Pharmacokinetic–Pharmacodynamic Modeling Of Doxycycline Hydrochloride-florfenicol Injection Against Actinobacillus Pleuropneumoniae And Haemophilus Parasuis In Swine

Porcine respiratory diseases caused by Haemophilus parasuis and Actinobacillus pleuropneumoniae have brought serious losses to the pig industry at home and abroad for the high morbidity and mortality.The combination of florfenicol（FF）and Doxycycline hydrochloride（Dox·HCl）has been used as one of the important clinical drugs for the strong antibacterial activity of Haemophilus parasuis and Actinobacillus pleuropneumoniae.In order to scientifically regulate the application of the combination of Dox·HCl and FF for Actinobacillus pleuropneumoniae and Haemophilus parasuis and avoid the development of drug resistance,it was particularly important to predict and formulate a reasonable drug regimen by Pharmacokinetics-pharmacodynamics（PK-PD）.And it can also provide a reference for the study of compound drugs PK-PD model.The dosing regimen of Doxycycline hydrochloride-florfenicol（Dox·HCl-FF）injection prepared by previous study for Actinobacillus pleuropneumoniae and Haemophilus parasuis was formulated by PK-PD model,in order to achieve the best therapeutic effect and avoid the emergence of drug resistance.It provided a new drug regimen for porcine contagious pleuropneumonia and the diseases caused by Haemophilus parasuis.And it can make full use of the clinical values of Dox·HCl-FF injection to promote the healthy development of the breeding industry.1 Pharmacodynamic of FF,Dox·HCl,Dox·HCl/FF against Actinobacillus pleuropneumoniae and Haemophilus parasuis,respectivelyPharmacodynamic of FF,Dox·HCl and Dox·HCl/FF against Actinobacillus pleuropneumoniae The strains used in this study were 131 strains Actinobacillus pleuropneumoniae kept in the laboratory.The minimum inhibitory concentration（MIC）of FF,Dox·HCl and Dox·HCl/FF against 131 Actinobacillus pleuropneumoniae strains were determined by the agar dilution method recommended by the clinical laboratory standardization institute（CLSI）,obtaining the MIC₉₀of FF,Dox·HCl and Dox·HCl/FF for 131 Actinobacillus pleuropneumoniae strains were 8μg/m L,8μg/m L and 2μg/m L,respectively.Actinobacillus pleuropneumoniae near MIC₉₀was selected for serotype identification and virulence test in mice.Finally,Actinobacillus pleuropneumoniae with number BW1 was selected for PK-PD experiment.The MIC and Minmum bactericidal concentration（MBC）of FF,Dox·HCl and Dox·HCl/FF on Actinobacillus pleuropneumoniae BW1 in vitro and in ex vivo were determined by micro broth dilution recommended by CLSI,respectively.The growth and killing curves in vitro and ex vivo and the mutation prevention concentration（MPC）were determined by plate counting method.Post-antibiotic effect（PAE）and mutant selection window（MSW）of Actinobacillus pleuropneumoniae were measured after incubation with drugs at different concentrations for 1 h and 2 h.The results showed that the MIC of FF,Dox·HCl and Dox·HCl/FF on Actinobacillus pleuropneumoniae BW1 were 8μg/m L,8μg/m L and 2μg/m L,the MBC were 8μg/m L,8μg/m L and 4μg/m L,the MPC were 19.2μg/m L,19.2μg/m L and 6.4μg/m L,so the ranges of MSW were 8-19.2μg/m L,8-19.2μg/m L and2-6.4μg/m L,respectively.The PAE of FF,Dox·HCl and Dox·HCl/FF for 1 h and 2 h were 0.01-1.25 h and 0.28-1.39 h;0.19-1.34 h and 0.29-3.00 h;0.19-4.54 h and 0.73-6.07h,respectively.The killing curves of FF in vitro and ex vivo and PAE showed that the antimicrobial activity of FF on Actinobacillus pleuropneumoniae was concentration-dependent types,the antimicrobial activity of Dox·HCl on Actinobacillus pleuropneumoniae was certain time-dependent and concentration-dependent types.When FF and Dox·HCl were used in combination,the bactericidal effect increased with the increasing of the concentration,showing significant concentration dependence.Therefore,area under the concentration-time by MIC（AUC/MIC）was selected as the PK-PD parameter.Pharmacodynamic of FF,Dox·HCl and Dox·HCl/FF against Haemophilus parasuis The strains used in this study was 115 strains Haemophilus parasuis kept in the laboratory.The MIC of FF,Dox·HCl and Dox·HCl/FF against 115 strains Haemophilus parasuis were determined by the agar dilution method recommended by CLSI,obtaining the MIC₉₀for FF,Dox·HCl and Dox·HCl/FF against 115 strains Haemophilus parasuis were 4μg/m L,4μg/m L and 1μg/m L,respectively.Haemophilus parasuis near MIC₉₀was selected for serotype identification and virulence test in mice.Finally,Haemophilus parasuis with number 55 was selected for PK-PD experiment.The MIC and MBC by micro-broth dilution method,MPC,MSW and PAE by plate counting method of FF,Dox·HCl and Dox·HCl/FF for Haemophilus parasuis 55 in vitro and ex vivo were determined by CLSI.The MIC of FF,Dox·HCl and Dox·HCl/FF for Haemophilus parasuis 55 were 4μg/m L,4μg/m L and 1μg/m L,MBC were 8μg/m L,8μg/m L and 2μg/m L in vitro and ex vivo.The MPC are 12.8μg/m L,12.8μg/m L and 4.8μg/m L,so the ranges of MSW were 4-12.8μg/m L,4-12.8μg/m L and 1-4.8μg/m L,respectively.The PAE for 1 h and 2 h were 0.05-1.18 h and 0.16-3.46 h;0.32-1.73 h and 0.87-2.18 h;0.67-3.08 h and 1.76-5.54 h,respectively.The killing curves of FF in vitro and ex vivo and PAE showed that the antimicrobial activity of FF on Haemophilus parasuis was concentration-dependent types,the antimicrobial activity of Dox·HCl on Haemophilus parasuis was certain time-dependent and concentration-dependent types.When FF and Dox·HCl were used in combination,the bactericidal effect increased with the increasing of the concentration,showing significant concentration dependence.Therefore,area under the concentration-time by MIC（AUC/MIC）was selected as the PK-PD parameter.2 Pharmacokinetic of Dox·HCl-FF injection against Actinobacillus pleuropneumoniae and Haemophilus parasuis in the respiratory tract of swineThe pharmacokinetic of Dox·HCl-FF injection against Actinobacillus pleuropneumoniae in the respiratory tract of swine A total of 24 healthy weaned piglets about 25 kg were randomly divided into 4 groups,and 6 in each group.Infecting piglets with clinical isolates of Actinobacillus pleuropneumoniae to establish the artificial piglet infection model.The 4 groups were the healthy group of FF,the healthy group of Dox·HCl and the healthy group and the diseased group of Dox·HCl/FF.The model were established by infected swines with clinical isolates of Actinobacillus pleuropneumoniae.After intramuscular injection at a dose of 20 mg/kg b.w,plasma samples and PELF samples were collected at different time points.And the concentrations of free FF and Dox·HCl in plasma and PELF were determined by high performance liquid chromatography（HPLC）.Pharmacokinetic data was simulated by the first-order absorption two-compartment model in Winnonlin software,the results suggest that the time to reach peak concentration(T_max)in plasma of the unilateral FF healthy group and the compound FF healthy group and diseased group were 3.41±0.27 h,3.45±0.21 h and3.67±0.08 h,the peak concentration(C_max)were 4.13±0.11μg/m L,4.08±0.09μg/m L and4.09±0.05μg/m L,respectively,the area under the concentration-time curve for 24 h(AUC_24h)were 91.86±4.52 h·μg/m L,105.52±1.46 h·μg/m L and 115.05±1.88 h·μg/m L;The T_maxin plasma of unilateral Dox·HCl healthy group and compound Dox·HCl healthy group and diseased group were 1.86±0.14 h,1.97±0.06 h and 2.04±0.07 h,the C_maxwere3.63±0.14μg/m L,3.58±0.07μg/m L and 3.60±0.09μg/m L,respectively,the AUC_24hwere68.20±3.98 h·μg/m L,72.77±1.41 h·μg/m L and 73.29±1.05 h·μg/m L;For the unilateral FF healthy group and the compound FF healthy group and the diseased group in PELF,the T_maxwere 3.14±0.10 h,2.90±0.05 h and 3.07±0.01 h,and the C_maxwere 8.03±0.20μg/m L,8.87±0.08μg/m L and 8.67±0.07μg/m L,the AUC_24hof were 144.22±1.98h·μg/m L,172.75±2.52 h·μg/m L and 180.22±3.13 h·μg/m L;For the unilateral Dox·HCl healthy group and the compound Dox·HCl healthy group and diseased group,the T_maxof PELF were 2.19±0.05 h,2.72±0.03 h and 2.68±0.03 h,respectively,the C_maxwere7.68±0.07μg/m L and 7.91±0.09μg/m L and 7.99±0.05μg/m L,the AUC_24hwere133.26±4.43 h·μg/m L,126.96±3.70 h·μg/m L and 169.82±4.38 h·μg/m L.In plasma and PELF,there was no significant difference in pharmacokinetic parameters between FF and Dox·HCl in the unilateral healthy group and the compound healthy group and diseased group.The pharmacokinetic of Dox·HCl-FF injection against Haemophilus parasuis in the respiratory tract of swine A total of 12 healthy weaned piglets about 20 kg were randomly divided into healthy and diseased group,and 6 in each group.Infecting piglets with clinical isolates of Haemophilus parasuis to establish the artificial piglet infection model.After intramuscular injection at a dose of 20 mg/kg b.w,plasma and PELF samples were collected at different time point,and the drug concentrations in plasma and PELF were determined by the HPLC method.Pharmacokinetic data was simulated by the first-order absorption two-compartment model in Winnonlin software,the results suggested that the T_maxin the plasma of the compound FF healthy and the diseased group were 3.56±0.05 h and 3.85±0.13 h,respectively,the C_maxwere 4.18±0.05μg/m L and4.15±0.08μg/m L,the AUC_24hwere 104.38±3.29 h·μg/m L and 111.51±1.79 h·μg/m L;For the compound Dox·HCl healthy and the diseased group were 1.91±0.07 h and 2.16±0.05h,respectively,the C_maxwere 3.56±0.06μg/m L and 3.65±0.06μg/m L,the AUC_24hwere72.18±1.66 h·μg/m L and 74.39±2.11 h·μg/m L;The T_maxin PELF of the compound FF healthy and the diseased group were 3.39±0.06 h and 3.41±0.18 h,respectively,the C_maxwere 8.55±0.07μg/m L and 8.33±0.07μg/m L,the AUC_24hwere 159.73±3.61 h·μg/m L and162.92±2.59 h·μg/m L,respectively;For the compound Dox·HCl healthy and diseased group in PELF,the T_maxwere 2.99±0.07 h and 3.08±0.05 h,the C_maxwere 7.35±0.03μg/m L and 7.24±0.04μg/m L,the AUC_24hwere 145.43±2.89 h·μg/m L and 152.34±1.06h·μg/m L.In plasma and PELF,the pharmacokinetic parameters of compound FF and Dox·HCl in healthy and diseased groups were not significantly different.3 Integrating PK-PD model and formulating optimal dosageIntegrating PK-PD model and formulating optimal dosage for Dox·HCl-FF injection against Actinobacillus pleuropneumoniae In order to obtain the parameters of the healthy and diseased group,the AUC_24h/MIC valus of the healthy group and the diseased group were fitted with the numbers of Actinobacillus pleuropneumoniae in ex vivo by the inhibited sigmoid E_maxmodel.For the compound FF,when E was 0,-3 and-4,the corresponding AUC_24h/MIC of bacteriostasis,sterilization and eradication were 3.94 h,15.99 h and 28.63 h（compound FF of healthy group）,5.61 h,18.83 h and 32.68 h（compound FF of diseased group）;For the compound Dox·HCl,when E was 0,-3 and-4,the AUC_24h/MIC were 0.78 h,10.32 h and 24.06 h（compound Dox·HCl of healthy group）,7.42 h,19.64 h and 31.08 h（compound Dox·HCl of diseased group）.For the higher and better PK-PD parameters and antibacterial effect,the datas of the diseased group were selected.The doses of prevention,treatment and eradication of Actinobacillus pleuropneumoniae of the diseased group were 1.37 mg/kg,4.59 mg/kg and 7.99 mg/kg（compound FF of diseased group）,1.92 mg/kg,5.08 mg/kg and 8.04 mg/kg（compound Dox·HCl of diseased group）by the dose formula.Integrating PK-PD model and formulating optimal dosage for Dox·HCl-FF injection against Haemophilus parasuis In order to obtain the parameters of the healthy and diseased group,the AUC_24h/MIC valus of the healthy and diseased group were fitted with the numbers of Haemophilus parasuis in ex vivo by the inhibited sigmoid E_maxmodel.For the compound FF,when E was 0,-3 and-4,the AUC_24h/MIC of bacteriostasis,sterilization and eradication were 7.95 h,21.90 h and 34.38 h（compound FF of healthy group）,7.96 h,23.09 h and 38.58 h（compound FF of diseased group）;For the compound Dox·HCl,when E was 0,-3 and-4,the AUC_24h/MIC were 9.05 h,22.42 h and 33.54 h（compound Dox·HCl of healthy group）,9.27 h,23.73 h and 37.70 h（compound Dox·HCl of diseased group）.For the higher and better PK-PD parameters and antibacterial effect,the datas of the diseased group were selected.The doses for prevention,treatment and eradication of Haemophilus parasuis of the diseased group were 1.07 mg/kg,3.11 mg/kg and 5.21 mg/kg（compound FF of diseased group）,1.34mg/kg,3.42 mg/kg and 5.43 mg/kg（compound Dox·HCl of diseased group）by the dose formula.The final dosing regimen of Dox·HCl-FF injection for Actinobacillus pleuropneumoniae and Haemophilus parasuisIn order to better cure the diseases of Actinobacillus pleuropneumoniae and Haemophilus parasuis,the larger treating dose of Dox·HCl/FF against these two bacteria was selected,that is,the dosage regimen of Dox·HCl/FF for Actinobacillus pleuropneumoniae.In order to achieve to the therapeutic effect and to save the dosage,the less dose of Dox·HCl/FF,namely the dose of FF,was selected as the final dose regimen,then the bacteriostatic,bactericidal and eradication dose were 1.37 mg/kg b.w,4.59 mg/kg b.w and 7.99 mg/kg b.w.PK-PD model based on the growth kinetics of bacteria can predict the efficacy of different dosage regimens.The expected efficacy can be achieved after 24 hours interval and 2 days of continuous administration.