| Objective: Tosufloxacin nanoemulsion(TFLX-NE) was prepared. The acute toxicity, pharmacological efficacy and quality of TFLX-NE were evaluated, which provided a new way for veterinary clinical treatment of bacterial diseases.Methods:(1) The oil phase, surfactant and cosolvent were selected according to the drug loading, stability of NE and the pseudo-ternary phase diagrm, designed optimum formula, prepared TFLX-NE.(2) The type of TFLX-NE was indentified by staining and dilution method determines. The configuration was measured by TEM magnified 30 000 times, and particle size of TFLX-NE was measured by LPSA. The drug content was determined through High Performance Liquid Chromatography(HPLC). The stability and shelf life were tested by stress testing, accelerated testing and long-term testing.(3) The acute toxicity of TFLX-NE was evaluate by LD50 and MTD testing.(4) Microtiter broth dilution method recommended by CLSI was adopted to determine the antibacterial activity of TFLX-NE with streptococcus agalactiae, Staphylococcus aureus, Pasteurella multocida, Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Salmonella paratyphi-A.(5) The pharmaceutical efficacy was evaluated by gavage freely to the model chicks infected with Salmonella typhimurium containing different concentrations of TFLX-NE.Results:(1) The TFLX-NE was light yellow, clear and transparent liquid, and the optimum formula of it was composed of tosufloxacin(1.36 wt%), origanum oil(3.24 wt%), lactic acid(2.60 wt%), RH-40(25.98 wt%), distilled water(66.82 wt%).(2) The structure pattern of TFLX-NE was O/W, which could be infinitely diluted by water. The shape of this nanoemulsion droplet was spherical, equally distributed, without adhesion, the particle size between 6.50~28.21 nm, with an average particle size of 16.16 nm. Its stability was good,the shelf life was 24 months under the condition of 25℃±2℃ and relative humidity of 60%±10%. The best detection wavelength was 277.60 nm. As the concentrations of tosufloxacin ranged from 3μg·mL-1 to 100μg·mL-1, the concentrations and peak areas showed good linear relationship; the average recovery rate and relative standard deviation of TFLX-NE were 99.33%±1.24% and 1.25%, respectively, the precision of this TFLX-NE was well, the drug content was 100.03% of the labeled amount.(3) The LD50 of TFLX-NE was greater than 10 000 mg·kg-1, the MTD of this nanoemulsion was 39.72g·kg-1;(4) The MICs of TFLX-NE against Streptococcus agalactiae, Staphylococcus aureus, Pasteurella multocida, Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Salmonella paratyphi-A were 0.25μg·mL-1~2.00μg·mL-1 and about 2~4 and 2~8 fold stronger than that of its API and Levofloxacin Hydrochloride respectively; the MBCs were 0.25μg·mL-1~4.00μg·mL-1 and about 2 ~ 8 fold stronger than that of its API and Levofloxacin Hydrochloride.(5) Successfully established avian paratyphoid disease models, the clinical results showed the death rate of infected chicks of TFLX-NE high, medium and low dose group(80mg·L-1、60mg·L-1、40mg·L-1), tosufloxacin API group decreased by 23.33%, 36.66%, 30%, 16.66%, which compared with infected group, and the relative weight gain rate increased by 18.57%, 47.44%, 35.15%, 18.97%. The data showed that mediun dose(60mg·L-1) of the TFLX-NE could be applied as clinical recommended dosage.Conclusion: The test was successfully prepared TFLX-NE, which had good stability, long storage time, high security, it could effectively resist veterinary clinical isolated gram-positive and gram-negative bacteria, which could effectively treat the sick chicks infected by Salmonella typhimurium. |
PDF Full Text Request