| With the rapid development of intensive pig production in recent years, E. coli disease prevalence become more and more serious. This disease could cause severe diarrhea in the piglet, induce the higher morbidity and mortality. In the sick feeder pigs, the symptoms were decreased appetite, degraded feed conversion and impaired growth. E. coli disease could cause great economic losses for the farmers. Cyadox is a novel antimicrobial growth-promoter of the quinoxalines. Lots of researches had been demonstrated that cyadox has low toxicity, good safety, and wide antimicrobial spectrum. Some studies showed that cyadox can promote the growth of swine, chicken and fish, reduce diarrhea frequencies, and prevent E. coli infection in piglets and broilers. Consequently cyadox has good prospects for development and application. At present there is not a scientific dose for treating the E. coli diarrhea. Therefore, this research studied Pharmacokinetics and Pharmacodynamics of cyadox in swine ileum content by using T Cannula of pig, and established a ex vivo PK-PD model to analyze the relation between the ileum content concentration of cyadox and the germicidal efficacy of cyadox in ileum content. These results formulated the reasonable regimen for treatment of E. coli diarrhea and provided a theoretical basis for rational clinical use of cyadox, decreased the bacterial drug resistance and extended the using time in clinical practice.1In vitro antimicrobial activity of cyadox against cvcc223The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cyadox and its main metabolites against cvcc223were evaluated by Broth microdilution method, which recommended by Clinical and Laboratory Standards Institute (CLSI). The results showed that cyadox was active against cvcc223under anaerobic condition. The MIC was1μg/mL, and the MBC was8μg/mL. Therefore, cyadox was the antibacterial agents with a higher activity, could be used for treating E. coli infection.Metabolites had no antibacterial activity except the Cy9, and the MIC of Cy9against cvcc223was also1μg/mL. The susceptibility testing of the combination of cyadox and Cy9against cvcc223were evaluated by the checkerboard method. The combined sensitivity index FIC=0.5/1+0.5/1=1expressed that the interaction was additive action.The mutant prevention concentration (MPC) of cyadox against cvcc223was evaluated by agar culture method. The results showed that the minimal concentration of agar plate in which there was no bacteria growth was20μg/mL. This concentration was considered MPC.According to the results of the minimum inhibitory concentration (MIC), we formulated a series of MH broth in which the concentration of cyadox were32MIC、16MIC、8MIC、4MIC、2MIC、1MIC and1/2MIC. After inoculation of bacteria, removed100μL respectively at0.5、1、2、3、6、8、12、18、24h to count bacteria. Take the hour as abscissa and bacterial number as ordinate to draw the killing curves. Enrofloxacin that belongs to concentration dependent antibiotic was used as a positive control. The results showed that bactericidal activity of cyadox is increasing with the concentration raising. The curves tend of cyadox were similar to Enrofloxacin. Therefore cyadox also belonged to concentration dependent antibiotic, The parameters fitting PK-PD equation were AUC/MIC and Cmax/MIC.2Pharmacokinetics of cyadox in swine plasma and ileum content12health castrated crossbred piglets weighing-20kg were randomly divided into2groups with6replications each. T Cannula models were established in the ileum of12pigs by fistula operation. The first group was normal group. The second group was infected group, belonged to diarrhea models.Plasma samples and ileum content samples were collected at0.5、1、2、3、4、6、8、12、24h after oral administration of Cyadox at30mg/kg bw. The plasma was2mL, and the ileum content was3-4mL in every time point. These samples were detected by high performance liquid chromatography (HPLC). The results showed that the concentration of cyadox in plasma was lower than1μg/mL, no bacteriostatic activities. We cannot use these data to build PK-PD model.Ileum content cyadox concentration fitted the delayed two-compartment model after single oral administrations in two groups. Cyadox metabolism in ileum content was quickly. By Winnonlin software computing and processing, the main pharmacokinetic s parameters as follows:Tmax is1.82-2.02h; T1/2α is0.52-0.62h, T1/2β is1.05-1.08h; Cmax is32.96-34.83μg/mL; T>MIC is7.48-8.18h;AUC is105.0-115.8h·μg/mL. 3In ex vivo antimicrobial activity of cyadox against cvcc223The minimum inhibitory concentration (MIC) of cyadox against cvcc223were evaluated by Content microdilution method. The minimum concentration of cyadox was1μg/mL in96-well microplate after culture for12hours under anaerobic condition. Considering the stability of cyadox in ileum content was not as good as in MH broth, we shortened the incubation time. The results showed that the bacterial growth was inhibited in ileum content containing cyadox1μg/mL.Add MH broth and aseptic ileum content into24-well plates. After inoculation of bacteria, removed100μL respectively at0.5、1、2、3、4、6、8、12、18、24h to count bacteria. Take the hour as abscissa and bacterial number as ordinate to draw the growth curves. The results showed that the growth curve in ileum content agree well with that in MH broth. E. coli cvcc223grew normally in ileum content.Filtered ileum content from different time points, inoculated with cvcc223, and then placed into the anaerobic incubator. Removed20μL respectively in1、2、4、8、12、24h to count bacteria. Take the hour as abscissa and bacterial number as ordinate to draw the killing curves. The results were as follows:The change of bacterial number was obvious mostly within high concentration of cyadox (>10μg/mL) at4hours. Bacteria had been completely killed at high concentration groups. The change of bacterial number was obvious within low concentration of cyadox (2-4μg/mL) at8hours. It just meant cyadox has germicidal action at low concentrations. Bacteria recovered growth at12hours at some groups. It reflects the concentration of cyadox was lower than MIC in these groups. Bacteria recovered growth at24hours at all groups. It reflects the concentration of cyadox was lower than MIC in all groups.4Risk assessment of resistanceAdd MH broth containing different concentrations of cyadox into aseptic bottles. After inoculation of bacteria, removed100μL respectively at adequate time to count bacteria on agar plates containing4MIC cyadox. The results were as follows:It was not easy for resistance strains to enrich in MH broth that the concentration of cyadox was greater than8μ/mL. It was easy for resistance strains to enrich in MH broth that the concentration of cyadox was equal to4uμg/mL after12hours. When the concentration of cyadox were1μg/mL and2μg/mL, resistance strains were most easily enriched in MH broth at6-8hours. The growth of0.5μg/mL group and blank group was similar, and resistance strains enriched generally appeared at24hours. The results showed that resistance strains were most easily enriched in MH broth when the concentration of cyadox near MIC. Calculating the metabolism of cyadox in ileum, we known the concentration of cyadox was lower than1μg/mL after12hours. The time that concentration of cyadox was between1μg/mL and2μg/mL was less than2hours. Therefore the emergence of resistance in1-2μg/mL group and blank group was similar.It is suggested that resistant subgroup most likely to enrich in the range of the MIC concentration, and exposed the bacteria for6-8hours. Calculating the metabolism in ileum after oral administration of cyadox30mg/kg bw, if taken drug thrice a day, the cyadox concentration at8hours was higher than the MIC concentration, and no antibiotic resistance produce theoretically. If taken drug two times a day, the cyadox concentration at8hours was closing in MIC concentration and exposed2hours approximately, the cyadox concentration was higher than4μg/mL at other time, and theoretically no antibiotic resistance produce also. If taken once a day, persistence time of low concentration was longer, and existed resistance risk theoretically.5Fitting PK-PD model and formulating dosage regimenWe had known that cyadox belonged to concentration dependent antibiotic, The parameters fitting PK-PD equation were AUC/MIC and Cmax/MIC.PK and PD data assayed by WinNonlin soft indicated that the PK-PD model of cyadox was Emax model. The relationship between ex vivo AUC/MIC、Cmax/MIC and antibiosis effect were handled using Hill equation. The results showed that the value of AUC/MIC/h was equal to Cmax/MIC essentially when E=0、-3、-4(inhibition、 sterilization eradication). In order to connect with the administration equation, we choose ex vivo AUC/MIC as simulation parameter. Final PK-PD equation decided as follow:. The value of ex vivo AUC/MIC/h were86.56、171.28、243.74respectively when E=0、-3、-4(inhibition、sterilization、eradication). Connected with the administration equation: could get the dosage X (mg/kg/d). When the MIC of cyadox against clinical bacteria was1μg/mL, E=0、-3、-4, the value of X were22.58、44.67、63.58mg/kg/d. Calculating the metabolism of cyadox in ileum, we known the concentration of cyadox was lower than1μg/mL after12hours. So an optimum dosage regimen was that taken45mg/kg twice a day. Refer to the result of the risk assessment of resistance test, dosage regimen which was2-3times per day orally could avoid antibiotic resistance produce. In order to avoid the emergence of resistant strains in clinical treatment, administration once a day was not recommended. Combined with clinical management, preventive dose regimens for23mg/kg bw oral administration, two times a day. Groups fed dosing regimens for0.9g/kg mixed feed, two times a day. Therapeutic dose regimens for45mg/kg bw oral administration, two times a day. Groups fed dosing regimens for1.8g/kg mixed feed, two times a day. Eradication dosing regimens for65mg/kg bw oral administration, two times a day. Groups fed dosing regimens for2.6g/kg mixed feed, two times a day.In summary, this topic is the first time to confirm that cyadox belongs to concentration-dependent agent and ex vivo PK-PD model was established successfully by the T Cannula in pig’s ileum. The fitting PK-PD equation that described cyadox treating the E. coli diarrhea was built. This topic got the best oral dose and the evaluation of resistance in clinic. These results gave scientific guidance for the reasonable use of cyadox in E. coli diarrhea, in order to reduce occurrence and transmission of drug resistance of bacteria. |
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