In Vivo Pharmacokinetics And Pharmacodynamics(PK/PD)Modeling And PK/PD Cutoff Study Of Florfenicol Against Pasteurella Multocida In Ducks

Pasteurella multocida is the causative agent of fowl cholera and has a significant economic impact on the poultry industry.Recently,the most commonly used method for controlling P.multocida infections is chemotherapy.Florfenicol is approved solely for animal use and plays an important role in livestock and poultry industry owing to the good antimicrobial activity,excellent pharacokinetic properties and less toxicity.Though,there are in vitro and ex vivo PK/PD studies of florfenicol against P.multocida,no in vivo PK/PD modeling was reported.In this study,we carried out the in vivo PK/PD modeling of florfenicol against P.multocida in ducks.The aims of this study were to obtain the best PK/PD parameters,optimize the dosage regimen,maximize the efficacy of florfenicol,and reduce the production of florfenicol resistant P.multocida.At the same time,we combined PK/PD modeling results with MIC distribution around the world using Monte Carlo Simulation in order to calculate a reasonable group dosage regimen and establish the PK/PD cutoff values of florfenicol against P.multocida1.In vitro pharmacodynamics.The MIC ranges of florfenicol against twelve P.multocida were 0.125-32 ?g/mL in broth and 0.25-32 ?g/mL in duck serum.The MIC ranges of cefquinome,gentamicin,tilmicosin and enrofloxacin were 0.25-128 ?g/mL,4-8 ?g/mL,1-128 ?g/mL and 0.25-2 ?g/mL,respectively The MBC and MPC ranges of florfenicol were 0.25-64 ?g·mL and 0.2-64 ?g/mL,respectively.The MBC of florfenicol was 2×MIC and the MPC was 1.6?2×MIC.The in vitro time-killing curves showed that when the initial bacterial concentration was 106 CFU/mL,2×MIC reached the bactericidal effect,while 4×MIC reached the bactericidal effect,with the initial bacterial concentration of 108 CFU/mL.2.Disease model establishment.AP.multocida infection model was established in six-week-old Gao you ducks.The scheme of infection model was tracheal inoculated with?102 CFU/mL bacteria suspension at a volume of 200 ?L.After 12 h inoculation,the bacterial burden in liver and lung was about 106?107 CFU/liver or CFU/lung..3.Pharmacokinetics of florfenicol in healthy and P.multocida infected ducks.168 infected ducks were used to study the florfenicol PK in serum and lung tissue at the oral doses of 1,15,30 and 60 mg/kg b.w.,respectively.At the same time,the PK of florfenicol in serum and lung tissue was also studyed in 52 healthy ducks at the oral dose of 30 mg/kg b.w..The concentration of florfenicol in serum and lung tissues were determined by high performance liquid chromatography?HPLC?,and the PK analyses were performed using a non-comartmental model provided in the WinNonlin software.Comparisons between groups were examined using independent sample t test provided in SPSS software.The protein binding of florfenicol in serum of P.multocida infected ducks was measured using equilibrium dialysis over the range of 1-20 ?g/mL.PK results showed that florfenicol have a good linear relationship between the doses and Cmax or AUC0₂4h at the dose range of 1?60 mg/kg b.w.?in serum?or 1?30 mg/kg b.w.?in lung tissue?.The maximum concentrations?Cmax?was lower in infected ducks?13.88±2.70 ?g/mL?vs.healthy control animals?17.86 ± 1.57 ?g/mL?.In contrast,the mean residence time?MRT:2.35 ± 0.13 vs.2.27 ± 0.18 h?and elimination half-life?T1/2?:1.72 ± 0.22 vs.1.62 ± 0.07 h?were similar for healthy and diseased animals,respectively.As a result,the area under the concentration curve for 0-12 h?AUC0-12h?for florfenicol in healthy ducks was significantly greater than that in infected ducks?49.47 ± 5.31 vs.34.52 ± 8.29 ?g·h/mL?.The PK differences of florfenicol in lung tissues between the two groups were in accordance with the serum PK differences.The concentration of florfenicol in lung tissues was approximately 1.2-fold higher than that in serum both in infected and healthy ducks indicating that florfenicol is effective in treating respiratory tract infections in ducks.The protein binding of florfenicol in serum of P.multocida infected ducks was about 10%.4.In vivo pharmacodynamics of florfenicol against P.multocida in ducks,PK/PDmodeling and optimizing dosage regimen.135 six-week-old Gaoyou ducks were divided into three grops,and infected with 0825Y1,0901 J1 and JY160110,respectively.In each groups,animals were divided into 9 subgroups.florfenicol was oral administrated to each subgroup at the doses of 0,1,2,5,8,10,15,20 and 30 mg/kg b.w.twice daily for three successive days.After the last administration,ducks were sacrificed humanly and one side of lung and liver were collected to calculate the burden of P.multocida in organs.The changes of bacteria in organs were integrating with PK/PD parameters Cmax/MIC and AUC0-24h/MIC by sigmoid Emax model with WinNonlin software,and the PK/PD parameters required for bacteriostasis and bactericidal effect were calculated.The dose needed to achieve bactericidal effect was calculated.The results showed that PK/PD parameters Cmax/MIC and AUC0-24h/MIC were both the optimal PK/PD parameters,and the correlation coefficients were about 0.9.For the sensitive strain 0825Y1,when the AUC0-24h/MIC values reached 117.54 and 108.19,Cmax/MIC values reached 23.76 and 21.61,it showed bactericidal effect in the liver and lung.For the florfenicol sensitive but gentamicin intermediary and enrofloxacin resistant strain 0901J1,when the AUC0-24h/MIC values reached 78.39 and 54.30,Cmax/MIC values reached 15.62 and 10.70,it showed bactericidal effect on the liver and lung.However,for the resistant strain JY160110,when the AUC0-24h/MIC values reached 2.03 and 2.06,Cmax/MIC value reached 0.42,it showed 1-Log10 kill effect on the liver and lung.It is obviously that PK/PD parameter values of florfenicol against P.multocida vary from different strains,but the values showed little difference using different organs as target.For the bacteria 0825Y1 and 0901J1,the daily dose to achieve bactericidal effect was 41 mg/kg b.w.,while for JY160110,the daily dose to reduce 1-Log10 kill was 64mg/kg b.w..5.Predicted the population doses and establish COPD by Monte Carlo Simulation.MIC distributions of florfenicol against 4314 P.multocida from 1987 to 2015 in different regions around the world were collected from NCBI database.And the MIC distribution maps of globe,the mainland of China and Taiwan district were made.Under different MIC distributions,we calculated the target attainment rate?TAR?at the recommended daily dose,group daily doses reach 50%TAR,90%TAR?or 85%TAR?,and analyzed PK/PD cutoff values.The target attainment rates were over 85.98%and 66.84%in the mainland of China and globe at the recommended daily dose,and was over 30.09%in Taiwan In the mainland of China,the daily doses achieve 50%TAR and 90%TAR were 21 and 52 mg/kg b.w.;in globe,the daily doses achieve 50%TAR and 85%TAR were 33 and 90 mg/kg b.w.;While in Taiwan,the daily dose reached 50%TAR was 84 mg/kg b.w..The cutoff values of florfenicol against P.multocida at the existing using daily dose?40 and 60 mg/kg b.w.?and predicted daily doses by Monte Carlo Simulation?52 and 90mg/kg b.w.?were 0.25 ?g/mL,4 ?g/mL,0.5?g/mL and 4 ?g/mL.