| Bovine mastitis is a common and economically important disease that affects the health of cow mammary glands,and has plagued the healthy development of the dairy industry for a long time.Currently,antibiotic or synthetic antimicrobial drug therapy remains one of the most commonly used options for the prevention and treatment of bacterial mastitis.As a third-generation fluoroquinolone veterinary drug,marbofloxacin has broad-spectrum antibacterial activity and has been approved by Europe and China for the treatment of bovine mastitis.Up to now,there is a lack of domestic marbofloxacin injection for the treatment of bovine mastitis on the market.Therefore,it is urgent to carry out the research and development of generic Marbofloxacin injection in clinical practice,and further improve the guarantee of drug supply in our country,which is of great significance for my country to become a pharmaceutical power.In this study,marbofloxacin injection produced by a domestic pharmaceutical company was used as the test preparation,and its antibacterial activity against mastitis-causing pathogens was investigated to evaluate the application prospect of domestic similar products in the treatment of bovine mastitis.In this experiment,25pathogenic bacteria（Staphylococcus aureus,Streptococcus and Escherichia coli）that cause mastitis in dairy cows were isolated and identified from a dairy farm in Jilin Province,and clinical drug susceptibility tests were carried out.The test results showed that clinically isolated strains were generally sensitive to marbofloxacin monomer and domestic marbofloxacin injection,and the sensitive strains accounted for 84%and 96%respectively,indicating that the generic preparation can effectively inhibit the development of infectious mastitis.In this study,the original drug Marbofloxacin Injection developed by the French company Ceva was used as a reference preparation to investigate the pharmacokinetic parameters and relative bioavailability and bioequivalence evaluation of the two preparations.A high performance liquid chromatography（HPLC）method was established to detect the content of marbofloxacin in bovine plasma.Ofloxacin was used as the internal standard substance,and 2 times the volume of methanol was added to the plasma to precipitate proteins.After high-speed centrifugation,the supernatant was passed through a 0.22μm filter,and the sample was separated by a Shimadzu 5μm C18 chromatographic column.The sample volume was 20μL,the column temperature was 30°C,and the elution was isocratic（p H3.0 formic acid solution:acetonitrile:water=76:15:9（v/v/v））.The complete method validation results show that the method has good specificity and resolution and there is no interference from impurity peaks nearby.Marbofloxacin had a good linear relationship within the concentration range of 0.1-5μg/m L（R≥0.9998）.The detection and quantitation limits of the method were 0.05μg/m L and 0.1μg/m L,respectively.The recoveries of low,middle and high concentrations of plasma samples were:92.27～117.83%,92.5～101.45%,93.46～103.47%,respectively,and the relative standard deviations were all≤7.18%.The added samples remained stable under room temperature,freeze-thaw and long-term cryopreservation conditions,and the coefficients of variation were all≤7.11%.The analytical method meets the requirements of the Pharmacopoeia"Guiding Principles for the Validation of Quantitative Analysis Methods of Biological Samples",and can stably and accurately detect the content of marbofloxacin in bovine plasma.In this experiment,a single-dose,double-period,two-treatment,randomized,cross-controlled design was used for bioequivalence research.A total of 18 healthy dairy cows were included as experimental animals and randomly divided into two groups.In the first cycle,cows were injected with 2 mg/kg marbofloxacin injection test preparation or reference preparation,and the second cycle test was carried out after 7 days of washing period.After pretreatment of blood samples,plasma concentrations were determined using an established HPLC method.According to the non-compartmental model,the pharmacokinetics module and bioequivalence module of DAS3.0 software were used to calculate the pharmacokinetic parameters and evaluate the bioequivalence of the two preparations respectively.The AUC（0-t）of the reference preparation and the test preparation are6.27±1.62h·mg/L and 6.50±1.50 h·mg/L respectively,and the AUC（0-∞）are 6.61±1.67h·mg/L respectively and 6.99±1.78 h·mg/L,Tmax were 0.72±0.12 h and 0.71±0.10 h,and Cmax were 2.16±1.08 mg/L and 2.23±1.26 mg/L,respectively,with no significant difference.The 90%confidence intervals of the geometric mean ratios of the Cmax,AUC（0-t）and AUC（0-∞）of the two preparations after logarithmic transformation were:84.58～114.62%,98.42～110.90%,98.64～113.57%.The results were all in line with the equivalence interval requirements（80.00%～125.00%）for bioequivalence in the"Technical Guidelines for Bioequivalence Tests of Veterinary Chemicals by Plasma Concentration Method".No animal adverse reactions and abnormal deaths occurred throughout the experimental period.In summary,the marbofloxacin injection produced by a domestic pharmaceutical company not only has universal antibacterial activity against the main pathogenic bacteria that cause mastitis in cows,but also has similar absorption,distribution,elimination process and safety in cows as marbofloxacin injection developed by a French company Ceva.The test preparation and the reference preparation are bioequivalent,and this study provides a marketable domestic generic preparation for the clinical treatment of bovine mastitis. |
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