was a three-compartment mammillary model with first-order absorption, transit compartments, and linear elimination. The estimated absolute oral bioavailability was low (36%). It was found that breed significantly influenced not only absorption but also the elimination of lasalocid. This study revealed that lasalocid absorption and elimination varied between the two breeds. This variability in pharmacokinetics may result in breed-related differences in drug residue accumulation in eggs, and ultimately, the risk associated with consumer exposure to drug residues may also vary.

In vitro comparison of the dermal penetration of three different topical formulations containing lasalocid.

BACKGROUND: Topical antimicrobial preparations are of utmost importance in treating suspected and confirmed meticillin-resistant Staphylococcus pseudintermedius (MRSP) infections due to the increasing incidence of widespread resistance to systemic antimicrobials. Lasalocid is active against MRSP in vitro and this may become an important topical antimicrobial for the treatment of canine pyoderma. HYPOTHESIS/OBJECTIVES: To determine effects of various formulation types on penetration and retention of lasalocid applied to canine skin in vitro. ANIMALS: Normal canine skin was collected from the thorax of five dogs that had been euthanized on the basis of health and/or intractable behavioural issues. METHODS: Solution, lotion and ointment containing 2% lasalocid were applied to ex vivo canine skin. Transdermal penetration was assessed for a 24 h period and retention of lasalocid was assessed at the conclusion of the study. RESULTS: The solution had significantly higher skin retention of lasalocid and proportion of applied dose retained in skin than lotion and ointment (Tukey-Kramer Honest Significant Difference test, P < 0.01). Lasalocid could not be detected in the receptor fluid of any Franz cell at any time point. CONCLUSIONS AND CLINICAL IMPORTANCE: Lasalocid was not identified in the receptor fluid of any sample, indicating that systemic absorption of the active ingredient in vivo is unlikely. Lasalocid may be useful in the treatment of MRSP infections if in vivo studies support safety and efficacy.

Development of Intramammary Drug Delivery Systems: Novel In Vitro Release Method.

The aim of this study was to develop an in vitro release model for intramammary drug delivery system (IMDS) evaluation. This study was the first to establish an in vitro-in vivo correlation with investigation of an IMDS containing lasalocid. Three different methods including the standard United States Pharmacopeia dissolution method with apparatus 2, a modified United States Pharmacopeia method using a dialysis bag, or a specifically designed enhancer cell system, were assessed for the release study. Full cream milk and water were selected as the release media. In vivo evaluation was carried out by administering lasalocid IMDS into the udder of lactating Holstein dairy cows. Milk samples were collected and analyzed at selected time points after treatment. Dissolution data were fitted to various kinetic models. The results indicated that the release rate of lasalocid from IMDS was controlled by factors other than diffusion, which could include the sedimentation of lasalocid to the interface and the wetting of lasalocid particles by water at the interface of oil in the formulation and release media. The results obtained in vivo and in vitro were consistent. The in vitro assessment supports formulation design for early stage development and potentially for in vivo performance analysis.

Transfer of the coccidiostats monensin and lasalocid from feed at cross-contamination levels to whole egg, egg white and egg yolk.

Recent legislation has addressed the unavoidable carry-over of coccidiostats and histomonostats in feed, which may lead to the presence of residues of these compounds in eggs. In this study, laying hens received cross-contaminated feed at a ratio of 2.5%, 5% and 10% of the therapeutic dose of monensin and lasalocid for broilers. The eggs were collected during the treatment and depletion period and were analysed using liquid chromatography-tandem mass spectrometry. The different egg matrices were separated and analysed during the plateau phase. High lasalocid concentrations, which exceeded the maximum residue level, and low monensin concentrations were found in whole egg. Plateau levels were reached at days 7-9 for lasalocid and at days 3-5 for monensin. For lasalocid, the highest concentrations were measured in egg yolk; residue concentrations in egg white were very low.

Determination of lasalocid residues in the tissues of broiler chickens by liquid chromatography-tandem mass spectrometry.

Lasalocid is a polyether ionophoric coccidiostat used for the prevention of coccidiosis in poultry at a prescribed concentration and during a certain time interval. Due to a public health concern about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the levels of lasalocid residues in the edible tissues of broiler chickens (breast muscle, thigh muscle, heart, liver, gizzard, kidneys and skin/fat) fed commercially produced feed containing 100 mg kg⁻¹) of lasalocid in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) with triple quadrupole. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.47 and 1.44 µg kg⁻¹, respectively. The average recovery based on the matrix-fortified calibrations for chicken tissues ranged between 79% and 98%. Lasalocid was found to accumulate in the liver, followed by the heart, skin/fat, kidneys, thigh muscle and gizzard. The lowest concentrations of lasalocid residues were found in the breast muscle. On day 5 of the WP, residue concentrations of lasalocid did not decline below the LOQ of the method, but were far below the maximum residue level (MRL) established for lasalocid in poultry from 20 to 100 µg kg⁻¹ by European Commission Regulation (EU) No. 37/2010. The results confirmed that the WP established for lasalocid is sufficient to ensure the decline of its residues in the tissues of broiler chickens to the safe residue level.

Avaliação de ionóforos pela técnica da perda do potássio celular e produção de gases in vitro / Ionophores evaluation by intracellular potassium depletion and in vitro gas production

Dois estudos foram realizados com vacas lactantes utilizadas como unidade experimental e doadoras de líquido ruminal, sendo as populações de bactérias utilizadas para avaliar a ação de níveis crescentes de lasalocida e monensina na resistência à perda de potássio intracelular, e para produção de gases in vitro. A perda de potássio (Kmax) da lasalocida foi menor para a população de bactérias obtidas do líquido de rúmen de vacas submetidas a dietas com monensina, óleo de soja e monensina mais óleo de soja (19,4 a 25,4 por cento) quando comparada com a perda de potássio em vacas submetidas a dietas sem ionóforo e óleo de soja (30,1 por cento). O mesmo ocorreu para a perda de potássio da monensina, em que o menor valor foi de 6,5 por cento para monensina mais óleo e o maior, de 29,5 por cento, para o controle. Necessita-se de alta concentração de monensina (Kd= 2,3µM), porém baixa de lasalocida (Kd= 0,2µM) para causar a metade da perda máxima de potássio intracelular da população de bactérias do rúmen de vacas submetidas a dietas com monensina. As populações de bactérias de vacas submetidas às dietas com monensina foram sensíveis à lasalocida. As amostras incubadas com própolis produziram menor volume de gases (12,9ml/100g de MS)

Two studies were carried out with lactating cows as experimental units and ruminal fluid donors. The ruminal bacteria population was used to evaluate the action of increasing levels of lasalocid and monensin on resistance of intracellular potassium depletion and in vitro gas production intracellular depletion potassium (Kmax) of lasalocid was lower to ruminal bacteria population obtained from rumen of cows fed diets with monensin, soybean oil and monensin plus soybean oil (19.4 to 25.4 percent) when compared to cows fed with control diet (30.1 percent). The same occurred for intracellular depletion potassium (Kmax) of monensin, in which the lowest value was 6.5 percent to monensin plus soybean oil and the greatest was 29.5 percent to control. High monensin concentration (Kd= 2.3µM) and low lasalocid concentration (Kd= 0.2µM) were necessary to cause half of maximum potassium depletion in ruminal bacteria population from cows fed diet with monensin. The ruminal bacteria population from cows feed diet with monensin were sensible to lasalocid. In vitro gas production showed the lowest volume when diets were incubated with propolis (12,9ml/100g of DM)

Inhibition of resistance plasmid transfer in Escherichia coli by ionophores, chlortetracycline, bacitracin, and ionophore/antimicrobial combinations.

Medicinal feed additives bacitracin, chlortetracycline (CTC), laidlomycin, lasalocid, and salinomycin inhibited the transfer of multiresistance-conferring plasmid pBR325 (Tet(r) Amp(r) Cp(r), 6.0 kb) into selected gram-negative strains with the use of an in vitro model. High concentrations of ampicillin-sensitive competence-pretreated Escherichia coli HB 101 cells were exposed to 10% (v/v) of 1:10 dimethyl sulfoxide/agent : water containing test mixtures for 0.5 hr prior to plasmid addition and transforming conditions. Transformation was inhibited for all antimicrobials and showed a positive association wich higher concentration. Additional testing of ionophore compounds separately and in combination with bacitracin, chlortetracycline, lincomycin, roxarsone, tylosin, and virginiamycin at representative feed concentrations demonstrated 80.6% to >99.9% inhibition (P < 0.001) of resistance transfer. Bacitracin alone inhibited transformation within the range of 50-500 ppm. No increase in resistance transfer was observed when poultry-derived and reference gram-negative isolates having low or no transformation efficiency were additionally tested. The results suggest that these compounds, at relevant concentrations used in animal feed, may interfere with cell envelope-associated DNA uptake channels or other transformation competence mechanisms. Through these mechanisms, ionophores and cell membrane-interactive feed agents such as CTC and bacitracin may act to inhibit resistance transfer mechanisms within poultry and livestock.

Development of an ELISA for lasalocid and depletion kinetics of lasalocid residues in poultry.

Polyclonal antibodies against the coccidiostat, lasalocid, were raised in sheep. The antisera were applied in an enzyme-linked immunosorbent assay (ELISA) for lasalocid, which was validated for chicken serum, liver and muscle. Bridge homology in the ELISA was overcome by absorbing unspecific antisera onto a conjugate between salinomycin and chicken serum albumin, which was immobilized onto Biosilon beads. The described assay is highly specific for lasalocid, and is capable of detecting lasalocid at concentrations of less than 0.15 ng/g, depending on the tissue. Residual concentrations of lasalocid in broilers fed medicated feed containing 90 mg/kg lasalocid were measured during a 7 day withdrawal period. The half-life of lasalocid was 11, 36 and 41 h in serum, liver and muscle, respectively. Lasalocid concentrations in liver were approximately 10 ng/g 7 days withdrawal of the medicated feed.

Effect of ionophorous anticoccidials on invasion and development of Eimeria: comparison of sensitive and resistant isolates and correlation with drug uptake.

Prophylactic levels of three ionophorous antibiotics, monensin, salinomycin, and lasalocid, were administered to groups of chickens and turkeys. All three ionophores markedly inhibited invasion of cecal tissues by sporozoites of ionophore-sensitive (IS) Eimeria tenella. Monensin and salinomycin also reduced invasion in turkeys by sporozoites of E. adenoeides, but lasalocid only minimally inhibited invasion. Invasion of ceca of monensin-medicated chickens was significantly greater by sporozoites of ionophore-resistant (IR) E. tenella than of the IS isolate. Concomitant experiments showed significant differences in [14C]monensin accumulation among IS and IR isolates of E. tenella. The decreased uptake of monensin by the IR isolates appeared to be accompanied by a decrease in responsiveness to the activity of monensin as well as to two other ionophores, salinomycin and narasin in cell culture. The amount of monensin, salinomycin or narasin required to inhibit development of E. tenella by 50% was 20 to 40 times higher for the IR isolates than for the IS ones. Collectively, the data suggest that differences in ionophore accumulation by IS and IR isolates of E. tenella might reflect differences in membrane chemistry and that these differences are responsible for the expressions of resistance that were observed in these studies. This expression of resistance appears to be common to all ionophores tested.