| In the past decades,which Streptococcus suis infections have been regarded as the major and world-wide problems in the swine industry.The upper respiratory tract,tonsils and nasal cavities is the S.suis nature habitat,which can cause meningitis,arthritis,pneumonia,and septicemia,not only in animal but also in human.When we treatment the Streptococcus suis,the first choice drug is penicillin.The drug-resistance is more and more serious,along with increasing number of macrolides are used in clinical.Acetylkitasamycin is a 16 ring macrolides,but it have not been widely used in veterinary clinical.At present,the drug in the veterinary use is not standard and not achieve good treatment,which is associated with emergence of drug resistance.In order to reduce this phenomenon,PK-PD mechanism is introduced into dosage consultation.Which reflect the relationship between engine body,the bacterial and the drug.In this research,take the sample from the pulmonary by electronic fiberscope and study pulmonary PK,in-vitro PD,ex-vivo PD,and combine ex-vivo data with in-vivo PK data to establish PK-PD model.At last verification of the dosage.1.Antimicrobial activity of acrtylkitasamycin against S.suisWith reference to the Clinical and Laboratory Standards Institute(CLSI),broth microdilution method was used to evaluated acetylkitasamycin and its five main components(A6A7,A5',A4A5,A1A3,A13)against the S.suis,the minimal inhibitory concentration(MIC)and the minimal bactericidal concentration(MBC).Analysis the results revealed that acetylkitasamycin and its five main components were active to S.suis,The MICs were 1 ?g/mL in the broth and pulmonary epithelial lining fluid(PELF),and the MBC were 2 ?g/mL.At the same time,seven main metabolite kitasamycin(A7,A6,A5,A4,A1,A3,A13)have a lower sensitive to S.suis than acetylkitasamycin,which MICs were 2 ?g/mL and MBCs were 8 ?g/mL respectively.The mutant prevention concentration(MPC)of acetylkitasamycin against S.suis was tested by agar method and the result was 5 ?g/mL.The bacterial of S.suis was exposed in different concentration of acetylkitasamycin,removing the drug after 1h and 2 h incubation,and then compute the PAE of acetylkitasamycin against S.suis.The PAEs of S.suis exposed to 2 MIC and 4 MIC acetylkitasamycin for 1h were 1.59 h and 1.88h respectively.The PAEs of S.suis exposed to 2 MIC and 4 MIC acetylkitasamycin for 2 h were 2.33 h and 2.96 h respectively.Which showed that acetylkitasamycin has a long PAE.According to the MIC values,the bacterical killing curves of acetylkitasamycin against S.suis were evaluated at different concentrations of acetylkitasamycin(0 MIC,1/2 MIC,1 MIC,2 MIC,4 MIC,8 MIC,16 MIC,32 MIC,64 MIC)in broth,the bacterial activity of acetylkitasamycin is increasing with the prolongation of time,and aslo with concentration increasing the bactericidal enhanced.Taken the sample from the pulmonary by the electronic fiberscope at the different time(0.5 h,1 h,2 h,4 h,6 h,8 h,12 h,24 h,36 h,48 h,72 h,96 h),the sample were filtered and together with S.suis in incubator.Then the killing curve were draw.The results showed that the killing curve in PELF very similar to in broth.Both in healthy and infected swine,acted out powerful bacterial effect at 1 h,2 h,4 h,6 h,8 h,12 h point,at other points revealed slight or no bacterial effects.At last,the parameter AUC/MIC was confirm as the PK-PD parameter for acetylkitasamycin against S.suis.2.Pharmacokinetics of acetylkitasamycin in swine plasma and pulmonary epithelial lining fluidIn this trial,12 healthy 20 kg weaned piglets were randomly assigned to one of two groups:one healthy and one infected with S.suis which were used to establish pneumonia models.Both of the two groups were given 50 mg/kg acetylkitasamycin by oral.Plasma and PELF samples were collected at different time points.The plasma were sampled about 5 ml,at the time 0.25 h,0.5 h,0.75 h,1 h,2 h,3 h,4 h,6 h,8 h,10 h,12 h,24 h.And the pulmonary epithelial lining fluid were collected by electronic fiberscope,at the time 0.5 h,1h,2 h,4 h,6 h,8 h,12 h,24 h,36 h,48 h,72 h,96 h.The samples were detected by LC-MS/MS,the remaining were used to determine the ex-vivo antibacterial activity of acetylkitasamycin.Which revealed that the concentration of acetylkitasamycin in pulmonary were much higher than in plasma,and also the PELF concentration directly reflect the interaction between the drug and the bacterial.So the concentration of acetylkitasamycin in pulmonary which suitable for setting up ex-vivo PK-PD models.Acetylkitasamycin in the pulmonary fitted the first order two-compartment model after oral administration.Through the Winnonlin software analog computation,the main PK parameters were:Tmax was 4.26 h-4.44 h,Cmax was 2.79 ?g/mL?2,81?g/mL,AUC was 61.78 h·?g/mL?63.46 h·?g/mL,T1/210 was 6.12 h?6.43 h.3.Making rational dosage regimen and fitting PK-PD modelAs described before,the best PK-PD parameter to describe the characteristics of acetylkitasamycin bactericidal was AUC/MIC which determined by ex-vivo and in-vitro killing curve.Winnonlin software was used to analysis PK and PD data.The relationships between different drug concentration and bacterial decreased and the parameter AUC/MIC were handled by Hill equation.The final Hill equation described as follow:E=3.28-7.58×C2.01/19.812.01+C2.01.When the E=0(inhibition),-3(sterilization),-4 eradication),the values of AUC/MIC(h)were 17.31?43.37?96.81 respectively.Combine with the equation:Dose=CL×AUC/MIC fu×F×MIC.When the MIC of acetylkitasamycin against clinical bacterial was 1?g/mL,the values of Dose were 15.62,39.14,87.36 mg/kg.On the basis of the results in-vitro and ex-vivo killing curve.Along with time prolonged,the antimicrobial effect enhanced,and also when the concentration was higher than 2 MIC,the bacteriostatic was obviously.Combined with the software MlxPlore and the values of T>MIC(16.7h),T>MPC(10.13 h),PAE=2.96 h.Given once daily,the drug concentration is around 1 MIC and most of the time was in the MSW,which can not avoid resistance.So if shorter dosing interval and given twice daily,the concentration of acetylkitasamycin were higher than 4 MIC within 24 h and above the MPC.At last,the preventive regimens were 15.62mg/kg b.w oral twice daily.Therapeutic regimens were 39.14 mg/kg b.w oral twice daily.Eradication regimens were 87.36 mg/kg b.w oral twice daily.In consideration of large scale farming,we choose mixed feeding dosage regimen.In accordance with groups feeding dosing equation D=d×n/W,d was the dose calculated,n was 2,W was(50 g/kg/d)weight and feed conversion ratio.The optimum dose regimen was 1.56 g/kg for mixed feeding,twice a day.4.Model ValidationChoosing the MIC value was 1 ?g/mL S.suis to infect piglets.According to the PK-PD model dose regimens 39.14 mg/kg b.w oral twice daily.The results showed that the piglets protection reached 66.7%.In conclusion,this research describes the characteristics of acetylkitasamycin antibacterial activity and PK characteristics in plasma and pulmonary in healthy and infected swine.This study gets the best dosage regimen and avoids resistance in clinic,and offers scientific guidance for acetylkitasamycin uses. |
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