Pharmacokinetic-Pharmacodynamic Modeling Of Cyadox Against Clostridium Perfringens And Clinical Efficacy In Broiler

Necrotic enteritis is a gastrointestinal infectious disease with intestinal mucosa haemorrhagic necrosis caused by Clostridium perfringens, with a considerably high incidence and mortality, which caused severe damage in poultry breeding. As a new candidate of quinoxalines, cyadox shows excellent bactericidal activity against Clostridium perfringens , the concentration in digestive tract was much higher than that in blood after broilers were dosed with oral administration, and it has little side effect and short withdrawal period, proved that cyadox is suitable to treat necrotic enteritis. This project researched the pharmacodynamics of cyadox against Clostridium perfringens and the pharmacokinetics of intestinal tract about broiler, based on PK-PD modeling formulated the dosage regimen. According to the dosage regimen, the randomized controlled trial was launched to provide theoretical basis and basic practice of clinical application.1. Pharmacodynamics of cyadox against Clostridium perfringensThe minimal inhibitory concentration (MIC) of 61 Clostridium perfringens isolates from broilers to cyadox was determined by agar dilution method according to the CLSI guideline. The MIC50 and MIC90 were calculated by SPSS 18.0 software, MIC and MBC of cyadox against Clostridium perfringens in vitro and ex vivo were determined by microdilution method about the selected strain. The mutant prevention concentration(MPC) of cyadox against Clostridium perfringens was evaluated by agar spread plate.Exposed to cyadox at concentration of 1 and 4 x MIC for 1 h and 2 h, the post-antibiotic effect (PAE) of cyadox against Clostridium perfringens was determined by drug removal method. The time-kill curves were determined in vitro and ex vivo to evaluate the antibacterial activity of cyadox against Clostridium perfringensThe results showed that MIC of cyadox against Clostridium perfringens were ranging from 1 ?16 ?ig/mL, MIC50 was 2.11 ?pg/mL, MIC90 was 4.40 ?g/mL. Based on the MIC data and pathogenicity, Clostridium perfringens cvcc2030 was selected. Cyadox shows the same antibacterial effect in vitro and ex vivo, the MIC of cyadox against Clostridium perfringens cvcc2030 in broth and ileum content were 2 ?g/mL and 2 ?g/mL, the MBC were 4 ?g/mL and 4 ?g/mL, respectively. The MPC of cyadox against Clostridium perfringens cvcc2030 was 20 ?g/mL. Exposed Clostridium perfringens cvcc2030 in different concentration of cyadox 1 h and 2 h, the PAE of Clostridium perfringens exposed cyadox for 1 h and 2 h were range from 0.88 ?1.24 h, 1.01 ?1.89 h,respectively.The results of in vitro time-kill curves demonstrated the antibacterial activity of cyadox against Clostridium perfringens was concentration-dependent, which consistent with ex vivo time-kill curves study.2. Pharmacokinetics study of cyadox in broiler ileum content110 healthy broilers, 4 weeks, were randomly divided into two groups. One of the groups was infected with Clostridium perfringens cvcc2030 to establish necrotic enteritis model. Both of two groups broilers received cyadox 30 mg/kg by oral administration.Ileum content samples were collected at pre-determined times (0.5 h, 0.75 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h and 24 h) after cyadox administration. Ileum content samples were detected by HPLC with ultraviolet detection to determine the concentration of cyadox.Ileum content cyadox concentration was fitted by Winnonlin 5.2 software. The main PK parameters of healthy group and infected group as follow: AUC were 410.75 h ?g/mL and 361.82 h ?g/mL, Cmax were 172.89 ?g/mL and 151.76 ?g/mL, T1/?, were 5.69 h and 5.82 h, CL/F were 72.44 mL/kg/h and 82.27 mL/kg/h, MRT were 2.59 h and 2.49 h,respectively. There were no significant differences between health broilers and infected broilers. After oral administration, the absorption and distribution of cyadox in broilers intestinal were rapid.3. PK-PD model integration and optimal dosage formulationUsing the Sigmoid Emax equation to integrate the relationship between ex vivo AUC/MIC24h and the logarithm of Clostridium perfringens concentration's variety, to calculate the PK/PD parameter AUC24h/MIC. The antibacterial effect of cyadox was quantified for three levels of this equation: bacteriostatic effect (E = 0), bactericidal effect(E = -3), and eradication effect (E = -4). Equilibrium dialysis method was used to get the free drug proportion in broiler intestinal tract, combined with dosage equation:Dose =Cl×(AUC/MIC)BP×MIC/F×fu, the bacteriostatic, bactericidal activity and bacterialeradication dosage were calculated.The equation derived from ex vivo time-kill curves were:(?). In ileum content, AUC24h/MIC values needed forbacteriostatic, bactericidal and eradication were 27.71 h, 78.93 h and 165.14 h,respectively. The ratio of free drug in broiler intestinal tract was 0.47. combined with dosage equation, for this experiment tested strains, the daily dose was predicted: 9.70 mg/kg (bacteriostatic), 27.63 mg/kg(bactericidal) and 57.81 mg/kg (eradication). In order to facilitate clinical administration, converted predicted dose to feed mixed dose were 100 mg/kg, 300 mg/kg and 600 mg/kg, respectively.4. Randomized controlled trial210 healthy broilers, 3 weeks, were randomly divided into six groups(n=35/group):blank control group, negative control group, positive control group, cyadox low-dose group(150 mg/kg feed mixed), cyadox middle-dose group(300 mg/kg feed mixed), and cyadox high-dose group(600 mg/kg feed mixed). The therapeutic indexes, growth performance, intestinal damage and Clostridium perfringens colony number were counted of each group.The mortality rate of cyadox in low-dose group, middle-dose group, high-dose group were 17.14%, 11.43%, 8.57%, respectively; effective rate was 77.14%, 85.71%, 91.43%,respectively; cure rate were 71.43%, 85.71%, 91.43%, respectively; average daily gain were 53.24 g, 62.64 g, 65.74 g, respectively; feed/gain ratio were 2.19, 2.00, 1.94,respectively; lesion scores were 0.97, 0.69, 0.54, respectively; Clostridium perfringens colony number (LogCFU/g) were 6.25, 5.45, 4.67, respectively. There was no significant difference between the cyadox middle-dose group and high dose group. Considering all factors, when the MIC ? 2?g/mL, 300 mg/kg feed mixed was recommended to treatment necrotic enteritis in broiler.In summary, this study elucidated the antimicrobial activity of cyadox against Clostridium perfringens and the pharmacokinetic characterization of cyadox in healthy and infected broilers intestinal tract, established the ex vivo PK-PD model, formulated the dosage regimen to prevent, treat, eradicate necrotic enteritis, and then launched the randomized controlled trial to validate the dosage regimen. It was of great significance to control necrotic enteritis and enrich the clinical application of cyadox.