1. 14.6.2016 г.1 PHARMACOKINETICS OF ENROFLOXACIN IN DUCKS WITH STEATOSIS AFTER FORCE-FEEDING Neno Bratoev, Lubomir Lashev Department of Pharmacology, Physiology and Physiological Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria Bulgaria

2. 14.6.2016 г.2Introduction Enrofloxacin (EFC) is second generation broad spectrum fluoroquinolone is developed for exclusive use in veterinary medicine, active against many Gram-positive and Gram-negative microorganisms. Its disposition has been studied in many avian species and some interspecies differences were pointed out. In the animals organism it is partly transformed to ciprofloxacin – also substance with high antimicrobial activity Although the opinion that when carried out according to professional standarts and on a small scale, force-feeding does not induce diet-related pathological changes, as the steatosis is reversible during this period the birds have changed physiology like increased body weight, triglyceridaemia, high ASAT and lipase blood levels. Such type of changes can define changes of the drug disposition in case of disease and necessary treatment The aim of our study was to investigate the pharmacokinetics of enrofloxacin and its main metabolite ciprofloxacin in force-fed ducks

3. 14.6.2016 г.3 Material and methods Animals Twelve 11-week-old male and female healthy ducks (sterile hybrid between male Muscovy ducks (Cairina moschata) and female Peking ducks (Anas platyrhynchos), weighing 2.91-3.94 kg and twelve 13-weeks-old ducks of the same breed, but at the end of the force feeding, weighting 4.05-5.45 kg were included in the experiment The animals were housed in groups of 6 birds each. During this period observations were carried out twice per day. Food and water were provided ad libitum. The food was withheld from 18 hours before treatment until 8 hours after it Drug Enrofloxacin hydrochloride 5% solution for i.v. injection (Baytril ® 5% injectable solution, Bayer Animal Health, Germany) was used. Oral treatment was made using the same solution after dilution with distilled water to concentration of 2.5%

4. 14.6.2016 г.4 Study design The ducks were allocated to four groups. Two groups (6 normal just before the fattening and 6 force-fed at the end of the period) were given a single i.v. dose of 10 mg/kg of b.w. in the right brachial vein. The other two groups containing 6 ducks each (not fed and at the end of the force fattening) were administered with the same dose enrofloxacin directly into the crop using a plastic tube attached to syringe. Blood samples (each of 1.0 ml) (without anticoagulant) were obtained from the left brachial vein at the times: after i.v. administration - 0.083, 0.25, 0.5, 1, 3, 6, 9, 12, 18 and 24 h. and after p.o. administration - 0.25, 0.5, 1, 3, 6, 9, 12, 18 and 24 hr. The samples were centrifuged for 15 minutes at 1800g. The obtained serum was stored at -20° C until analysis. Material and methods

5. 14.6.2016 г.5 Analytical method Plasma concentrations of enrofloxacin and its metabolite ciprofloxacin were assayed by a HPLC method. The system consisted on a Hipersil Spherisorb ODS-2(C18)-250 x 4.6- mm 5 µm column, a Surveyor LC Pump Plus, A Surveyor fluorescence detector, and a Surveyor Autosampler Plus (Thermo Fisher Scientific Inc., USA) The limit of quantification (LOQ) of EFL and CIP were found to be 0.01 µg mL-1. The limit of detection (LOD) was determined to be 0.005 µg mL-1 for both compounds Pharmacokinetic analysis The pharmacokinetic analysis was carried out by specialized software WinNonlin 6.2.1 (Pharsight, USA) Statistical analysis Descriptive statistics was performed with Statistica for Windows (Statistica 6.0.1, USA). Data were presented as mean ± SD

6. 14.6.2016 г.6 Results The followed biochemical parameters (ASAT, ALAT, LDH, Cholesterol, Bilirubin, Triglycerides), characteristic for the liver function, presented on the next slide show typical for steatosis findings - higher values compared to control birds

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9. 14.6.2016 г.9 Results The serum concentrations after i.v. injection presented two- compartment model of distribution and elimination The oral administration was followed by a one-compartment of absorption and elimination

10. 14.6.2016 г.10 Table 1. Selected pharmacokinetic parameters (Mean±SD) of enrofloxacin in ducks after single i.v. injection at a dose of 10mg/kg t 1/2  (h) t 1/2  (h) k 10 (h -1 ) k 12 (h -1 ) Cl B (l/kg/h)Vss(l/kg) AUC 0-inf (  g/ml.h ) MRT(h) Beforefeeding4.28 ±0.65 0.47 ± 0.36 0.47 ± 0.14 0.98 ± 0.56 0.258 ± 0.049 1.272 ± 0.146 39.84 ± 6.87 4.99 ± 0.45 Afterfeeding5.80 ± 1.22 0.52 ± 0.36 0.23 ± 0.04 0.74 ± 0.50 0.189 ± 0.022 1.436 ± 0.211 53.45 ± 6.24 7.65 ± 1.29 The force-fed birds show slower distribution, higher volume of distribution and longer elimination half life, respectively longer presence of enrofloxacin in the organism of the treated birds Results

11. 14.6.2016 г.11 Table 2. Selected pharmacokinetic parameters (Mean±SD) of enrofloxacin in ducks after single intraingluvial administration at a dose of 10mg/kg – compartment analysis. t 1/2abs (h) t 1/2el (h) AUC 0-inf (  g/ml.h) Tmax(h)Cmax (  g/ml) F(%) Beforefeeding0.46 ±0.32 8.71 ± 3.0 25.35 ± 5.52 1.83 ± 0.74 1.75 ± 0.17 59 AfterFeeding0.53 ± 0.24 6.59 ± 7.94 34.44 ± 16.43 1.77 ± 0.53 4.06 ± 1.66 64.4 Results The absorption (t 1/2 abs ) of enrofloxacin in the force-fed ducks was slower Maximal serum concentrations (Cmax) and bioavailability (F) were higher in fattening ducks The elimination (t 1/2el ) is shorter in ducks after force-feeding

12. 14.6.2016 г.12 Table 3. Selected pharmacokinetic parameters (Mean  SD) of enrofloxacin and its metabolite ciprofloxacin in ducks after single intraingluvial administration at a dose of 10mg/kg – Noncompartment analysis Substance t 1/2 el (h) C max (  g/ml) T max (h) AUC 0-inf (  g/ml.h) MRT(h)F(% BeforefeedingEnrofloxacin7.42 ±1.62 1.79 ± 0.17 323.49 ± 4.1 11.63 ± 2.19 53.3 Ciprofloxacin7.54 ± 1.75 1.82 ± 0.18 2.58 ± 1.02 24.24 ± 4.21 11.9 ± 2.35 AfterfeedingEnrofloxacin13.8 ± 3.82 4.25 ± 1.21 1.25 ± 0.88 30.18 ± 3.62 9.80 ± 1.36 53 Ciprofloxacin21.59 ± 5.81 0.67 ± 0.20 5.13 ± 4.35 13.73 ± 3.01 31.7 ± 9.7 No significant difference between both groups using noncompartment analysis The bioavailability (F) was not influenced by feeding. The relations AUC cipro /AUC enro lead to the idea that metabolism of enrofloxacin in fed ducks is lower Results

13. 14.6.2016 г.13 Discussion The force-fed ducks distribute better enrofloxacin in the organism compared to normal ducks and eliminate it slower than in normally fed. The differences mentioned are not always statistically significant but could be a reason for longer enrofloxacin retention in force-fed ducks compared to normal The force-fed ducks distribute better enrofloxacin in the organism compared to normal ducks and eliminate it slower than in normally fed. The differences mentioned are not always statistically significant but could be a reason for longer enrofloxacin retention in force-fed ducks compared to normal The force-feeding does not affect significantly the oral enrofloxacin absorption, elimination and bioavailability. On the base of the CFC concentrations could be accepted that quality of metabolite is lower, respective the metabolism level is also lower The force-feeding does not affect significantly the oral enrofloxacin absorption, elimination and bioavailability. On the base of the CFC concentrations could be accepted that quality of metabolite is lower, respective the metabolism level is also lower

14. 14.6.2016 г.14 Conclusion The force-feeding of ducks can result in slower absorption, better distribution and slower elimination of enrofloxacin The force-feeding of ducks can result in slower absorption, better distribution and slower elimination of enrofloxacin The enrofloxacin bioavailability after oral administration and ciprofloxacin serum concentrations values are not significantly changed The enrofloxacin bioavailability after oral administration and ciprofloxacin serum concentrations values are not significantly changed

15. 14.6.2016 г.15 Thank you for attention !