Pharmacokinetic-Pharmacodynamic Modeling Of Cyadox Against Salmonella Pullorum In Broiler

Salmonellosis is a systemic disease responsible for important economic losses in poultry breeding, which can causes high chick morbidity and mortality. With the rapid development of intensive chicken farming, Salmonella infection is getting worse and worse. Quinoxalines, including cyadox, are used to treat diseased animals. With broad-spectrum antibacterial activity for livestock and fish,cyadox can prevent and control animal diseases, also improve bioavailability of feed and the production performance. However, a scientific and reasonable dosage regimen is not yet established. PK-PD modeling can reflect the relationship between the host, drug and pathogens, which has the ability to achieve clinical efficacy and to minimiza the selection and spread of resistant pathogens. The purpose of the present study was to estimate pharmacokinetic-pharmacodynamic (PK-PD) surrogates for bacteriostasis, bactericidal activity and bacterial elimination against cvcc528, using a PK-PD approach for cyadox in broilers after oral administration. With WinNonlin software to construct ex-vivo PK-PD model, we get a reasonable dosage regimen of cyadox to treat salmonella diarrhea in broilers. It also provide a theoretical basis for reasonable application of cyadox in clinical.1Antibacterial activity of cyadox against Salmonella cvcc528The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of cyadox and its major metabolites(Cy1, Cy2, Cy4, Cy6, Cy9, Cy12) were individually estimated in MH broth and in ileum content against cvcc528that served as test organism in all experiments. The test method was recommended by clinical and Laboratory Standards Institute (CLSI). The MIC and MBC of cyadox in MH broth were2μg/mL and4μg/mL. All its main metabolites only Cy9has antibacterial activity. And the MIC of Cy9is2μg/mL in broth. The MIC of cyadox in ileum content is2μg/mL.According to the MIC of cyadox in MH broth, We prepared1/2MIC,1MIC,2MIC,4MIC,8MIC,16MIC,32MIC of cyadox with broth for bactericidal experiments, inoculated same bacteria to the broth. After1,2,4,6,8,12,24hours under anaerobic conditions we get100μL, which was gradient diluted by saline then count. The result shows that the bacteriostatic curves of cyadox is similar with the known concentration-dependent antibiotic fluoroquinolone. Meanwhile the bactericidal activity increases with increasing concentration of the cyadox, The time to kill all bacteria gets shorter. This indicates that the bactericidal type of cyadox against cvcc528is concentration-dependent.Assessment of the intracellular antibacterial activity of cyadox. RAW264.7cells were diluted to1×106cells in24-well culture plates. Which were incubated with salmonella. One hour later, culture plates were incubated with gentamicin to kill extracellular and then built infected cells system. Incubated with2μg/mL,8μg/mL,20μg/mL cyadox,4h,8h,24h later, intracellular bacteria were lysed by cell lysates, gradient diluted and then count to get antibacterial curve. The result shows that compared with the blank control group cyadox significantly inhibit the intracellular growth of Salmonella cvcc528, There is not proliferation of intracellular bacterias when we add20μg/mL cyadox to RAW264.7cells. And the inhibition activity increased with the increasing of the concentration of cyadox.Post-antibiotic effect (PAE) of cyadox against Salmonella is estimated by removal of drug methods. Salmonella was incubated with1MIC,2MIC,4MIC of cyadox. After1and2hours, we get ride of drug by highly diluted method. The colony-forming unit (CFU) per milliliter were determined at different time. After that we establish recovery growth kinetic curves of salmonella to calculated the PAE of the cyadox. The result shows that cyadox does not have PAE, when the concentration of cyadox is4MIC, after4hours it completely killed all bacterias, by which we detected none of salmonella after reconstruction. The mutant prevention concentration (MPC) of cyadox is determined by agar method. Preparation of agar plates containing series concentration of cyadox, we concentrated Salmonella to1010CFU/mL, inoculated1010CFU bacteria on the plates, cultured72hours. The result shows that, the lowest drug concentration on agar plates without bacterial growth under anaerobic conditions namely the mutant prevention concentration (MPC) of cyadox against cvcc528minimum is40μg/mL.Ileum content samples from100broilers that had been administered cyadox orally collected at different time points were filtered and sterilized. It was incubated with same amount of salmonella. At0,1,2,4,8,12,24h we count the bacterias. The result shows that in the high concentration group (>10μg/mL), the CFU of which has changed significantly. After cultured in vitro for4hours, there is no detectable bacteria. The CFU of low concentration group (2～10μg/mL) has declined after cultured12hours. When the drug concentration is lower than the MIC(2μg/ml), some bacterias were suppressed, and the growth rate of salmonella was slightly lower than the blank control group, indicating that the drug concentration of these groups can not inhibit the growth of salmonella. There is no bacteria rebound phenomenon happened in our experiment.2Pharmacokinetic study of cyadox in broiler ilum content and plasma100broilers,30days old, were randomly divided into two groups with50replications each. One group were infected with cvcc528, which belongs to diarrhea model. Cyadox was administered orally at a dose rate of30mg/kg bw. Blood samples and ileum content samples were collected prior to each treatment and at0.5,1,2,3,4,6,8,12,24,48h, We use high performance liquid chromatography(HPLC) to detect the samples. The result shows that the maximum concentration of cyadox in plasma is0.016μg/mL, which does not yet reach the MIC of cyadox (MIC=2μg/ml). Therefore, it does not satisfy the requirement of the model. The concentration of cyadox in ileum contents of healthy broilers at each time were:205.87,234.26,293.81,49.4,13,2.15,0.59,0.42,0.36μg/mL,. The concentration of cyadox in the ileum contents of infected broilers were:57.02,110.58,157.52,53.54,17.31,3.71,2.37,0.74,0.4μg/mL。The metabolic processes of cyadox in ileum both correspond to one compartment model. The metabolism of cyadox is fast in the intestine of broilers, The time to achieve the highest concentration of cyadox(Tmax) is1.08～1.32h, Tm is0.62～0.67h, The highest concentration of cyadox(Cmax) is143.28to283.34μg/mL, T>MIC is5.23～6.36h, Area under the curve(AUC) is379.22to691.84h.μg/mL. As it can be seen the Cmax and AUC in healthy broilers is slightly larger than the corresponding value in infected broilers. That maybe caused by the changes of intestinal flora and related drug metabolizing enzyme activity after chickens infected with Salmonella.3Risk assessment of resistanceInoculated of same amount of salmonella into MH broth, which contains1,2,4,8,16,32,64～g/mL of cyadox. The concentration of bacterias is109CFU/mL, cultured in the anaerobic incubator, At different time,100μL of each group were incubated on the agar plates which contains8μg/mL(4×MIC) cyadox. The result shows that in the blank group and the drug concentration of which group is lower than MIC (2μg/mL), lots of salmonella appears drug resistance mutants. The most intensive drug-resistance mutants appeared in groups, of which the drug concentration is in the range of MIC (2μg/mL) to the MPC (40μg/mL). However, when the drug concentration is bigger than the MPC(40μg/mL), the salmonella are no longer to generate drug-resistance, Cyadox killed all bacterias. It illustrates that the drug concentration must reach to MPC on the pathogenetic location in order to inhibit drug to get drug-resistance, which does not quite fit with the MSW theory..Refer to the drug concentration in the ileum contents of broilers, If the administration frequency is2to3times within24hours, the time of cyadox concentration higher than MPC is about40%. It can quickly kill the drug-resistant bacterias. It is only about three hours when the concentration of cyadox is between the MIC and MPC, it will not produce resistant mutants theoretically. When administered once a day, the time is shorter when the concentration of cyadox exceed MPC, meanwhile low concentration of cyadox will contact longer with Salmonella. The risk of resistant enriched.4Fitting PK-PD model and dosage regimenAccording to the result of antibacterial experiment of cyadox against cvcc528, Cyadox belongs to concentration-dependent antibacterial drugs. Therefore, we reference parameters Cmax/MIC and AUC/MIC to fit PK-PD equation.Sigmoid Emax model was quoted to simulate relationships between Cmax/MIC AUC/MIC and ex-vivo antimicrobial efficacy. The results showed that the value of Cmax/MIC is equal to AUC/MIC When E=0,-3,-4. To connect with the administration, we choose AUC/MIC as simulation parameter. The final PK-PD fitting equation is E=2.72-7.3*C1.63/335.931.63+C1.63.When E=0,-3,-4(bacteriostatic, bactericidal and eradication) the corresponding value of ex vivo AUC/MIC values were244.02,739.66,1509.99. According to dosage equation X=d×AUCexvivo/MICtested/AUCinvivo/MICtarget,we can caculate optimal dose X (mg/kg/d). When MIC of caydox is2μg/mL, The corresponding value of X namely dosage is21.16,64.14,130.95mg/kg/d. For prevention of Salmonella, the dosage is20mg/kg bw; Therapeutic dosage is65mg/kg bw administered, For eradication of bacteria and to avoid the generation of resistant bacteria, the dosage is130mg/kg bw administered. As intensive broiler farming developed, we converted the dosage to mixed feeding, groups administered formula is D=d×n/W, For21days old broilers, the value of W is approximately120g/kg/d, D is groups mixed feeding dosage. If the MIC of cyadox against clinical isolates is2μg/mL, the best groups mixed feeding dosage is1.08g/kg, twice daily, as therapeutic dosage.In summary, this study clarified the antibacterial activity of cyadox against Salmonella pullorum is obviously concentration-dependent and weak time-dependent, It reveals the intestinal dynamic characteristics of cyadox in healthy broilers and Salmonella infected broilers. We built at the first time the PK-PD model of cyadox against Salmonella pullorum, in consideration of the risk of clinical resistance mutations select. We identified the optimal dosage regimen of cyadox to prevent and treat the Salmonella pullorum infection in the clinical, which reduces the development of drug resistance in the treatment of Salmonellosis and saves the cost for farmers.