| Bacterial pneumonia in pig was a respiratory diseases with complex etiology and great harm to current intensive pig industry,and often characterized by secondary infection and mixed infection with a variety of pathogenic bacteria,so a single drug treatment was often difficult to effectively control the pig bacterial pneumonia in clinic.Combination therapy use the drug interactions can be appropriately raise curative effect and reduce the generation of drug-resistant bacteria.Doxycycline hydrochloride(DoxHcl)and florfenicol(FF)combination therapy were selected by the minimum inhibitory concentration(MIC)and combined chemosensitivity test of ceftiofur hydrochloride,danofloxacin mesylate,tylosin tartrate,DoxHcl,tilmicosin phosphate and FF against streptococcus suis,actinobacillus pleuropneumoniae and haemophilus parasuis.However,the efficacy of DoxHcl and FF were not satisfactory because of the abuse and misuse in clinic.In addition,DoxHcl and FF had a poor bioavailability and short elimination half-time in swine,so a higher doses and multiple dosing were given in clinic,its great inconvenience was brought to veterinary workers.In order to improve the therapeutic effect of DoxHcl and FF,hydroxypropyl-β-cyclodextrin,polyvinylpyrroliddone and hydroxypropyl methyl cellulose and saturated solution stirring combined with high pressure homogenization method were used to prepare a compound suspension of DoxHcl and FF,and its pharmacokinetic-pharmacodynamic(PK-PD)model against streptococcus suis cvcc607 was did in this subject,to develop a reasonable dosage regimen for clinical use.A new and efficient preparation was provided and to be an effective solution to the treatment of pig pneumonia in this study.1.Selected of combination therapyThe MIC of ceftiofur hydrochloride,danofloxacin mesylate,tylosin tartrate,DoxHcl,tilmicosin phosphate and FF against 6 Streptococcus suis strains,1 Actinobacillus pleuropneumoniae strain and 6 Haemophilus parasuis strains isolated from pigs were evaluated by the broth microdilution method according to the relevant standards formulated by Clinical and Laboratory Standard Institute(CLSI),then the checkboard method was used to evaluated combined chemosensitivity test of 10 drug combinations against different bacteria,and the drug combination with synergistic or additive interaction was selected,the drug combination with close pharmacokinetic parameters was selected,combined with the disposal process of antibiotics in pigs.The MIC results demonstrated that DoxHcl and FF are both with strong antibacterial activity,the MIC of DoxHcl is 0.125～1 μg/mL,and that of FF is 0.25～1 μg/mL.The chemosensitivity results showed that DoxHcl and FF show synergistic or additive interaction against different bacteria,and DoxHcl and FF have close pharmacokinetic parameters and no incompatibility in vitro,so DoxHcl and FF were chosen to develop the compound preparation for treatment of bacterial pneumonia in pigs.2.Preparation of DoxHcl and FF compound suspensionDoxHcl and FF compound suspension were preapred by using the technology of saturated solution stirring combined with high pressure homogenization.Single factor experiment was used to selected a best content of Hydroxypropyl-β-cyclodextrin,Polyvinylpyrroliddone and hydroxypropyl methyl cellulose.The inclusion rate was measured by high-performance liquid chromatography(HPLC).The preparation releasing rate in PBS buffer was detected by the "Kaplan Law" of Veterinary Pharmacopoeia.The compound suspension evaluation index were evaluated according to "People’s Republic of Veterinary Pharmacopoeia" edited in 2010,at the same time,the acceleration test and long term stability evaluation were did in this compound suspension.The result shown that the best ration of HP-β-CD DoxHcl and FF was 3:1:1,the best content of PVP was 5%and HPMC was 0.25%,the inclusion rate was 45.28%of DoxHcl and 89.69%of FF,the release rate of DoxHcl and FF were significantly delayed by the preparation in PBS buffer,and the appearance of suspension was ivory yellow;the pH value of suspension was 5;the sedimentation rate was 0.999;the re-dispersed and syringeability were fine;and its no stimulation and hemolysis in the suspension site of target animal.The acceleration test and long term stability evaluation shown that the suspension had a good stability stored in the dark.3.Pharmacokinetic-pharmacodynamic(PK-PD)modelingHigh-performance liquid chromatography(HPLC)detection method of DoxHcl and FF in serum and lavage fluid were established respectively by studying the linear,sensitivity,specificity,recovery rate,accuracy and stability.Swine were inoculated subcutaneously about 1.2×109 CFU/mL with 3～5 mL to established experimental model of swine streptococcus suis.Serum and lavage fluid samples were collected at different time points after intramuscular administration at a dose rate of 0.2 mL/kg.Dates of the drug concentration were analyzed by Winnonlin pharmacokinetic software.The antibacterial effect of DoxHcl and FF combination therapy in lavage fluid were tested by the broth microdilution method,according to the results of antibacterial effect,the in vitro bacterial killing curves were established to determine the rate and extent of bacterial killing by DoxHcl and FF combined therapy against streptococcus suis in broth and lavage fluid to determine the antimicrobial type.Filtered lavage fluid samples and inoculated with streptococcus suis cvcc607 to draw the killing curves.Reference parameters AUC/MIC to fit PK-PD equation and established a PK-PD model by Winnonlin software to prepare a dosage regimen.The pharmacokinetic results shown that DoxHcl and FF were all in accordance with one-compartment open model in healthy swine and streptococcus suis infected swine.The main lung PK parameters of DoxHcl and FF in streptococcus suis infected swine were as follows:the absorption half life time(T1/2ka)was 0.290±0.054 h and 0.178±0.069 h;the elimination half life time(T1/2β)wais 18.074±1.967 h and 13.462±2.387 h;the area under concentration time curve(AUC)was 64.069±5.452 h·μg/mL and 59.973±8.007 h·μg/mL;the peak concentration(Cmax)was 2.296±0.084 μg/mL and 2.913±0.177 μg/mL;the mean residence time(MRT)was 25.001±5.369 h and 28.532±6.913 h;the clearance rate(CL)was 0.031±0.002 L/h/kg and 0.033±0.004 L/h/kg.The minimum inhibitory concentration(MIC)of DoxHcl and FF in lavage fluid were 0.125 μg/mL and 0.25 μg/mL and it was same to the dates of culture medium in vitro,and the killing curves shown that the combined therapy belong to concentration dependent antibacterial agents.The dosage regimen was as follows:for prevention of pig pneumonia,the dosage is 3.8 mg/kg b.w.;therapeutic dosage is 20.01 mg/kg b.w.administered,for eradication of bacteria and to avoid the generation of resistant bacteria the dosage was 39.56 mg/kg b.w.administered.So the dosage regimen of the suspension was 0.019 mL/kg-0.198 mL/kg and the dosing administered at intervals of 78.787 h.In summary,we had successfully prepared a compound suspension of DoxHcl and FF with preparation evaluation indexes meeting the requirements.The research of the PK-PD model against streptococcus suis evcc607 was did in this subject,and a reasonable dosage regimen was developed a new and efficient preparation was provided and to be an efective solution to the treatment of bacterial pneumonia in pigs in this study. |
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