Studies On Preparation, Preparation, And Bioavailability Of Compound Tilmicosin-florfenicol Nanoemulsion

In this research,we made nanoemulsion as carrier of compatibility of Timicosion and Florfenicol.The formulations of Compound Timicosion and Florfenicol Nanoemulsion(Tilmicosin - Florfenicol Nanoemulsion) were optimized by protract the pseudoternary phase diagrams to prepare Tilmicosin - Florfenicol Nanoemulsion. Then the stability , safety , vitro antibacterial effect , pharmacy of Tilmicosin - Florfenicol Nanoemulsion were studied providing a theoretical basis for it in the veterinary clinical application.1. Analytical method establishment of Tilmicosin - Florfenicol NanoemulsionThe content determination of Tilmicosin and Florfenicol analytical methods were respectively established by ultraviolet-visible spectrophotometer. The results showed that two good linearity were obtained by Tilmicosin in the range of 5～50μg·mL^-1 and by Florfenicol in the range of 1～50μg·mL^-1, and the average recovery, relative standard deviation(RSD), RSD of the with-in-day precision, RSD of the day-to-day precision of Tilmicosin and Florfenicol were (98.37±1.12)% and (97.55±1.37)%, 1.13% and 1.40%, 0.55% and 1.23%,1.17% and 1.70%, respectively. The analytical methods of High Performance Liquid Chromatography (HPLC) which established had high recovery rate, good repetitiveness and precision in this research. It could offer a good specificity method of content determination of Tilmicosin - Florfenicol Nanoemulsion.2. Preparation and quality evaluation of Tilmicosin - Florfenicol NanoemulsionThe effect of formation of the nanoemulsion factors was investigated by pseudo-ternary phase diagrams. The composition of nanoemulsion was determined initially. The optimal match of Tilmicosin and Florfenicol was determined by Kirby-Baueer test. The prepared prescription of Tilmicosin - Florfenicol Nanoemulsion was ascertained finally. The structure type of Tilmicosin - Florfenicol Nanoemulsion was judged by centrifugation and staining method.Its appearance and particle diameter distribution was investigated by transmission electron microscope and laser particle size analysator respectively. Through photostability testing by strong light,accelerated testing and long-term testing verify its stability.The final prepared prescription of Tilmicosin - Florfenicol Nanoemulsion was w(Tilmicosin)= 11.07%, w(Florfenicol)= 1.11% , w(dimethyl sulfoxide)= 1.11% , w(citric acid)=1.64% , w(tween-80) =24.93%,w(ethanol)= 13.29%,w(propylene glycol)= 8.86%, w(IPM)= 5.32%, ,w(H2O)= 31.02%. The prepared Tilmicosin - Florfenicol Nanoemulsion was the type of oil-in-water and the liquid was amber, transparent and homogeneous. The nanoemulsion drop presented spherical shape, and the drop size averaged 15.4 nm with a polydispersity index of 0.08. Tilmicosin - Florfenicol Nanoemulsion had well stability and the expiration date was 18 months. The results showed that the quality of Tilmicosin - Florfenicol Nanoemulsion was stable,achieving requirement of clinical medication.3. Safety evaluation of Tilmicosin - Florfenicol NanoemulsionThe safety of Tilmicosin - Florfenicol Nanoemulsion was evaluated by acute toxicity test and skin irritation test. The median lethal dose(LD50) of Tilmicosin - Florfenicol Nanoemulsion was 2043.7mg.kg^-1, so it belong to Low toxicity drug.Serious irritation didn't appear when Tilmicosin - Florfenicol Nanoemulsion was used on complete and damaged skin of rabbits respectively.The results showed that Tilmicosin - Florfenicol Nanoemulsion was safe by oral and intramammary administration.4. Combination antibacterial effect of Tilmicosin - Florfenicol Nanoemulsion in vitroCombination antibacterial effect of Tilmicosin - Florfenicol Nanoemulsion against three clinical strains in vitro was investigated by single drug minimum inhibitory concentration(MIC) determination, combination susceptibility test and Kirby-Baueer disc diffusion test. The results showed that synergistic effect was generated when Tilmicosin and Florfenicol were combined. MIC of the mixed Tilmicosin and Florfenicol against Staphylococcus aureus was both 1/4 times compared with single drug respectively, MIC against Streptococcus agalactiae was 1/4 and 1/8 times, and MIC against Escherichia coli was both 1/4 and 1/16 times. Each of fractional inhibitory concentration(FIC) index was not more than 0.5. The results suggested that combination antibacterial activity of Tilmicosin - Florfenicol Nanoemulsion against three pathogenic bacteria was strong.5. Therapeutic efficacy of Tilmicosin - Florfenicol Nanoemulsion against chronic respiratory disease of chicken The high, middle and low dose of Tilmicosin - Florfenicol Nanoemulsion and Tilmicosin– Florfenicol solution was respectively dosed by drinking freely to treat chronic respiratory disease of chicken. The results indicated that cure rate of Tilmicosin - Florfenicol Nanoemulsion in high, middle and low dose group was 93.3%,83.3% and 33.3% respectively, and effective rate was 96.7%,93.3% and 66.7% respectively. The cure rate and effective rate of Tilmicosin– Florfenicol solution was63.3% and 73.3% respectively. The results of the statistical analysis showed the therapeutic efficacy of high and middle dose group were both obviously better than low dose group and Tilmicosin– Florfenicol solution group(P<0.05). Clinical recommended dose was middle on these grounds when Tilmicosin - Florfenicol Nanoemulsion was used to treat chronicPasteurella disease of chicken. Administration dose could increase fairly for serious one. The prepared Tilmicosin - Florfenicol Nanoemulsion had a high safety,good stability and high-performance,That could provide a new type of nano-antibacterial drugs for veterinary clinical medication.6. Studies on bioavailability of Tilmicosin - Florfenicol NanoemulsionThe content determination of Tilmicosin and Florfenicol analytical methods were respectively established by High Performance Liquid Chromatography. The results showed that two good linearity were obtained by Tilmicosin in the range of 0.05～10μg·mL^-1 and by Florfenicol in the range of 0.05～5μg·mL^-1, and the average recovery, relative standard deviation(RSD), RSD of the with-in-day precision, RSD of the day-to-day precision of Tilmicosin and Florfenicol were (98.29±1.80)% and (99.33±1.20)%, 1.83%and 1.21%, 0.40% and 0.35%,1.14%and1.06% respectively. The analytical methods of High Performance Liquid Chromatography (HPLC) which established had high recovery rate , good repetitiveness and precision in this research.Healthy adult chicken was used for subjects, and compound Tilmicosin - Florfenicol solution was used to compare. We do a research of bioavailability. Experimental results show that bioavailability of compound Tilmicosin - Florfenicol Nanoemulsion in animals is 116.61% for Tilmicosin and 109.83% for Florfenicol.It explain the drug- delivery system of nanoemulsion promoted the take-up of medicine and enhance the bioavailability.