Study On Pharmacokinetics And Pharmacodyamics Relationship Of Gamithromycin Against Pasteurella Multocida From Cattle

Pasteurella multocida,an opportunistic pathogen,which can be found all over the world.There is no host specificity in animals.When the host's immunity is compromised due to various reasons,this pathogen begins to attack the host and causes serious diseases.Pasteurella multocida can cause a variety of diseases in livestock,poultry and human,including bovine hemorrhagic septicemia,rabbit Pasteurellosis,swine atrophic rhinitis,swine pulmonary disease,avian typhoid and various kinds of human inflammation.It is an important zoono sis which has the potential for infecting a variety of animals and humans.Pasteurellosis is a common disease in cattle,which mainly causes loss of appetite,dyspnea,fever,cough and other symptoms.The mortality rate of pneumonia in cattle caused by Pasteurellosis is increasingly high.Pasteurellosis usually occurs easily,causing serious economic losses to the cattle industry.Gamithromycin is a new generation of macrolide antibiotics,which is approved by many foreign food and drug administrations for clinical treatment of bovine respiratory diseases,including EMEA,HPFB and FDA.As a clinical first-line drug,its rational use to reduce drug-resistant strains has been a concern.As genes resistance of Pasteurella multocida to macrolides are constantly detected and reported,the production of a large number of resistant strains not only threatens the l ife and health of livestock,but also endangers human health.Therefore,the optimization of the clinical dosage of the drug and reducing the production of resistant strains is a huge problem for veterinary researchers.At present,using PK/PD as a common method for making clinical rational drug plan,is often used in the dose optimization of clinical first-line drugs.However,there are few reports on PK/PD of gamithromycin against Pasteurella multocida in China.Therefore,the ex vivo PK/PD model and the in vivo PK/PD model of gamithromycin against Pasteurella multocida were established in this study,which were used to determine the PK/PD parameters(AUC/MIC,C_max/MIC and T>MIC)which is closely related to antibacterial activity and were used to calculate the dose by formula which can not only produce bactericidal effect,but also reduce the ge neration of drug resistance.This would better serve as a guide in the clinical use of veterinary drugs and reduce the production of drug-resistant strains.The main contents and results are as follows:1.Establishment of the analysis methods of gamithromycinIn this paper,a high-performance liquid chromatography?HPLC?with tandem mass spectrometry detection?HPLC-MSMS?method for the determination of gamithromycin in a variety of biological samples?cattle serum,transudate,exudate and mice plasma?was established,which can detect the concentration of gamithromycin rapidly and accurately.The sample was pretreated by protein precipitation method,and the mobile phase was eluted by 0.1%formic acid water and acetonitrile gradient.The absolute recovery rate of gamithromycin in four kinds of body fluids was 94.05?115.92%and the inter-assay and intra-assay variation as measured by%RSD were all<10%;the limit of detection?LOD?of biological samples in cattle was 1 ng/ml,and the limit of quantification?LOQ?of biological samples in cattle was 2 ng/ml;the LOD of plasma samples in mice was 5 ng/ml,and the LOQ of plasma samples in mice was 10 ng/ml.Compared with the methods reported in the literature,this study supplemented the de tection methods of gamithromycin in mice plasma,cattle transudate and exudate for the first time,which laid a foundation for the study of ex vivo PK/PD model of gamithromycin in cattle and in vivo PK/PD model of gamithromycin in mouse.2.In vitro efficacy test?1?The MIC values of gamithromycin in MH II broth,cattle serum,transud ate,exudate and mice plasma were measured by 96 well plate double dilution method.The results showed that the average MIC values in MH II broth were 20 times higher than those in serum.MIC values of Pasteurella multocida NM-5-7 in serum,transudate,exudate of cattle was 0.031?g/m L.?2?The results of in vitro time course of gamithromycin against Pasteurella multocida NM-5-7 showed that the antimicrobial activity of gamithromycin was concentration dependent.When the drug concentration was below 4×MIC,the antibacterial effect of gamithromycin increased along with the increase in drug concentration;when the drug concentration was above 4×MIC,the increase of drug concentration no longer made its antibacterial activity significantly enhanced.?3?In vitro PAE test showed that gamithromycin had a long PAE to Pasteurella multocida NM-5-7.When the bacteria was exposed to 1×MIC,2×MIC,4×MIC and 8×MIC of gamithromycin solution for 1h,the PAE values in vitro were 0.84h,1.10h,1.27h and 1.44h,respectively.3.The ex vivo PK/PD modeling of gamithromycin against Pasteurella multocida in cattleFollowing the establishment of tissue cage model and acute inflammation model in six healthy cattle,a randomized crossover design was used,in which 6mg/kg of gamithromycin was injected intravenously and subcutaneously,respectively.The serum,transudate,exudate samples were collected at the predetermined time point,and were measured by HPLC-MSMS.The concentration time data of the three biological samples were processed by Win Nolin software.After a single dose of intravenous administration,the main pharmacokinetic parameters in serum were 5.52?g·h/m L for AUC_1ast,35.27h for t_1/2,54.33 L/kg for Vd,1066.67m L/h/kg for Cl.The T_max in transudate and exudate were 4.5h and 5.5h,C_maxin transudate and exudate were 0.09?g/m L and 0.11?g/m L,respectively.After a single dose via subcutaneous administration,the results showed that the concentration-time date of serum,transudate,exudate were consistent with noncompartmental model.The main pharmacokinetic parameters in serum were 48.30h for t_1/2,6.23?g·h/m L for AUC_1ast,1.00h for T_max,0.43?g/m L for C_max,112.95%for F.The T_max in transudate and exudate were 9.5h and 7.0h,C_maxin transudate and exudate were 0.05?g/m L and0.11?g/m L,respectively.The PK/PD model of gamithromycin against Pasteurella multocida was successfully established for the first time by combining the ex vivo PD results in serum,transudate and exudate of cattle with the PK parameters of cattle.The minimum AUC ₂₄/MIC values for inhibition of Pasteurella multocida in serum,transudate and exudate were 6.4h,4.07h and 6.4h,respectively.The values of AUC₂₄/MIC for bactericidal effect in cattle serum and exudate were 90.32h and127.37h,respectively.While the value of AUC₂₄/MIC value for bacterial eradication in serum was443.15h.According to the MIC₉₀ reported in the literature,the minimum dosage of cattle in serum for bacterial eradication was 13.45mg/kg b.w.4.The in vivo PK/PD modeling of gamithromycin against Pasteurella multocida in miceOn the basis of the successful establishment of murine lung infection model by endotracheal intubation into Pasteurella multocida NM-5-7,16 groups of different doses of gamithromycin were used by subcutaneous administration.The plasma samples of mice were collected at the predetermined time point and drug concentration were determined by HPLC-MSMS.The concentration-time date of mice plasma was processed by Win Nolin software.At the same time,three mice were killed concurrently,and the lung homogenate was taken for bacterial colony count.Three mice in the control group were killed before administration and 24 hours after administration.The results showed that the AUC in mice plasma were 0.86?8.42?g·h/m L,the C_maxwere 0.55?5.69?g/m L,T_maxwere 0.17?0.25h,t_1/2were 8.04?13.64h,respectively.Combining the PK parameters with the MIC of gamithromycin against Pasteurella multocida in mice plasma,the correlation between f AUC_0-24/MIC and?log CFU/lung within 24h was obtained by using inhibitory effect I_maxmodel.The average f AUC_0-24/MIC in the lungs of mice required for the antimicrobial effect,1-log,2-log and bactericidal effect was 56.77h,90.18h,143.06h and239.44h,respectively.Combined with the MIC₉₀ reported in the literature,the minimum clinical dosage needed to achieve the bacteriostatic and bactericidal effects was calculated to be 2.10 mg/kg b.w.and 8.86mg/kg b.w.by extrapolation to cattle.The results of this study can provide a scientific basis for the formulation of dosage regimen for the treatment of clinical Pasteurella multocida infection with gamithromycin,this would assist achieve the treatment effect and reduce the production of drug-resistant strains,scientific and rational use which would prolong the life of the drug.