The Study On Diclazuril Nanoemulsion

In this research, we selected nanoemulsion as carrier of diclazuril. The formula of diclazuril nanoemulsion were optimized by protracting the pseudoternary phase diagrams. Then the stability, safety, effect and bioavailability of diclazuril nanoemulsion were studied, providing a basis for diclazuril nanoemulsion in the veterinary clinical application.1. Preparation and quality evaluation of diclazuril nanoemulsionThe optimal formula of diclazuril nanoemulsion was determined by pseudo-ternary phase diagrams. Then diclazuril nanoemulson was prepared. The structure type of diclazuril nanoemulsion was judged by centrifugation and staining method. Its appearance and particle diameter distribution were investigated by transmission electron microscope and laser particle size analysator respectively. Its stability was verified through photostability testing by strong light, accelerated testing and long-term testing. The results showed that the final prepared prescription of diclazuril nanoemulsion was w(diclazuril)=5%, w(Tween-80)=30%, w(ethanol)=5%, w(ethyl acetate)=3.9%, w(Dimethylformamide)=2.5%, w(distilled water)=53.6%. The type of prepared diclazuril nanoemulsion was oil-in-water and the liquid was yellow, transparent and homogeneous. The nanoemulsion drop presented as spherical shape and the average drop size was 14.0 nm with a polydispersity index of 0.064. Diclazuril nanoemulsion had good stability and the period of validity was 21 months. So the quality of diclazuril nanoemulsion had achieved the requirement of clinical medication.2. Analytical method establishment of diclazuril nanoemulsionThe analytical methods of content determination about diclazuril nanoemulsion were established by ultraviolet-visible spectrophotometer. The results showed that a good linearity of diclazuril was obtained in the range of 5～25μg·mL^-1.The average recovery, relative standard deviation(RSD), RSD of the with-in-day precision and RSD of the day-to-day precision of diclazuril were (99.18±1.74)%, 1.75%, 0.24% and 0.54%. The established analytical methods of content determination about diclazuril nanoemulsion had good specificity, high recovery rate, good repetitiveness and precision. It could offer a good specificity method of content determination about diclazuril nanoemulsion.3. Safety evaluation of diclazuril nanoemulsion The safety of diclazuril nanoemulsion was evaluated by acute toxicity test. The results showed that median lethal dose(LD50) of diclazuril nanoemulsion was 2751 mg·kg^-1, 95% confidence interval of LD50 was 2561～2984 mg·kg^-1. So diclazuril nanoemulsion was low toxicity drug.4. Study on effectiveness of diclazuril nanoemulsion aiming at chicken coccidiosisThree hundreds 18-day-old chickens were randomly divided into high, middle, low dose group of diclazuril nanoemulsion, diclazuril solution group, negative control group and positive control group. Each chicken of the experimental group and positive control group was inoculated 0.5mL Eimeria coccidium oocyst suspension(including 1.0×105 oocysts).In contract, the negative control group was given equivalent physiological saline. After 48h, high, middle and low dose group of diclazuril nanoemulsion was separately given 2.0,1.0,0.5 mg·kg^-1 diclazuril by drinking water and diclazuril solution group was given 1.0 mg·kg^-1 diclazuril by drinking water. The drug administration last for 5d. The negative and positive control group were given drinking water normally. Relative weight gain rate, livability, cecum lesion value and oocyst value were determined at eighth day after infection. Then ACI(anticoccidial index) was calculated. The results showed that ACI of diclazuril solution group was 153.86, so diclazuril solution was an inefficient anticoccidial drug. However, ACI of high, middle and low dose group of diclazuril nanoemulsion were 194.34, 195.81 and 186.35 respectively. ACI of diclazuril nanoemulsion was greater than 180, so diclazuril nanoemulsion was efficient anticoccidial drug.5. Study on bioavailability of diclazuril nanoemulsion in chickenThe content analytical method of diclazuril in blood plasma was established by high performance liquid chromatography (HPLC) and the bioavailability of diclazuril nanoemulsion in chicken was studied. The results showed that chromatographic peak of diclazuril was uniform and symmetrical and the retention time of diclazuril was 4.670 min. A good linearity of diclazuril in blood plasma was obtained in the range of 2.5～40μg·mL^-1. The average recovery, relative standard deviation(RSD), RSD of the with-in-day precision and RSD of the day-to-day precision of diclazuril in blood plasma were(96.83±0.68)%, 0.70%, 0.59% and 0.54%. The established analytical method of HPLC about diclazuril in blood plasma had good specificity, high recovery rate, good repetitiveness and precision and it could offer a precise analytical method for the study on bioavailability of diclazuril nanoemulsion in chicken. The drug-time data of diclazuril nanoemulsion and diclazuril solution in chicken were according with two-compartment model with first order absorption. AUC of diclazuril nanoemulsion and diclazuril solution were (417.9333±18.6409)μg·h/mL and ( 174.8786±29.8342 )μg·h/mL. With respect to diclazuril solution, the relative bioavailability of diclazuril nanoemulsion was 238.98%.So diclazuril nanoemulsion had a high bioavailability.The prepared diclazuril nanoemulsion had a high drug loading rate, good stability, high safety, strong effectiveness and high bioavailability and it would provide a new type of nano-anticoccidial drug for veterinary clinic.