Preparation And Preliminary Research Of Liver-Targeted With Nitroxynil Liposomes

Objective:Fascioliasis is a zoonotic parasitic disease, the ruminants is most common, especially cattle, sheep. Many provinces were regional epidemic and occor throughout the year in China, especially in summer and autumn. Cattle and sheep are most serious infect, the sheep is 30% to 50% generally, in some areas up to 100%, cattle 30% to 60% generally in some areas as high as 90%. Animals in the disease process will emaciate, developmental disorders, decreased cattle production ability, dairy milk yield decrease, meat and wool production is reduced and lower quality, resulting in huge economic losses to the livestock industry.Nitroxynil was researched and development in the late 1960s of an effective expulsion of fasciola hepatica synthetic drug by the French hrone merry urban institute, and in 1987 imported into our country used as veterinary clinical medicine. Nitroxynil is highly efficient to adult stage of F. hepatica and F. gigantica, while the drug is highly bound to plasma proteins in the blood (binding rate> 97%), rarely distributed within the organization, so that can not sufficient to kill the immature larvae in the liver parenchyma with the recommendation concentration. Intramuscularly and subcutaneously injected are the preferred ways in clinical. However, in the administration of the site will appear inflammatory swelling, and animal appear head shaking, walking shaking, tachypnea, even death.It is a long time that liposome use as drug carrier systems to improve drug targeting, treatment, and reduce the side effects, like anti-cancer drugs. Liposome without modified inject into the animal body intravenous, which was recognized as the foreign body by macrophage so that has the natural targeting, and mainly consumed by phagocytic cells in the liver and spleen, so that is the ideal drug carrier for treatment of the reticuloendothelial system in liver parasitic diseases and the parasite disease.In this study, preparation nitroxynil liposome using liposome encapsulated technology, aim to increase the bioavailability, reducing toxicity, extend the cycle life, and to explore its clinical significance.1 Preparation of Nitroxynil liposomeMethod:Prepared nitroxynil liposome by film homogenized. Phospholipids to cholesterol weight ratio (A), drug to lipid ratio (B), the inclusion time (C), ultrasonic temperature (D) as four factors make the orthogonal experiment L9(34) in a small scope, inclusion rate as the evaluation index to chose the best prescription.Result: The influence degree of the factors:B>A>D>C. The best prescription: phospholipids to cholesterol weight ratio of 2:1, drug-lipid ratio of 3:1, ultrasonic time:6min, inclusion temperature:40℃. After repetitive verify the prescription inclusion rate was 50.05 ± 1.62%. Conclusion:Nitroxynil liposome has good inclusion rate and reproducibility preparation by the best prescription, have good reproducibility.2 The quality evaluation of nitroxynil liposome.Method：Assay of nitroxynilHPLC conditions:A Column: Phenomenex-C18 (4.6mm×250 mm) was used. the mobile phase:acetonitrile-0.01 mol/L phosphate pH 6.5 (25:75, V/V), flow rate:1.5mL/min, column temperature:40℃, the detection wavelength: 271nm, the injection volume was 10μL. Result: By methodology validation this HPLC condition was suitable to assay nitroxynil, and had a good peak time and interval time reasonable, good reproducibility and precision. Standard curve was y=25250x-173662, R2= 0.9999, linearity range of 10μg/mL-400μg/mL. Method: Determination the liposome particle size by dynamic light scattering with Zetasizer Nano particle size analyzer.Result: Nitroxynil liposome particle size between 200-300nm as normally distributed. Method:Determination inclusion rate: For the determination of liposome entrapment efficiency was selected by the orthogonal experimental:ratio of gel column internal diameter to glucanpacking height (A), sample loading (B), elution flow rate (C) as the factors, determination by HPLC, to calculate the inclusion rate.Result: Nitroxynil liposome was well-separated by use 100-300μm of Sephadex G-50 as a chromatographic matrix, ratio of gel column internal diameter to glucan packing height (A): 2:7, sample loading (B):1.0mL, elution flow rate (C):1mL-min-1. As elution profile showed that liposomes elution time was 4-15min. The effluents were collected, 10% Trixton-100 liquid broken solution, determination by HPLC.Conclusion: This method prepared nitroxynil liposome was uniform particle size distribution. And used dextran gel column separation nitroxynil liposome, the peak time is suitable, could effectively separate nitroxynil liposome and plasma nitroxynil.3 The pharmacokinetics study of nitroxynil liposome in rabbitsMethod: The Pharmacokinetics characteristics of nitroxynil liposome were compared with pharmacokinetics characteristics of nitroxynil injection in rabbits in this study. Healthy New Zealand rabbits were divided into 2 groups that one group for nitroxynil liposome and the other for nitroxynil injection, injected the same dose (8.8mg/kg) in ear marginal vein, take blood samples after injected in 5min, 10min, 15min, 30min, 45min, 1h, 1.5h, 2h, 4h, 8h,12h, 24h, 36h. The drug concentration of nitroxynil plasma was determination by HPLC, and drug concentration-time datas were calculated using 3p97.Results: HPLC standard curve for blood:y = 27547x-31146, R2 = 0.9995. The linearity range was 6.25μg/mL-400μg/mL. The drug concentration-time dates of nitroxynil liposome and nitroxynil injection were fit with two compartment model.The main pharmacokinetic parameters of nitroxynil injection in rabbit: A=14.44±1.25μg/mL, α=3.10±0.37/h, B=15.50±2.88μg/mL, β=0.031±0.0028/h, V (c)=0.29±0.00083 (mg/kg) /(μg/mL), T1/2α= 0.22±0.0028h; T1/2β=22.21±2.48h, AUC= 501.18±35.76 (μg/mL)*h, CL(s)=0.018±0.0007 mg/kg/h/(μg/mL).The main pharmacokinetic parameters of nitroxynil liposome in rabbit: A=8.85±0.7μg/mL, a=1.80±0.25/h, B=13.08±1.22μg/mL, β=0.0155±0.002/h, V (c)=0.4±0.025 (mg/kg) /(μg/mL), T1/2a=0.38±0.011h, T1/2β=44.83±0.29h, AUC=851.30±48.37(μg/mL)*h, CL(s)=0.0103±0.0009 mg/kg/h/(μg/mL)。 The elimination half-life (T1/2β) nitroxynil liposome, the central compartment volume of distribution (Vc), there was a significant difference (p<0.01) compared with the nitroxynil injection.Conclusion: Nitroxynil liposome was significantly longer elimination half-life (T1/2β), volume of distribution of the central compartment (Vc) increases, that showed Nitroxynil liposome increased bioavailability, sustained-release and immediate-release effect in rabbits. 4 Preliminary study of the liver-targeting of Nitroxynil liposomeMethods: Healthy KM mice were divided into 2 groups that one group for nitroxynil liposome and the other for nitroxynil injection, injected the same dose in caudal vein, take liver samples after injected in 15min,30min,45min, Hi, 2h, 4h, 8h, 12h, 24h. The drug concentration of plasma was determination using HPLC, and drug concentration-time dates were calculated.Results: HPLC standard curve for Liver: y=26156x+36621, R2=0.9996.The linearity range was 6.25μg/mL-400(μg/mL. Nitroxynil injection and nitroxynil liposome were measured in liver AUC 注=87.00 (μg/mL)*h, AUC 脂=197.29 (μg/mL)*h, calculated relative liver uptake re=2.27.Conclusion: Relative uptake rates are targeted tissue preparations commonly used measure, when Re>1, indicating that the preparation has liver-targeting, nitroxynil liposome liver re=2.27, there was a significant difference (p<0.01) compared with the AUC 注 and AUC 脂, showed that nitroxynil liposome have good targeting.Total conclusion: The prescription of nitroxynil liposome chosed in this study is reasonable, and the nitroxynil liposome has a good pharmacokinetic character and liver-targeting, that can effect improve nitroxynil side effect and bioavailability.