Influence of dexamethasone and gamithromycin on the acute phase response in LPS-challenged calves

Introduction : Lipopolysaccharide (LPS) is a potent inducer of the bovine acute phase response and has been widely used in research to provoke acute inflammation. An intravenous challenge with LPS elicits the endogenous synthesis and release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). These cytokines initiate fever and stimulate the hepatic production of acute phase proteins, such as Serum Amyloid A (SAA). Regarding the fact that immunomodulating drugs are able to influence this acute phase response, the aim of the present research was to study the potentials of dexamethasone and gamithromycin in a standardized LPS-inflammation model. Dexamethasone was applied as a positive control, due to its major anti-inflammatory effects. The novel azalide gamithromycin on the other hand, was selected since macrolide antibiotics have been reported to exert immunomodulatory effects. Furthermore, the combination of both drugs was studied for possible additive and/or synergistic effects. Materials and Methods : A standardized and reproducible inflammation model was developed by challenging twelve 4-week-old calves intravenously with a single dose of LPS (E. coli serotype O111:B4, 0.5 µg/kg body weight (BW)). Three control animals on the other hand received an equivalent volume of 0.9% NaCl. Rectal body temperature was measured and plasma samples were collected at several points in time until 72h p.a. These samples were analyzed using ELISAs for TNF-α, IL-6 and SAA. As part of the immunomodulation study, eighteen different calves were randomly divided in three groups, each group consisting of six calves. The groups received a single bolus of respectively 0.3 mg/kg BW dexamethasone i.m. (Dexa 0.2%®, Kela), 6 mg/kg BW gamithromycin s.c. (Zactran®, Merial) and the combination of both drugs. At Tmax of the drug (time at which the maximum plasma concentration is reached) the LPS-bolus was administered, followed by a similar experimental design as for the inflammation model. Results and Conclusions : In comparison with the results obtained in LPS-administered animals which did not receive any treatment, dexamethasone and the combination of dexamethasone and gamithromycin significantly inhibited the release of TNF-α, IL-6 and SAA after an LPS-challenge. The administration of gamithromycin solely did not affect the cytokine and acute phase protein concentrations. Regarding the course of the body temperature, neither dexamethasone, nor the combination had a major influence, while gamithromycin alone induced a remarkable delay of the maximum body temperature. In other words, these results demonstrate the possible additive effect of a combined administration of an antibiotic with a corticosteroid in the acute phase of a bacterial infection, which could contribute to a better clinical condition of the animal

Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in plasma of lipopolysaccharide-challenged pigs

Introduction : Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and IL-6. In research, multiplex assays currently are a very popular tool for the simultaneous detection of biomarkers of infection and inflammation. Specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single factor analysis, yet for multi-parameter analyses, this approach is time-consuming and expensive. Cytometric bead array (CBA) is a flexible, bead-based flow cytometric application for the simultaneous detection of various soluble proteins of interest. The aim of the present study was to develop and validate a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6. The results were compared to commercial ELISA kits. Materials and Methods : Four pigs with a mean body weight (BW) of 24.9 kg were intravenously challenged with 15 µg ultrapure LPS/kg BW (Escherichia coli serotype O111:B4). Plasma was isolated and stored at -70 °C until analysis. Capture antibodies were covalently coupled to the surface of beads with unique fluorescence intensities (Becton Dickinson Biosciences). Detection antibodies were conjugated with R-Phycoerythrin (R-PE). A mixture of beads was firstly incubated with an appropriate standard mixture. Subsequently, a mixture of detection antibodies, either directly or indirectly conjugated to R-PE, was added to accomplish the desired sandwich format. The samples were finally analyzed on a BD FACSArrayTM Bioanalyzer. ELISAs were purchased from R&D Systems. Results : Table 1 shows the validation parameters of the developed CBA 3-plex assay and the commercial ELISAs. Following an in vivo LPS challenge, similar plasma concentration-time profiles were observed for all cytokines with CBA and ELISA. Discussion : This is the first CBA study describing a validated multiplex protocol for the simultaneous measurement of the major porcine pro-inflammatory cytokines TNF-α, IL-1β and IL-6. In research, ELISAs are still considered as the gold standard for determination of secreted proteins in serum or plasma, however, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of cytokine profiles in small porcine plasma volumes

Pharmacokinetics of dexamethasone in broiler chickens

Dexamethasone (DEX) is a synthetic derivate of cortisol and is one of the most potent glucocorticoids in man and animal. It is well known as an anti-inflammatory drug in many species. In poultry, however, data on the use of DEX are scarce. DEX would be a possible candidate-drug to influence mediators like cytokines and acute phase proteins in a lipopolysaccharide (LPS) inflammation model. First of all, it is important to determine the pharmacokinetics to investigate the immunomodulating properties of this drug. Data on disposition of DEX in broilers are unknown. The aim of this study was to investigate several pharmacokinetic parameters (area under the curve, AUC; absorption and elimination rate constant, kabs and kel; half-life of absorption and elimination, t1/2abs and t1/2el; volume of distribution, Vd; clearance, Cl; maximum plasma concentration at 0 h; maximum plasma concentration, Cmax; time to Cmax, Tmax and absolute bioavailability, F) of DEX in broiler chickens. The DEX experiment was performed as a two-way cross-over design, each group containing three 4-week-old broiler chickens and with a wash-out period of 96 hours. The animals received a bolus of 0.3 mg/kg body weight DEX intravenously (IV) in the wing vein or intramuscularly (IM) in the pectoral muscle. Blood (1 ml) was sampled by venipuncture from the leg vein before (time 0) and post-administration (0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12 and 24 hours). Plasma was stored at ≤ -15°C until analysis. Quantitation of DEX in plasma was carried out using in-house developed and validated LC-MS/MS methods. The pharmacokinetic parameters were analyzed using WinNonlin, version 6.2.0 (Pharsight). The pharmacokinetics of DEX were analyzed according to a one-compartmental model (see Table 1). After IM administration, the maximum plasma concentration was reached fast at 0.37 h. In contrast to many other species, t1/2el of the corticosteroid was very short. The clearance in chickens was 1.26 L/h.kg, which is higher than for mammals. The Vd amounted to 1 L/kg and is equal to values found in other species. In chickens, a bioavailability of 100% after IM administration of DEX could be calculated, indicating a complete absorption of the drug. The pharmacokinetics hereby presented, will be very useful in an in-house developed LPS-inflammation model in broiler chickens

Pharmacokinetics of gamithromycin in pigs

Objectives : Gamithromycin, a 15-membered semi-synthetic macrolide antibiotic of the azalide subclass, has recently been developed for the treatment and prevention of bovine respiratory disease (BRD). Besides the anti-infectious properties, macrolides have frequently been reported to be able to influence various inflammatory processes, such as the production of pro-inflammatory cytokines and mediators. The aim of this study was to determine the pharmacokinetic (PK) parameters of gamithromycin in pigs, whereafter the disposition of the antibiotic can be used in further research to investigate its immunomodulating properties in a porcine lipopolysaccharide (LPS) inflammation model. Materials and Methods : Twelve male pigs, with a mean body weight (BW) of 24.4 kg were randomly divided in two groups. The animals received a single injection of 6 mg/kg BW gamithromycin (ZACTRAN®, Merial), either intravenously (IV) in the ear vein (n = 6) or subcutaneously (SC) (n = 6). Blood was collected from the jugular vein into EDTA tubes before administration; on day 0 at 15, 30 and 45 minutes and 1, 2, 4, 6, 8, 10, 12 and 24 h post administration (p.a.) and once daily from day 2 to day 14 p.a. (24 h intervals). Plasma was isolated and stored at ≤ -15°C until analysis. Quantitation of gamithromycin in the plasma samples was performed using an in-house developed and validated LC-MS/MS method. The pharmacokinetic parameters were analyzed using the software program WinNonlin (Pharsight). Results and Conclusions : The area under the plasma concentration-time curve (AUC0→∞), absolute bioavailability (F), half-life of absorption and elimination (t1/2abs and t1/2el, respectively), volume of distribution (Vd), clearance (Cl), maximum plasma concentration (Cmax) and time to Cmax (Tmax) were determined and critically compared to the PK parameters in cattle and foals1,2. Results and conclusions will be presented at the congress. References 1. Huang et al., 2010, J. vet. Pharmacol. Therap. 33(3), 227-237 2. Berghaus et al., 2012, J. vet. Pharmacol. Therap. 35(1), 59-6

Pharmacokinetics of gamithromycin in broiler chickens

Gamithromycin (GAMI) is a 15-membered semi-synthetic macrolide, which is effective against bovine respiratory disease. Since this drug is for this moment only registered for cattle, data on other species are rare. Moreover, it is stated that macrolides, such as azithromycin and clarithromycin, should have immunomodulating capacities (Kanoh and Rubin, 2010). GAMI could have a positive effect on respiratory disorders in chickens, for instance in infection of Mycoplasma gallisepticum. Therefore, it is important to determine the pharmacokinetics to investigate the immunomodulating properties of GAMI in a lipopolysaccharide inflammation model. Data on the disposition of this drug in broilers are unknown. The aim of this study was to investigate several pharmacokinetic (PK) parameters (area under the curve, AUC; absorption and elimination rate constant, kabs and kel; half-life of absorption and elimination, T1/2abs and T1/2el; volume of distribution, Vd; clearance, Cl; maximum plasma concentration, Cmax and time to Cmax, Tmax) of GAMI in healthy broiler chickens. The experiment was performed as a parallel study with 12 4-week-old female broiler chickens. Six animals received an intravenous (IV) bolus of 6 mg/kg BW in the wing vein. The other six were injected with the same dose, subcutaneously (SC) in the neck. Blood (1 ml) was sampled by venipuncture from the leg vein before (time 0) and after administration (0.08, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12 and 24 hours and once daily on day 2 till day 14 for GAMI). Plasma was stored at ≤ -15°C until analysis. Quantitation of GAMI in plasma was carried out using an in-house developed and validated LC-MS/MS methods. The pharmacokinetic parameters were analyzed using WinNonlin, version 6.2.0 (Pharsight). The results show similar pharmacokinetic profiles of GAMI after IV and SC administration. Chickens clear GAMI faster than mammals and Vd is quite similar, which leads to a shorter T1/2el. Furthermore, there is a complete and fast absorption as well as a fast elimination of GAMI following SC administration in broiler chickens. References: Kanoh, S., Rubin, B.K., 2010. Mechanisms of Action and Clinical Application of Macrolides as Immunomodulatory Medications. Clinical Microbiology Reviews 23, 590-615