Clinical Breakpoint And Physiological Based Pharmacokinetic/Pharmacodynamic Model Of Cefquinome Against Haemophilus Parasuis

Haemophilus parasuis（H.parasuis）is a common pathogen in the respiratory tract of piglets.It invades piglets and causes symptoms such as fibrinous serositis,arthritis,and meningitis,which endangers the development of the breeding industry.In veterinary clinic,extensive agricultural use of antibiotics poses a risk of increasing antimicrobial resistance,which has been one of the main public health burdens.Cefquinome is the fourth-generation cephalosporin antibiotic solely for animals.It can effectively treat pig respiratory diseases caused by H.parasuis,and has excellent application prospects in veterinary clinic.In order to monitor the sensitivity of H.parasuis to cefquinome and ensure the antibacterial effect of cefquinome,this study carried out the optimization of dosage regimen and the establishment of clinical breakpoint to guide rational use and protect clinical potent of cefquinome.In this study,firstly,the wild cutoff,pharmacodynamic cutoff respectively determined by Monte Carlo simulation and population pharmacokinetic model,and clinical cutoff were established.The basic process of determination for clinical breakpoint in veterinary medicine was proposed,and the clinical breakpoint of H.parasuis to cefquinome were established.Secondly,the physiological based pharmacokinetic（PBPK）model including the pulmonary interstitial fluid sub-compartment and the semi-mechanistic pharmacodynamic model based on the inoculum effect was constructed,respectively.The PBPK/PD model was established to formulate the clinical dosage regimen and withdrawal period at different infection stages.Finally,the dosage regimen and withdrawal interval were evaluated by hollow fiber infection model and clinical infected-animal trail.1.The establishment of clinical breakpoint of cefquinome against H.parasuisThe MIC distribution of cefquinome against 344 H.parasuis was imputed into ECOFFinder,the epidemiological cut-off value of CEQ against H.parasuis was 1μg/m L.MIC,MBC and MPC of cefquinome against HPS42 were 1,2 and 6.4μg/m L.The PAE exposed cefquinome for 1 h was 0.18～0.4 h,and the PAE exposed cefquinome for 2 h was 0.32～0.67h.For the HPLC method,the LOD is 0.03μg/m L and LOQ is 0.05μg/m L,the recovery ranged within 88%-96%,and CV is less than 13%.The PK parameters of cefquinome in healthy and H.parasuis infected pigs,derived from non-compartmental analysis.The PK parameters of cefquinome in the serum of healthy and diseased pigs were calculated by Win Nonlin.Cefquinome was rapidly absorbed within 0.25 h from the injection site and reached the C_max.The C_max in the diseased pig was twice as that in the healthy group.The AUC,T_1/2,and MRT of cefquinome in the healthy and diseased pig sera were 8.61±2.68 and 15.52±4.07 h×μg/m L;3.52±0.81 and 3.19±0.92 h;and 4.61±0.68 and 3.59±0.88 h,respectively.The PK/PD parameters determined from the integration with in vivo PK data and the ex vivo MIC values.The PK/PD parameters,AUC_24h/MIC,were fitting into the inhibitory sigmoid E_maxmodel.With the bactericidal effect,the target endpoint for PK/PD index(AUC_24h/MIC)in serum is 41 h.A populational pharmacokinetic model was established after the assessment of diagnostic graph,visual predictive curve and boostrap.By Monte-carlo simulation and pop-pharmacokinetic,the pharmacodynamic cutoff was determined as 0.125μg/m L.In the clinical experiment,42 pigs were divided into six diseased groups（n=6）,which were infected by the above strains,and the control group（n=6）.The physiological parameters,including body temperature,mental state,and respiratory symptoms were monitored to adjudicate the administration cefquinome.The cure rate of each group after administration of cefquinome（2 mg/kg once daily for 3 days）was noted to calculate clinical cutoff by Windo W,CART and nonlinear regression fit.The clinical cutoff was set as 4μg/m L.Overall,refer to the document of clinical breakpoint determination from CLSI and EUCAST,a standard process was proposed about the determination of clinical breakpoint.The clinical breakpoint of cefquinome against H.parasuis was 1μg/m L.2.The establishment of PBPK/PD model of cefquinome against H.parasuisThe flow rate of 1μL/min had the best in vitro RR（27.59%±0.10%）.At the flow rate,the delivery（%）was steady（CV<11%）.The in vitro RR of microdialysis and retrodialysis was similar for different flow rates,a finding that indicates retrodialysis can be adopted to investigate the in vivo RR.After probe acclimatization,a sample with 0.75μg/m L CEQ was driven by the pump as flow rate of 1μL/min.The dialysate was collected every 15 min.The in vivo RR was 28.8%±2.74%.The PK parameters of CEQ in dialysate were calculated by Phoenix version 8.3.CEQ was absorbed into the lung and reached the maximum concentration of 2.48±0.46μg/m L at 1.25 h.The elimination half time(T_1/2λ)was 1.16±0.07 h,and the mean residence time was 3.01±0.19 h.The AUC of the concentration-time curve was 8.34±1.73μg*h/m L.Compared with a previous study,the AUC_dialysate to AUC_plasma ratio after intramuscular injection was 0.96.A six-compartment PBPK model,including plasma,liver,kidney,lung,muscle,and the rest of the organs.Each compartment is defined by a tissue weight and tissue blood flow rate.All compartments（except lung）are assumed to be perfusion limited and well stirred.The lung compartment is represented as a permeability-limited model comprising three sub-compartments,namely lung blood,lung interstitial fluid,and lung tissue.The present PBPK model was developed based on Berkeley Madonna（Version 10.1.3）.The chemical-specific parameters,such as tissue/plasma partition coefficient,were used as initial values in the model calibration and the“final model parameters”need to be adjusted and optimized by comparing simulations and observed PK data.The current model was validated by comparing the results of the simulation with the previously available data.Based on World Health Organization guidelines,model simulations are available within a twofold range of the measured values.Not all observed PK data used for validations were used for calibration.The goodness of fit between the simulation and observations was analyzed with linear regression for both calibration and validation;R² was calculated.R²≥0.75 is regarded as a general criterion for good prediction.The mean absolute percentage error（MAPE）was also used to validate the model.A local sensitivity analysis was performed for a discrete time point（24 h）to determine the most influential parameters on the area under the curves（AUCs）for the plasma,liver,kidney,and lung（including lung blood and lung interstitial fluid）CEQ concentrations.Cardiac output（QCC）,kidney flow rate（QKC）,renal clearance（Kurine C）,protein binding rate of lung tissue（PT）,the partition coefficients of different organs,the transfer constant from interstitial fluid to tissue（KTI）and from tissue to the interstitial fluid（KIT）was the sensitive parameters.Monte Carlo analysis was added to the PBPK model to estimate the sensitive of parameter uncertainty and individual variability.Only the sensitive parameters are simulated by Monte Carlo analysis.Physiological parameters are generally considered to have a normal distribution,and chemical-specific parameters are considered to have a lognormal distribution.The default coefficient of variation（CV）for partition coefficients（PCs）and transport constant rates are assumed to be 20%.The default CVs of other parameters were set as 30%.The lower bound（2.5 percentile）and upper bound（97.5 percentile）of the distribution were also set to constrain the range of random selection.A population PBPK model was established by Berkeley Madonna.And the withdrawal intervals can be predicted.In vitro time-killing curves of cefquinome against low-inoculum and high-inoculum H.parasuis,respectively.A semi-mechanism pharmacodynamic model based on inoculum effect by Phoenix software.The ability of different models and mathematical equations to describe the in vitro time-killing curves of different inoculums was evaluated.According to the value of AIC,BIC and-2loglikelihood,the best model was selected.It is a two-compartment model,including susceptible subcomparment and insensitive subcomparment.The growth rate is described in the form of capacity limitation,and the transfer rate of susceptible subcomparment to insensitive subcomparment is described as time correlation.The inoculum effect was described by the effect of start inoculum（SI）on EC₅₀ value.The model was successfully established after evaluated by diagnostic map,visual predictive curve and bootstrap.The PBPK/PD model was constructed by combining the semi-mechanism pharmacodynamic model based on inoculum effect and the PBPK model.It is predicted that the drug concentration of free cefquinome in pulmonary interstitial fluid changes at different doses,and the bacterial growth changes under drug exposure.It was used to design the dosage regimen for different infectious stage.The dosages of 3mg/kg/12h and 5mg/kg/12h are determined to respectively treat the infectious animal for the early and late stages of infection.The population physiological pharmacokinetic model predicted that when cefquinome was administered at 3mg/kg/12h and 5mg/kg/12h,the withdrawal period in pigs was 3 days.3.Hollow fiber infection model and clinical trial to evaluate the result of PBPK/PD model.Based on the PBPK model,the drug concentration-time curve of free cefquinome in pulmonary interstitium at different doses was predicted.Hollow fiber infection model was constructed by setting peristaltic pump rate,central chamber volume,injection drug volume and injection pump rate.The low and high inoculum of H.parasuis were respectively inoculated into hollow fiber culture barrel.The recommended dose and the PBPK/PD model predicted dose were used for administrated into the Hollow fiber infectious model,respectively.Samples were collected at different time points in the sampling channel,which was used for bacterial count and drug concentration determination.The hollow fiber infection model can accurately reproduce the drug concentration of free cefquinome in pulmonary interstitial fluid for different doses.The dose predicted by PBPK/PD model is better than the recommended dose about the bactericidal effect of H.parasuis at low and high inoculum.The infected animals were challenged by H.parasuis,and different infection stages of the animal model were established by different administration time.The 20 diseased animals were average divided into 4 groups.The animals were respectively administered with the recommended dose and the PBPK/PD model predicted dose.Combined with the clinical symptom score of the animals,the survival rate and cure rate,and the bacterial load after administration,the effect of dosage was evaluated.A HPLC-MS/MS method for the determination of cefquinome in pig edible tissues was established.The detection limit of cefquinome in pig liver,kidney,muscle and fat samples was 5 ng/m L,and the quantitative limit was 10 ng/m L.The concentration range of cefquinome in fat was 10～100 ng/m L,and the recovery rate was 83.1～117.98%.The range of drug concentration added in kidney was10～500 ng/m L,and the recovery rate was 71.0～119%.The recovery rate was 84.2～119%in the concentration range of 10～100ng/m L for muscle.The range of drug concentration added to pig liver was 10～200 ng/m L,and the recovery rate was 89.7～114%.The intra-assay coefficient of variation was less than 11.0%,and the inter-assay coefficient of variation was less than 12.8%.After the last administration for three days,pigs in each group were slaughtered to determine the drug concentration in different tissues.The drug concentration was lower than the maximum residue limit.The results showed that the withdrawal period predicted by the population physiological pharmacokinetic model was reliable.