PRUDENT USE OF FLUOROQUINOLONES IN AVIAN SPECIES: PHARMACOKINETICS OF FLUMEQUINE AND ENROFLOXACIN FOR PK/PD MODELLING IN TURKEY.

The importance of prudent and rational use of antimicrobial is important, not only to safeguard the efficacy of these drugs in humans and veterinary medicine but, even more so, to prevent the emergence and spread of undesirable resistance phenotypes in zoonotic pathogens as well as in commensal bacteria that can be transmitted between animals and humans. Even more importance and attention is now given to the prudent use of medically important antimicrobial drugs, referring to those drugs for human therapeutic use. The fluoroquinolones belong to this category. These are very potent antimicrobials and active against a wide range of pathogenic organisms and are well distributed in the body after administration. This class of antimicrobials has a therapeutic effect on most infections in different organs or tissues. Although it is rare that fluoroquinolones are the only available agent for treatment of a specific infectious disease, fluoroquinolones are important alternative medicinal products for a veterinarian to have as option for treatment. Fluoroquinolones have a unique mechanism of action not related to conventional antimicrobials, and therefore their efficacy should be retained as long as possible. The avian production increased enormously in the last 50 years and the European Union (EU) is one of the world's top producers in poultry meat and a net exporter of poultry products. In this production, turkeys that is considered a \u201cminor species\u201d, but it is important in the livestock production of Italy. Scarce data exist about the usage of antimicrobial drugs in turkey and even less is known about their efficacy. As the limited number of medicinal products authorized in this species, antimicrobial therapy is frequently carried out with the few products authorized or with drugs \u201cextra-label\u201d used with the consequence of increases of selective pressure and also with the possibility of cross-resistance within the same pharmacological group of compounds. The studies reported in this thesis aimed to revise the use of fluoroquinolones in turkey to maintain the efficacy and reduce the spread of resistance against E.Coli, the most common zoonotic avian pathogen. Pharmacokinetic(PK)/pharmacodynamics (PD) models are the best tool in order to select optimal dosage regimen. To confirm dosages used at farms and allow the integration of PK/PD data, the plasma concentrations in blood from healthy animals collected during treatment with flumequine and enrofloxacin, were determined. The first step was to optimize and validate a fast, simple, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS)/MS/MS method suitable for the detection of a wide range of concentrations of fluoroquinolones as those occurring in pharmacokinetic and residue depletion studies from several matrices. The first trial presents a sensitive and reliable confirmatory method for the extraction, identification, quantification of five fluoroquinolones . For the extraction and matrix clean-up of fluoroquinolones residues from all biological matrices, the Quick Easy Cheap Effective Rugged Safe (QuEChERS) methodology was adopted; only for plasma samples acetonitrile was used. The analyses were performed by (LC-MS. LC separation was performed on a C18 Kinetex column (100x2.1 mm, 2.6 \ub5m, Phenomenex, CA, USA) with gradient elution using ammonium acetate solution (10 mM, pH 2.5) and methanol containing 0.1% formic acid. Mass spectrometric identification was done using an LTQ XL ion trap (Thermo Fisher Scientific, CA, USA), with a heated electrospray ionization probe, in positive ion mode. The method was validated according to the European Legislation (decision 2002/657/EC) and EMA guideline (EMA/CVMP/VICH/463202/2009); selectivity, linearity response, trueness (in terms of recovery), precision (within-day repeatability and within-laboratory reproducibility), limit of detection, limit of quantification, decision limits, detection capability, absolute recovery and robustness were evaluated using turkey blank matrices. All data were within the required limits established for confirmatory methods except for flumequine which presented a recovery value slightly higher than 110% in muscle and intestinal content. For all fluoroquinolones, all the extraction rates were greater than 70% and limits of quantification ranged from 1.2 \ub5g/kg to 118.8 \ub5g/kg. This method was suitable for the identification and quantification of fluoroquinolones in plasma samples of turkeys treated for the purpose of second and third trials. In the second trial, the PK behavior of flumequine administered to 32 healthy turkeys as an oral bolus via gavage or as 5 days of 10-hours pulsed administration in drinking water were compared, using the EU authorized dose of 15 mg/kg and the double dose of 30 mg/kg. The MIC of 235 Escherichia coli field strains isolated from poultry were determined for PD to develop a PK/PD model. Blood samples were collected at established times over 24 h, and the obtained plasma was analyzed using the LC-MS/MS/MS method previously described. A monocompartmental model and a noncompartmental model were applied to the data to obtain the PK results. The maximum concentration (Cmax)/MIC50 and the plasma concentration-time curve from 0 to 24 hours (AUC0\u201324)/MIC50 ratios were, respectively, 0.67 \ub1 0.09 and 4.76 \ub1 0.48 and 1.18 \ub1 0.35 and 7.05 \ub1 2.40 for the 15 and 30 mg/kg bolus doses, respectively. After 10-hours pulsed administration of 15 mg/kg, values of Cmax/MIC50, 0.19 \ub1 0.02 on day 1 and 0.30 \ub1 0.08 on day 5 of therapy were obtained, the AUC/MIC50 ratios were 2.09 \ub1 0.29 and 3.22 \ub1 0.93 on d 1 and 5, respectively. Higher values were obtained with the doubled dose of 30 mg/kg: the Cmax/MIC50 ratios were 0.49 \ub1 0.11 on day 1 and 0.69 \ub1 0.18 on day 5; the AUC/MIC50 ratios were 5.15 \ub1 1.15 and 6.57 \ub1 1.92 on d 1 and 5, respectively. For both types of administration and both dosages, the Cmax/MIC50 and the AUC/MIC50 ratios achieved were significantly lower than the fluoroquinolones breakpoints usually considered for efficacy. The last trial involving 50 healthy turkeys, was conducted to evaluate the efficacy of enrofloxacin. As in the previous study, the effectiveness of different treatment schemes against E. coli was evaluated by a PK/PD approach, correlating the PK results with the MIC determined for 235 E. coli strains. In this study, 3 different oral treatments (a single oral gavage, 5 days of 10-hours pulsed water medication, and 5 days of 24-hours continuous water medication) and single parenteral (subcutaneous; SC) treatment using 2 different doses of enrofloxacin (i.e., the EU authorized dose, 10 mg/kg, and double the EU recommended dose, 20 mg/kg) were evaluated. Blood samples were collected at established times over 24 h. Plasma was analyzed using a LC-MS/MS/MS that was validated in house. A monocompartmental and a noncompartmental model were applied to the data to obtain the PK results. After gavage administration, the mean maximum concentration Cmax/MIC50 and area under the curve AUC0\u201324/MIC50 ratios were, respectively, 3.07 \ub1 0.62 and 7.01 \ub1 1.03 and 25.48\ub1 3.04 and 57.2 \ub1 3.73 for the 10 and 20 mg/kg doses, respectively. After SC administration of 10 mg/kg, Cmax/MIC50 and AUC0\u201324/MIC50 ratios were 3.45 \ub1 0.75 and 33.96 \ub1 7.46, respectively. After the administration of 10-h pulsed or 24-h continuous medicated water at 20 mg/kg, lower values of Cmax/MIC50 (10-h pulsed: 3.45 \ub1 0.7; 24-h continuous: 3.05 \ub10.48) and AUC0\u201324/MIC50 (10-h pulsed: 42.42 \ub1 6.17; 24-h continuous: 53.32 \ub1 5.55) were obtained. Based on these results, the European Union-recommended dosage of 10 mg/kg seems ineffective to achieve adequate drug plasma concentrations and even the 20 mg/kg by 10 h pulsed or continuous medicated water administration did not reach completely efficacious concentrations in plasma against colibacillosis. Although the results obtained were not completely encouraging, the medicated water should preferably be provided continuously. To conclude about the efficacy of enrofloxacin treatment against colibacillosis, target tissue concentration should be extensively considered