Non-genomic action of beclomethasone dipropionate on bronchoconstriction caused by leukotriene C4 in precision cut lung slices in the horse.

BACKGROUND: Glucocorticoids have been proven to be effective in the therapy of recurrent airway obstruction (RAO) in horses via systemic as well as local (inhalative) administration. Elective analysis of the effects of this drug on bronchoconstriction in viable lung tissue offers an insight into the mechanism of action of the inflammatory cascade occurring during RAO which is still unclear. The mechanism of action of steroids in treatment of RAO is thought to be induced through classical genomic pathways. We aimed at electively studying the effects of the glucocorticoid beclomethasone dipropionate on equine precision-cut lung slices (PCLS).PCLS were used to analyze ex-vivo effects of beclomethasone on inhibiting bronchoconstriction in the horse. The inhibiting effect was measured through instillation of a known mediator of inflammation and bronchoconstriction, leukotriene C4. For this, the accessory lobes of 13 horses subjected to euthanasia for reasons unrelated to the respiratory apparatus were used to obtain viable lung slices. RESULTS: After 30 minutes of PCLS incubation, beclomethasone showed to significantly inhibit the contraction of the bronchioles after instillation with leukotriene C4. The EC50 values of the two contraction curves (LTC4 with and without BDP) differed significantly from each other (p = 0.002). The possibility of a non-genomic rapid mechanism of action seems likely since transcriptional activities require a longer lag period. CONCLUSIONS: In human neuroendocrinology, high levels of glucocorticoids have been proven to function via a non-genomic mechanism of membrane receptors. The concentration of beclomethasone used on the lung slices in our study can be considered as high. This allows speculation about similar rapid non-genomic mechanisms of high-dosage inhaled glucocorticoids in the lower airways of horses. However, further assessment on a molecular basis is needed to confirm this.

The in vitro use of the hair follicle closure technique to study the follicular and percutaneous permeation of topically applied drugs.

Recent studies on follicular permeation emphasise the importance of hair follicles as diffusion pathways, but only a limited amount of data are available about the follicular permeation of topically applied drugs. This study examines the use of a hair follicle closure technique in vitro, to determine the participation of hair follicles in transdermal drug penetration. Various substances, with different lipophilicities, were tested: caffeine, diclofenac, flufenamic acid, ibuprofen, paracetamol, salicylic acid and testosterone. Diffusion experiments were conducted with porcine skin, the most common replacement material for human skin, in Franz-type diffusion cells over 28 hours. Different experimental settings allowed the differentiation between interfollicular and follicular permeation after topical application of the test compounds. A comparison of the apparent permeability coefficients of the drugs demonstrates that the percutaneous permeations of caffeine and flufenamic acid were significantly higher along the hair follicles. In the cases of paracetamol and testosterone, the follicular pathway appears to be of importance, while no difference was found between interfollicular and follicular permeation for diclofenac, ibuprofen and salicylic acid. Thus, the hair follicle closure technique represents an adequate in vitro method for gaining information about follicular or percutaneous permeation, and can replace in vivo testing in animals or humans.

Plasma and urine concentrations of marbofloxacin following single subcutaneous administration to cats.

The pharmacokinetic properties of marbofoxacin, a third generation fluoroquinolone, were investigated in 12 healthy adult cats after single subcutaneous (SC) administration of 2 mg/kg BW (Part I, n=8 cats) and 4 mg/kg BW (Part II, n=4 cats). In each part of the study blood and urine samples were collected before treatment and thereafter for 5 days. The plasma and urine concentrations of marbofloxacin were determined by HPLC with UV detection. Pharmacokinetic calculations were performed for each treated animal using an open one-compartment-model with first-order elimination after SC dosing. Marbofloxacin in plasma (means): Maximum concentrations (Cmax) of about 1.2 and 3.0 microg/ml were measured 2.3 and 4 hours (tmax) after dosing of 2 and 4 mg/kg BW, respectively. Elimination from the body was low with a total clearance (Cl/F) of approximately 0.1 l/h/kg for both dosages. The half-life (t 1/2) for this process was calculated with 8-10 hours. AUC increased almost proportional when doubling the dose, i.e., 19.77 +/- 6.25 microg * h/ml (2 mg/kg BW) and 51.26 +/- 11.83 microg * h/ml (4 mg/kg BW). Plasma kinetics measured were in accordance with data from literature. Marbofloxacin in urine (means): Maximum drug concentrations were detected 4 and 8 hours after dosing with 70 microg/ml (2 mg/kg BW) and 160 microg/ml (4 mg/kg BW), respectively. Inhibitory effects of the urinary matrix on the antimicrobial activity of the drug were taken into account when performing PK/PD calculations. However, a concentration-dependent bactericidal activity (Cmax/MIC > 8-10) which is claimed for fluoroquinolones was sufficiently met with focus on Escherichia (E.) coli (MIC90 0.5 microg/ml). In the same matrix a threshold value of 1.0 microg/ml was undercut 82 and 116 hours after SC dosing, respectively. Hence, a time-dependent bacteria killing kinetic (T > MIC) which may be of relevance for some Gram-positive germs like Staphylococcus spp. (MIC90 1.0 microg/ml) should be covered, too.

Tissue distribution of cloxacillin after intramammary administration in the isolated perfused bovine udder.

BACKGROUND: Various intramammary suspensions containing cloxacillin benzathine are registered for use in cattle as antibiotics for intramammary use at drying off. To ensure antibacterial efficacy, the glandular tissue concentration of an antimicrobial agent must be sufficient. Since the possibilities to measure concentrations in the different areas of the glandular tissue in vivo are very limited, it was the aim of the present study to examine the distribution of cloxacillin in vitro using the isolated perfused bovine udder. METHODS: Mammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. 600 mg cloxacillin benzathine were administered as Orbenin Extra Dry Cow by the intramammary route to six front and rear quarters each. Samples of glandular tissue--at different distances from and vertical to the teat right up to the udder base--were gathered from the treated quarters after 6 h. Perfusate was also sampled before and hourly after treatment for 6 h. The cloxacillin content of the tissue samples and perfusate samples was analysed by high performance liquid chromatography. RESULTS: The concentration of cloxacillin in the glandular tissue of front quarters measured 6 h after administration tended to decrease with increasing vertical distance from the teat. The decrease pattern of the concentration was not quite clear in rear quarters. A considerable variation in the tissue concentrations of cloxacillin was obvious, which reflects in vivo conditions. The concentrations measured in the perfusate samples were below the limit of quantification at all time points, indicating limited absorption of the antibiotic from the glandular tissue. CONCLUSION: After intramammary administration of the dry off product containing cloxacillin benzathine concentrations of more than 0.5 µg/g (MIC) were reached in all regions of the front and rear quarters.

Pharmacological studies of bronchial constriction inhibited by parasympatholytics and cilomilast using equine precision-cut lung slices.

Equine precision cut lung slices (PCLS) were used to evaluate the ability of several pharmacologic compounds, parasympatholytics atropine, ipratropium, tiotropium and phosphodiesterase (PDE) 4-inhibitor cilomilast, to inhibit methacholine (MCh) induced bronchial constriction. The study war performed using vital lung samples from 20 horses, harvested immediately after euthanasia and lung examination.Tissue samples from the cranial, caudal and accessory lobe were taken for histological examination. Viability in PCLS of 250 +/- 20 microm was confirmed by observation of cilia movement and bronchial contraction in response to the addition of increasing concentrations of MCh (10(-8) to 10(-4) mol/l). Washing in pure RPMI-medium resulted in bronchial reopening. After incubation (20 minutes in an RPMI-solution containing cilomilast (10(-5) mol/l), atropine, ipratropium or tiotropium (10(-7) mol/l) each group (randomized, placebo controlled) was treated with MCh again. After reopening the unaffected viability of the PCLS was confirmed by the addition of MCh. EC50 values were calculated from bronchial cross-sections for each contraction. EC50 values significantly increased after atropine, ipratropium and tiotropium addition. No difference was found for cilomilast and in control slices. Statistical analysis showed a large interindividual, but a small intraindivual variability. No correlation was found between the clinical examination, histological findings and PCLS' responsiveness. We were able to confirm Vietmeier's (2007) values for the responsiveness of equine PCLS to MCh and to proof the effect of several new bronchodilatators using a new technique that allows the assessment of several substances in the same horse.

Differential sensitivity of differentiated epithelial cells to respiratory viruses reveals different viral strategies of host infection.

To address the initiation of virus infection in the respiratory tract, we established two culture systems for differentiated bovine airway epithelial cells (BAEC). Filter-grown BAEC differentiated under air-liquid interface (ALI) conditions to generate a pseudo-stratified mucociliary epithelium. Alternatively, precision-cut lung slices (PCLS) from the bovine airways were generated that retained the original composition and distribution of differentiated epithelial cells. With both systems, epithelial cells were readily infected by bovine parainfluenza virus 3 (BPIV3). Ciliated cells were the most prominent cell type affected by BPIV3. Surprisingly, differentiated BAEC were resistant to infection by bovine respiratory syncytial virus (BRSV), when the virus was applied at the same multiplicity of infection that was sufficient for infection by BPIV3. In the case of PCLS, infection by BRSV was observed in cells located in lower cell layers but not in epithelial cells facing the lumen of the airways. The identity of the infected cells could not be determined because of a lack of specific antibodies. Increasing the virus titer 30-fold resulted in infection of the ALI cultures of BAEC, whereas in PCLS the ciliated epithelium was still refractory to infection by BRSV. These results indicate that differentiated BAEC are readily infected by BPIV3 but rather resistant to infection by BRSV. Disease caused by BRSV may require that calves encounter environmental stimuli that render BAEC susceptible to infection.

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study.

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.

The automated, accurate and reproducible determination of steady-state permeation parameters from percutaneous permeation data.

Procedures for the in vitro determination of percutaneous permeation with Franz diffusion cells are widely accepted. However, the calculation of relevant endpoints, such as the steady-state flux (J) and the permeation coefficient (P(app)), still depends on visual data inspection or an approximation of the steady-state flux as the maximum observed absorption rate. As both these approaches must be considered inappropriate, an automated and reproducible algorithm to analyse permeation data is presented. The method detects both lag-times and non-linear data resulting from substance accumulation in the acceptor compartment of static diffusion cells. It was evaluated by using simulated data, and data from experiments with caffeine and testosterone on bovine udder skin and human reconstituted epidermis (SkinEthic), which represent model barriers with high and low barrier strengths, respectively. It was shown that the algorithm is a suitable method for the identification of steady-state ranges in permeation data. If used on data generated with appropriate experimental approaches, it is a reproducible and time-saving alternative to the visual analysis of diffusion data.

A sulfadimidine model to evaluate pharmacokinetics and residues at various concentrations in laying hen.

Low level intake of drugs from the ingestion of contaminated feed may lead to residue problems in food animals. Sulfadimidine (SDD) was used as a model to determine the residue risk at various doses in laying hens. The drug was administered as a single intravenous injection (100 mg kg(-1) body weight, BW), as a single oral dose (100, 30, 10, 3, 1 mg kg(-1) BW) and via medicated feed for 7 consecutive days (30, 10, 3 mg kg(-1) BW). Drug levels were determined with HPLC-UV for plasma, yolk and albumen. Pharmacokinetic values, which were calculated using a first-order one-compartment model, residue levels and transfer rates into the eggs were found to be dose-dependent. Even low doses of 3 and 1 mg kg(-1) BW resulted in measurable residues in yolk and albumen 1 day after a single oral administration. After ingestion of medicated feed at 3 mg kg(-1) BW, mean drug levels at 0.14 +/- 0.01 microg g(-1) were found in albumen and at 0.09 +/- 0.01 microg ml(-1) in plasma. Generally, the residue levels in albumen and plasma were higher than in yolk. These findings demonstrate a residue risk for the consumer even after low level intake of drugs.

Comparison of bovine udder skin with human and porcine skin in percutaneous permeation experiments.

Rat and pig animal skin has been the most common replacement material for human skin for use in in vitro permeability experiments. Unfortunately, the permeability barrier of skin from laboratory animals is known to be relatively weak, due to significant follicular transport. Pig skin has been shown to be a suitable model for human skin. Unfortunately, it cannot be gathered from the regular slaughtering process, which makes it unsuitable for permeation experiments. We therefore studied the suitability of bovine udder skin, an untreated waste material of the butchering process, as a possible replacement material for use in in vitro permeability tests. We investigated the barrier strength of bovine udder skin against four different substances, and its histology and lipid profile, in comparison with pig skin and heat separated human epidermis. Pig and human skin were found to be equally permeable, whilst bovine udder skin seemed to exhibit a weaker, but less variable, barrier against caffeine, benzoic acid, testosterone, and flufenamic acid. The skin of all three species contained variable contents of the major lipid classes: cholesterol, ceramides, cholesterol ester, fatty acids and triglycerides. Morphological differences mainly comprised variations in the density of hair follicles. Based on these results, the amount of free fatty acids and triglycerides and the density of hair follicles seem to be important factors in the differences between the skin barriers in the three species.

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection.

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.