Georgios Delis

Application and validation of a LC/fluorescence method for the determination of amoxicillin in sheep serum and tissue cage fluid.

A LC method with fluorescence detection after pre-column mercury dichloride derivation was developed and validated for the quantitative determination of amoxicillin in sheep blood serum and tissue cage fluid at levels down to 100 and 200ng/mL, respectively. Spiked blood serum and tissue cage fluid samples were deproteinized, derivatized with mercury dichloride and extracted prior to reversed phase LC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 355nm and an emission wavelength of 435nm. Separation was carried out on a C(18) column with a mobile phase consisting of phosphate buffer, octanesulphonate sodium (OCT), and acetronitrile. A regression model using 1/concentration weighting was found the most appropriate for quantification. The intra-day precision for serum was 1.65-8.74% and for tissue cage fluid was 2.48-6.27%. The inter-day precision for serum was 0.39-3.57% and for tissue cage fluid was 0.44-2.54%. The overall precision over 3 days for blood serum using of 108 replicates was 1.70-9.44% and for tissue cage fluid using of 54 replicates was 2.51-6.76%. Studies of amoxicillin stability in blood serum and tissue cage fluid indicated that amoxicillin was stable after 4 weeks storage at -85 degrees C. The method was successfully applied for the determination of amoxicillin in blood serum and tissue cage fluid samples collected from rams after intravenous administration.

Bioavailability and pharmacokinetics of sulphadiazine, N4-acetylsulphadiazine and trimethoprim following intravenous and intramuscular administration of a sulphadiazine/trimethoprim combination in sheep.

The combination of sulphadiazine and trimethoprim is extensively used in farm animal species; however, there are no data concerning its pharmacokinetics after intramuscular administration in sheep. Twelve rams of the Chios breed were used to study the disposition of sulphadiazine, its metabolite N4-acetylsulphadiazine and trimethoprim after intravenous (i.v.) and intramuscular (i.m.) administration of a sulphadiazine/trimethoprim (5:1) combination in sheep. Sulphadiazine bioavailability (±SD) was 69.00%±10.51%. The half-life of the terminal phase (4.10±0.58 h afteri. v., and 4.03±0.31 h after i.m. administration) was significantly higher than the respective value for trimethoprim (0.59±0.19 h) afteri.v. administration. The maintenance of a constant plasma concentration ratio after i.v. administration was therefore impossible. The acetylation capacity in sheep, determined by the AUC ratio between N4-acetylsulphadiazine and the parent compound, sulphadiazine, was very low (less than 4%). The most remarkable finding of this study was that trimethoprim was not detected in sheep plasma after i.m. injection. In conclusion, according to the findings of the present study, following i.v. administration of the sulphadiazine/trimethoprim combination, trimethoprim can be considered as the limiting factor for any possible synergistic effect, and the i.m. route cannot be recommended in sheep.

Influence of the injection site on the pharmacokinetics of amoxicillin after intramuscular administration of a conventional and a long-acting formulation in sheep.

The pharmacokinetics of amoxicillin (AMX) were investigated in sheep following intravenous (i.v.) and intramuscular (i.m) injection, comparing two different drug formulations, a conventional and a long-acting AMX-trihydrate suspension. For the i.m. application two different injections sites, the neck area and the hind limb were used to identify possible differences in the kinetic parameters related to the site of injection. A three-compartment open model could best describe AMX disposition after i.v. administration. Data analysis after i.m. administration of the conventional suspension at both injection sites revealed the occurrence of a flip-flop phenomenon, clearly indicating that absorption of AMX is the rate-limiting step of its overall disposition. A moderate effect of the injection site was observed with a tendency for the neck area to be advantageous, mainly in terms of rate rather than extent of absorption. Injection of the long-acting formulation led to a focal depot formation, thus yielding lower but remarkably prolonged serum AMX levels reflected in the respective terminal half-lives. The concentration-time profile of AMX after administration of the long-acting formulation was less affected by the injection site, but the low serum levels justify its use only in cases in which a high susceptibility of the involved bacterial population is confirmed.

Peripheral distribution of amoxicillin in sheep and influence of local inflammation.

The pharmacokinetics of amoxicillin (AMX) in blood serum (SBS) and tissue cage fluid (TCF) was studied in sheep. Four tissue cages, prepared from silicone rubber tubing, were subcutaneously inserted in the neck area (two on each side) of the experimental animals and AMX was administered both intravenously (IV) and intramuscularly (IM) at the dose rate of 15mg/kg bodyweight. The impact of local inflammation on AMX distribution in TCF was studied after intra-cavity injection of a lambda carrageenan solution in one of the two tissue cages used after each administration. In contrast to the three-compartment AMX disposition after IV injection, two-compartment, absorption-limited pharmacokinetics was observed after IM administration. Non-inflamed and inflamed TCF data revealed, in all cases, the attainment of low, but prolonged concentrations and absence of an inflammation-induced effect on AMX penetration into and elimination from TCF.

Prevalence and mechanisms of resistance to fluoroquinolones in Pseudomonas aeruginosa and Escherichia coli isolates recovered from dogs suffering from otitis in Greece

The aim of this study was to investigate the prevalence and the implicated mechanisms of resistance against selected veterinary fluoroquinolones (enrofloxacin, marbofloxacin and pradofloxacin) among 101 Pseudomonas aeruginosa (n=75) and Escherichia coli (n=26) isolates collected from dogs suffering from otitis. Resistance ranged from 32.0% to 48.0% with differences not being considered statistically significant among the three agents or between the two bacterial species. However, individual MICs of pradofloxacin, the latest veterinary fluoroquinolone, were significantly lower than those of enrofloxacin, the oldest one, indicating an increased in vitro potency of the former antimicrobial. Pradofloxacin MIC90 was, additionally, the lowest (8μg/ml), in E. coli, or among the lowest (8μg/ml), in P. aeruginosa isolates. Resistance was in most cases associated with topoisomerase substitutions, with patterns GyrA:V73G in P. aeruginosa and GyrA:S83L+D87N/ParC:S58I+A86V in E. coli being reported for the first time in small animal isolates. Only 6.7% and 15.4% of P. aeruginosa and E. coli otitis isolates, respectively, carried plasmid-mediated quinolone resistance (PMQR) genes, which, moreover, contributed minimally to resistance. Efflux pump activity was additionally detected in resistant E. coli isolates, even those lacking topoisomerase substitutions or PMQR genes. The emergence of resistance in the canine otitis isolates seemed to be associated with previous, prolonged systemic fluoroquinolone administration. In any case, antimicrobial susceptibility testing should guide the selection of systemic FQs for the treatment of canine otitis.

Pharmacodynamic interactions of amikacin with selected β-lactams and fluoroquinolones against canine Escherichia coli isolates

Knowledge of in vitro antimicrobial interactions can serve as a guide for clinical application of combination antimicrobial regimens. The aim of the present study was to determine the pharmacodynamic interactions of amikacin with either amoxicillin/clavulanic acid, ceftazidime, enrofloxacin or marbofloxacin against clinical canine Escherichia coli isolates. Bactericidal activity of individual antimicrobials was assessed by use of static kill curves. Interactions between amikacin and each of the β-lactams or fluoroquinolones were subsequently analyzed by employing the fractional maximal effect method. Amikacin, compared with all other agents, displayed the most rapid and extensive bacterial killing, the lowest level (with respect to MIC) at which half the maximal effect was observed and the most linear concentration-effect relationship. The combinations of amikacin with amoxicillin/clavulanic acid or ceftazidime were completely synergistic in four and three out of the five investigated isolates, respectively, with additivity being sporadically observed. On the other hand, the combinations of amikacin with enrofloxacin or marbofloxacin yielded a mosaic of interaction types with no discernible pattern or differentiation between fluoroquinolone-susceptible and resistant isolates; synergy was only infrequently observed, mainly at increased fluoroquinolone concentrations. In conclusion, the combinations of amikacin with the two β-lactams were found to be more promising, in terms of synergy achievement, compared with the respective combinations with the two fluoroquinolones.

Pharmacokinetics and tolerability of aminosidine after repeated administrations using an optimal dose regimen in healthy dogs and in dogs with leishmaniosis

Optimisation of dose schedules of aminoglycosides is required in order to increase efficacy and prevent their toxicity. The objective of this study was to determine the pharmacokinetic profile and the safety of aminosidine in dogs with naturally occurring leishmaniosis and in healthy dogs after once daily administration. Six young-adult, male, healthy, Beagle dogs and 12 dogs with clinical signs of canine leishmaniosis without azotemia and proteinuria were included in the study. Diagnosis of the disease was confirmed by serology, parasitology and molecular techniques. Pharmacokinetics and evaluation of renal function after repeated (once daily for 21 consecutive days) subcutaneous administration of aminosidine, at the dose of 15 mg/kg b.w. in both the healthy and the diseased animals were compared. Concentrations of aminosidine were determined by high-performance liquid chromatography and pharmacokinetic analysis was performed by the non-compartmental method. No significant differences were observed between healthy and diseased dogs considering all pharmacokinetic parameters. In general, mean Cmax ranged between 46.41 and 54.32 μg/mL and between 38.69 and 40.73 μg/mL in healthy dogs and in dogs with canine leishmaniosis, respectively. No accumulation of the drug was observed in either group since total elimination of aminosidine and half-life lambda z were not modified throughout the administration period. Aminosidine was well tolerated in all dogs with no clinical and clinicopathological signs of nephrotoxicity. Once daily administration of high dose of aminoglycosides, resulted in effective serum concentrations and absence of nephrotoxicity.