| Haemophilus parasuis?H.parasuis?is a conditional pathogen of porcine upper respiratory tract,which is characterized by cellulosic pleurisy,arthritis and meningitis.The outbreak of the disease has brought huge economic losses in pig industry,coupled with the multiple resistance of H.parasuis to many antibiotics in recent years,so it is necessary to find a safe and effective antimicrobial agent to treat disease of H.parasuis infection.Aditoprim?ADP?is a new type of antibacterial synergist for animals.It has been reported that it has widely distributed and has high bioavailability in vivo.Early pharmacodynamic studies showed that it had an effect on both Gram-positive bacteria and Gram-negative bacteria.Compared with its similar drug trimethoprim?Trimethoprim,TMP?,the antibacterial activity of ADP was much stronger than trimethoprim.Sulfamethoxazole?SMZ?is a kind of intermediate-effect sulfonamides used in the whole body.It has a broad-spectrum antibiotic and widely used in clinic.It is widely used in the treatment of diseases in aquatic products,livestock and poultry.SMZ was absorbed rapidly in vivo and reached a high concentration in plasma and lung tissue.However,in recent years,the resistance of SMZ to pathogenic bacteria in respiratory tract and digestive tract has increased year by year.It was found that the antibacterial ability of the combination of ADP and SMZ was much stronger than single used.It reduces the resistance while reducing the dosage of single drug,which has a good development prospect.In this study,through the pharmacodynamic study of ADP and sulfamethoxazole and ADP-SMZ compound,and we found that the antibacterial effect of ADP was significantly lower than ADP-SMZ compound.Therefore,we studied the relationship of ADP-SMZ compound injection against H.parasuis by PK-PD model.Therefore,the reasonable administration scheme of ADP-SMZ compound injection was determined in this experiment.1 Pharmacodynamic Study of ADP,SMZ and ADP-SMZ CompoundWith reference to the agar dilution method recommended by CLSI,The minimum inhibitory concentration?MIC?of ADP,SMZ and ADP-SMZ compound against 104 strains,and the MIC50 of ADP,SMZ and ADP-SMZ against H.parasuis were obtained.It is 128?g/m L,greater than 512?g/m L and 32/640?g/m L.Thirteen sensitive strains were selected for serotyping of enterbacterial repetitive intergenic consensus-PCR?ERIC-PCR?,and mouse virulence tests were performed at the same time.120 healthy Balb/c mice of 16 g to 20 g were selected.Each strain was a group 1×107CFU.Observing the pathological changes and deaths of mice,and selecting the more pathogenic strains,H78 was finally selected for PK-PD experiments.The MIC and minimum bactericidal concentration?MBC?of ADP,SMZ and ADP-SMZ against H78 in broth and PELF were measured.with reference to the broth dilution method recommended by CLSI.The results showed that ADP in broth and PELF,The MIC in the solution was 4?g/m L and 2?g/m L,the MBC was 8?g/m L and 4?g/m L respectively;the MIC of SMZ in the broth was 512?g/m L and MBC was 1024?g/m L;The MIC of ADP-SMZ in broth and PELF was 2/10?g/m L and0.5/2.5?g/m L,and the MBC was 4/20?g/m L and 1/5?g/m L,indicating that the Antibodies or bacteriostatic factors or other components that can enhance the antibacterial activity of ADP and ADP-SMZ compound,so the antibacterial activity of ADP and ADP-SMZ compound in PELF is higher than the antibacterial activity in broth;measured by the plate coating method,respectively ADP and ADP-SMZ have minimal resistance to mutations?MPC?of H78 at 20?g/m L and 6.4/32?g/m L;H78was exposed to different concentrations of ADP and ADP-SMZ After 1 h and 2 h,determine the antibacterial effect?PAE?of H.parasuis incubation with ADP for 1 h and 2 h,respectively,0.02 h?0.20 h and 0.40 h?0.48 h;measure H.parasuis Incubate with ADP 1 h and 2 h of post-antibiotic effect?PAE?,respectively,The results were 0.27 h?0.63 h and 0.46 h?1.11 h.Then according to the MIC measurement results,set different concentrations of ADP and ADP-SMZ compound drugs to incubate with H78,and take 0.1 m L at different time points to count the bacteria on the appropriate dilution multiples to draw the ADP and ADP-SMZ compound in vitro bactericidal curve for H.parasuis,according to the drug concentration in PELF at different time points after ADP-SMZ compound administration,adding the corresponding concentration of drug to the blank PELF,and incubating with H78 Draw the sterilization curve in ex-vivo.The ex-vivo killing curve and the in vitro killing curve both increase with time,and the bactericidal effect is significantly enhanced,which has little relationship with the concentration,showing obvious time dependence.The final selected PK-PD parameter is AUC/MIC.2 Pharmacokinetics of ADP,SMZ and ADP-SMZ compoundA high performance liquid chromatography?HPLC?detection method for ADP-SMZ compound in plasma and PELF was established.For the ADP-SMZ compound injection,the pigs were divided into healthy and diseased groups,and pharmacokinetics of ADP-SMZ compound injection was carried out in six animals in each group.The diseased group needed to establish the disease model first,In the group,ADP-SMZ compound injection was administered intramuscularly at a dose of25 mg/kg b.w?calculated as SMZ?.Plasma and PELF samples were collected at different time points,measured by HPLC.The concentration showed that the drug concentration of ADP and SMZ in the plasma was significantly lower than that in the PELF,and the main site of H.parasuis in the pig was the lung,which would cause lung disease,so The pharmacokinetic parameters of ADP and SMZ in alveolar fluid were fitted by Winnonlin software,and PELF samples collected at different time points were used as cultures for ex-vivo pharmacodynamic tests in ex-vivo.Pharmacodynamic and pharmacokinetic data were fitted to establish a PK-PD model.Results:The area under the curve?AUC?of ADP in healthy pigs and diseased pigs was 49.14±0.52 h?g/m L and 49.94±0.91 h?g/m L,respectively,and the peak time(Tmax)was 2 h and 2 h,respectively.The concentrations(Cmax)were 6.78±0.11?g/m L and 6.84±0.25?g/m L,respectively.There was no significant difference in the pharmacokinetic parameters of ADP in the healthy and diseased groups.The area under the curve?AUC?of SMZ in healthy pigs and diseased pigs was 162.79±12.45h?g/m L and 166.16±12.67 h?g/m L,respectively.The peak time(Tmax)was 2 h and 2 h,respectively.(Cmax)were 25.44±2.51?g/m L and 26.35±2.04?g/m L,respectively.There was no significant difference in the pharmacokinetic parameters of SMZ in PELF of healthy and diseased pigs.ADP injection and SMZ injection were used as controls for ADP-SMZ.Pigs were divided into two groups,one group was injected with ADP injection?5 mg/kg b.w.?and the other group was injected with SMZ injection?25 mg/kg b.w.?.PELF samples were also collected at the same time points as the combination of ADP and SMZ,and then the concentrations of ADP and SMZ in the PELF at each time point were determined by HPLC.The results of the pharmacokinetic parameters of the two unilateral drugs are as follows:the AUC of ADP in healthy pigs is 44.82±0.52 h?g/m L,Tmax is 2 h,and the Cmax is 6.88±0.14?g/m L.The AUC of SMZ in healthy pig medicine was 199.41±10.14 h?g/m L,Tmaxwas 2 h,and the Cmax was 23.4±2.58?g/m L.3 Ex-vivo PK-PD Model of ADP-SMZ Compound Injection and Formulation of DosageAccording to the pharmacokinetic parameters,the best PK-PD parameters were selected to construct a ex-vivo PK-PD model of ADP-SMZ compound drugs against H.parasuis.The relationship between the AUC/MIC value and the logarithmic change of the bacteria in the curve test was fitted,and the AUC/MIC values under the antibacterial,bactericidal,and clearing effects in the diseased group were calculated to be 17.81,62.04,and 137.28 h.The dose calculation formulas were 6.23,21.71,and48.05 mg/kg bw for the purposes of prevention,treatment,and radical cure,respectively.Then use Mlx Plore software to predict the dosing schedule:once a day,3to 7 days.To sum up,Based on the Pharmacodynamic-Pharmacokinetic?PK-PD?synchronous model of H78 by ADP-SMZ compound injections,a dosing rigemin for ADP-SMZ compound injections was formulated.It provided a new therapeutic drug and a scientific and rational dosing regimen for the clinical treatment of H.parasuis. |
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