硝碘酚腈脂质体的制备及肝脏靶向性初步研究
发布时间：2018-07-25 08:57
【摘要】：肝片吸虫病是十分常见的人畜共患寄生虫病,普遍存在于牛、羊等反刍动物。在我国各省区呈区域性流行,一年四季均可发生,特别是夏秋季节非常普遍。牛、羊的感染率最高,羊的感染率可达30%～50%,甚至部分地区全部感染,牛的感染率有30%～60%,部分地区90%以上。肝片吸虫病会致使动物发育停滞,体重消减,耕牛生产力低下,乳牛产奶量减少,毛和肉产量减少并且品质降低,给畜牧业造成巨大的经济亏损。硝碘酚腈(Nitroxynil)是20世纪60年代末研制的高效抗肝片吸虫药,对肝片吸虫和大片吸虫的成虫期均都具有很高的疗效,然而药物与血浆蛋白具有高度结合力(结合率97%),使游离硝碘酚腈在组织内集聚量很少,以致在推荐剂量下,肝实质中的药物浓度不足以杀灭未成熟的幼虫。在临床中肌内注射与皮下注射为首选方式,然而在给药部位发生炎性肿胀,动物出现摇头、走路摇晃、呼吸急促甚至死亡等现象。脂质体作为药物的载体系统,将药物包封于脂质双分子层中,可以提高药物在机体内的靶向性,增强治疗效果,降低毒副作用,已经在医学临床中得到广泛的应用。脂质体双分子层生物膜主要由磷脂和胆固醇组成,未经修饰的脂质体经静脉给药进入动物机体中,被巨噬细胞作为“侵入者”吞噬而产生天然靶向性,主要由肝和脾中的吞噬细胞(网状内皮细胞)吞噬,因此脂质体是用于治疗肝寄生虫病、利什曼原虫病等肝脏内寄生虫病的理想药物载体。本试验使用脂质体包封技术制备硝碘酚腈脂质体,旨在提高其有效生物利用度、延长作用时间、降低药物毒性作用,并探究其临床使用意义。1硝碘酚腈脂质体的制备方法：采用薄膜超声法制备硝碘酚腈脂质体,以磷脂-胆固醇质量比(A)、药脂比(B)、超声时间(C)、水化温度(D)四个因素进行正交试验设计L9(34),以包封率为指标,筛选最佳工艺。结果：经试验数据分析,各因素对工艺的影响程度：BADC.最佳工艺处方为：磷脂-胆固醇质量比为2：1,药脂比为3：1,超声时间6min,水化温度40℃。经重复性验证该处方包封率为50.05±1.62%。结论：本试验筛选出的处方及工艺所制备的硝碘酚腈脂质体包封率较好、重现性好。2硝碘酚腈脂质体的质量评价2.1HPLC测定脂质体中硝碘酚腈含量方法的建立方法：色谱条件：流动相：乙腈-0.01 mol/L磷酸盐pH6.5(25:75, V/V);色谱柱：Phenomenex-C18 (4.6 mm x 250 mm,5μm);流速：1.5 mL/min;柱温：40℃；检测波长：271 nm；进样量：10μL。结果：经方法学验证,该色谱条件适合硝碘酚腈含量测定,出峰时间合适,峰形对称,与其他杂质峰分离度良好,重现性与精密度均符合要求。标准曲线为y=25250x-173662,R2=0.9999,线性范围：10-400μg/mL。2.2粒径的测定方法：使用Zetasizer Nano分析仪动态光散射法测定脂质体粒径大小。结果：硝碘酚腈脂质体粒径于200-300nnm呈正态分布。2.3包封率测定方法：以葡聚糖凝胶柱内径与填充高度比(A),加样量(B),流速(C)三个因素设计正交试验,筛选葡聚糖凝胶柱分离硝碘酚腈脂质体的最佳条件,收集脂质体流出液,使用HPLC测定硝碘酚腈含量,计算包封率。结果：使用100-300μm的葡聚糖凝胶G-50作为层析基质,葡聚糖凝胶柱内径与填充高度比：2：7,加样量：1.0mL,流速：1mL-min-1。洗脱曲线显示,脂质体洗脱时间为4-15min。收集流出液用10%Trixton-100溶液破乳,用HPLC测定硝碘酚腈含量。结论：本试验方法制得脂质体粒径分布较均匀,使用葡聚糖凝胶柱分离硝碘酚腈脂质体出峰时间合适,可以有效分离硝碘酚腈脂质体与游离硝碘酚腈。3硝碘酚腈脂质体在家兔体内药动学试验方法：选择健康家兔2组,比较耳缘静脉注射相同剂量(8.8mg/kg)的硝碘酚腈注射液与硝碘酚腈脂质体,分别于Smin, 10min,15min、30min、45min、1h、1.5h、2h、4h、 8h、12h、24h、36h采血,使用上述HPLC方法测定含量,用3P97软件分析药动学模型以及药动学参数。结果：HPLC绘制血液中硝碘酚腈标准曲线为：y=27547x-31146,R2=0.9995。线性范围均为6.25-400μg/mL。硝碘酚腈注射液和硝碘酚腈脂质体耳缘静脉注射后的血药浓度-时f间分布均符合—级吸收二室模型。硝碘酚腈注射液在家兔体内主要动力学参数为：A=14.44±1.25μg/mL, a=3.10±0.37/h, B=15.50±2.88μg/mL, β=0.031±0.0028/h, V (c)=0.29±0.00083 L), Tl/2a= 0.22±0.0028h; Tl/2β=22.21±2.48h, AUC=501.18±35.76 CL(s)=0.018±0.0007 mg/kg/h/(μg/mL)。硝碘酚腈脂质体在家兔体内主要动力学参数：A=8.85±0.71μg/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)/(pg/mL), Tl/2α= 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)。硝碘酚腈脂质体消除半衰期延长(T1/2β)、中央室分布容积(Vc)增大,与硝碘酚腈注射液比较有极显著差异(p0.01)。结论：硝碘酚腈脂质体在家兔体内消除半衰期显著延长、中央室分布容积增大,生物利用度升高,揭示其具有缓释作用。4硝碘酚腈脂质体的肝脏靶向性初步研究选择健康KM小鼠2组,尾静脉注射相同剂量的硝碘酚腈注射液与硝碘酚腈脂质体,分别于15min.30min、45min、1h、2h、4h、8h、12h、24h解剖小鼠,采集肝脏组织,使用上述HPLC分析方法进行含量测定。结果HPLC绘制肝脏组织标准曲线为：y=26156x+36621,R2=0.9996。线性范围为6.25～400μg/mL。硝碘酚腈注射液与硝碘酚腈脂质体肝脏中AUC注=87.00(μg/mL)*h, AUC脂=197.29(μg/mL*h,肝脏相对摄取率re=2.27。结论：硝碘酚腈脂质体中肝脏re=2.27,AUC 注与AUC脂差异极显著,表明具有良好的靶向性。总结：本课题筛选的硝碘酚腈脂质体制备工艺合理,所制备出的脂质体具有较好的药动学特征及肝脏靶向性,揭示其可能有效的改善硝碘酚腈的副作用与生物利用度。
[Abstract]:Fasciola hepatica is a common zoonotic parasitic disease, which is common in ruminant animals such as cattle and sheep. It has a regional epidemic in all provinces in China. It can occur throughout the year, especially in summer and autumn. The infection rate of cattle and sheep is the highest, the infection rate of sheep can reach 30% to 50%, even in some regions, and the infection rate of cattle is 3 0% to 60% and more than 90% in some areas. Fasciola hepatica caused stagnation of animal development, weight loss, low productivity of cattle, reduced milk production, reduced hair and meat production and reduced quality, resulting in huge economic losses to animal husbandry. Nitroxynil was a highly effective anti liver Fasciola medicine developed at the end of the 1960s, to the liver. The adult stage of Fasciola and tracerworms both have high curative effect, but the drug and plasma protein have high binding force (97%), so that the concentration of free nitliodiolitrile in the tissue is very small, so that the concentration of the liver parenchyma is not enough to kill the immature larvae at the recommended dose. Intramuscular injection and intramuscular injection in clinic Injection is the first choice, but there is an inflammatory swelling in the part of the drug, and the animals shake their heads, walk and shake, and breathe quickly or even death. As the carrier system of the drug, the liposomes are encapsulated in the lipid bilayer, which can improve the target of the drug in the body, enhance the therapeutic effect and reduce the side effects. It has been in medicine. The liposome biome biofilm is mainly composed of phospholipids and cholesterol. The unmodified liposomes are injected into the animal body through the vein, and the macrophage is phagocytic as a "intruder" to produce natural targeting, mainly by phagocytic (reticuloendothelial cells) in the liver and spleen. The body is an ideal drug carrier for the treatment of liver parasitology, Leishmania and other parasitic diseases in the liver. The liposomes were prepared by liposome encapsulation technology to improve the effective bioavailability, prolong the action time, reduce the toxicity of the drug and explore the clinical significance of the.1 nitliodiolitrile liposomes. Preparation method: using thin film ultrasonic method to prepare nitodipholonitrile liposome, using four factors of phospholipid - cholesterol mass ratio (A), drug fat ratio (B), ultrasonic time (C) and hydration temperature (D) orthogonal experiment design L9 (34). The optimum technology was screened by encapsulation efficiency. The result: the test data analysis, the influence degree of each factor to the process: BADC. most The best formula was as follows: the ratio of phospholipid to cholesterol was 2:1, the ratio of drug to lipid was 3:1, the time of ultrasound was 6min, and the hydration temperature was 40 degrees C. The encapsulation efficiency of the formulation was 50.05 + 1.62%. by repeatability: the encapsulation efficiency of the liposome of nitodipholonitrile prepared by this test was better, and the quality of the liposome of.2 was reproducible, and the quality of the liposome of.2 was reproducible. Evaluation of the method for the determination of nitriodiolitrile content in liposomes by 2.1HPLC: chromatographic conditions: mobile phase: acetonitrile -0.01 mol/L phosphate pH6.5 (25:75, V/V); chromatographic column: Phenomenex-C18 (4.6 mm x 250 mm, 5 mu m); flow rate: 1.5 mL/min; column temperature: 40 C; detection wavelength: 271: 10 micron results: methodological test The chromatographic condition is suitable for the determination of nitramiol nitrile content, the peak time is suitable, the peak shape is symmetrical, the separation degree is good with other impurity peaks, the reproducibility and precision meet the requirements. The standard curve is y=25250x-173662, R2=0.9999, and the linear range: 10-400 mu g/mL.2.2 particle size determination method: using the Zetasizer Nano analyzer dynamic light scattering method Results: the particle size of nitro iodide liposomes was determined by the positive distribution of.2.3 encapsulation efficiency in 200-300nnm: the optimum conditions for the separation of the liposomes from the dextran gel column were screened by three factors such as the internal diameter of the Sephadex column and the height of filling (A), the amount of addition (B) and the velocity of velocity (C). The content of NO3 iodide phenolitrile was measured by HPLC and the encapsulation efficiency was calculated by using HPLC. Results: the internal diameter of the dextran gel column was compared with the filling height using the 100-300 - M dextran gel, 2:7, 1.0mL, velocity: 1mL-min-1. elution curve showed that the elution time of liposome was 4-15min. collection of 10%Trixton-1 from the effluent. 00 solution demulsification and HPLC determination of nitriodiphenolitrile content. Conclusion: this method is made to make the particle size distribution of liposomes is more uniform and the time of separating nitro iodonitrile liposomes with glucan gel column is suitable. It can effectively separate the pharmacokinetics test method of nitodiolitrile liposomes and free nitodiiodolonitrile.3 nitro iodolonitrile liposomes in rabbits. 2 groups of healthy rabbits were selected to compare the nitodiiodolonitrile injection and nitnoiodolonitrile liposomes with the same dose (8.8mg/kg) in the auricular vein. Smin, 10min, 15min, 30min, 45min, 1H, 1.5h, 2h, 4h, 8h, 12h, blood sampling, and pharmacokinetic parameters were analyzed using the above-mentioned method. The standard curve of C to draw blood nitnoiodolonitrile is y=27547x-31146, and the linear range of R2=0.9995. is 6.25-400 mu g/mL. nitnol acetonitrile injection and niodolonitrile liposomes after the auricular vein injection, and the distribution of F is conformed to the level absorption two compartment model. The main kinetic parameters of nitramonitrile injection solution in rabbits are A=1 4.44 + 1.25 mu g/mL, a=3.10 + 0.37/h, B=15.50 + 2.88 mu g/mL, beta =0.031 + 0.0028/h, V (c) =0.29 + 0.00083 L), Tl/2a= 0.22 + 35.76. 8 + 1.22 g/mL, beta =0.0155 + 0.002/h, V (c) =0.4 + 0.025 (mg/kg) / (pg/mL), Tl/2 alpha = 0.38 + 0.011h, T1/2 beta = 44.83 + 0.29h. Significant difference (P0.01). Conclusion: the half-life of nitliodiolitrile liposomes in the rabbit was prolonged significantly, the volume of the central ventricular distribution was increased, the bioavailability was increased, and the liver targeting of the.4 nitliodiolitrile liposome was preliminarily studied in 2 groups of the healthy KM mice and the same dose of nitliodioliodide injection was injected into the tail vein. 15min.30min, 45min, 1H, 2h, 4h, 8h, 12h, 24h of mice were dissected with nitliodiolitrile liposomes to collect liver tissues and use the above HPLC analysis method to determine the content. Results HPLC drew the standard curve of liver tissue: y=26156x+36621, R2=0.9996. linear range was 6.25 to 400 micronnoolitrile injection and nitliodiolitrile liposomes. AUC injection of =87.00 (mu g/mL) *h, AUC fat =197.29 (mu g/mL*h, liver relative uptake rate re=2.27.), re=2.27. conclusion: the liver re=2.27 in nitliodiolitrile liposomes, AUC injection and AUC lipid are very significant, indicating that it has good targeting. The pharmacokinetic characteristics and liver targeting ability of the nitrite were found to be effective in improving the side effects and bioavailability of nitnocyanolitrile.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.795
本文编号：2143304
[Abstract]:Fasciola hepatica is a common zoonotic parasitic disease, which is common in ruminant animals such as cattle and sheep. It has a regional epidemic in all provinces in China. It can occur throughout the year, especially in summer and autumn. The infection rate of cattle and sheep is the highest, the infection rate of sheep can reach 30% to 50%, even in some regions, and the infection rate of cattle is 3 0% to 60% and more than 90% in some areas. Fasciola hepatica caused stagnation of animal development, weight loss, low productivity of cattle, reduced milk production, reduced hair and meat production and reduced quality, resulting in huge economic losses to animal husbandry. Nitroxynil was a highly effective anti liver Fasciola medicine developed at the end of the 1960s, to the liver. The adult stage of Fasciola and tracerworms both have high curative effect, but the drug and plasma protein have high binding force (97%), so that the concentration of free nitliodiolitrile in the tissue is very small, so that the concentration of the liver parenchyma is not enough to kill the immature larvae at the recommended dose. Intramuscular injection and intramuscular injection in clinic Injection is the first choice, but there is an inflammatory swelling in the part of the drug, and the animals shake their heads, walk and shake, and breathe quickly or even death. As the carrier system of the drug, the liposomes are encapsulated in the lipid bilayer, which can improve the target of the drug in the body, enhance the therapeutic effect and reduce the side effects. It has been in medicine. The liposome biome biofilm is mainly composed of phospholipids and cholesterol. The unmodified liposomes are injected into the animal body through the vein, and the macrophage is phagocytic as a "intruder" to produce natural targeting, mainly by phagocytic (reticuloendothelial cells) in the liver and spleen. The body is an ideal drug carrier for the treatment of liver parasitology, Leishmania and other parasitic diseases in the liver. The liposomes were prepared by liposome encapsulation technology to improve the effective bioavailability, prolong the action time, reduce the toxicity of the drug and explore the clinical significance of the.1 nitliodiolitrile liposomes. Preparation method: using thin film ultrasonic method to prepare nitodipholonitrile liposome, using four factors of phospholipid - cholesterol mass ratio (A), drug fat ratio (B), ultrasonic time (C) and hydration temperature (D) orthogonal experiment design L9 (34). The optimum technology was screened by encapsulation efficiency. The result: the test data analysis, the influence degree of each factor to the process: BADC. most The best formula was as follows: the ratio of phospholipid to cholesterol was 2:1, the ratio of drug to lipid was 3:1, the time of ultrasound was 6min, and the hydration temperature was 40 degrees C. The encapsulation efficiency of the formulation was 50.05 + 1.62%. by repeatability: the encapsulation efficiency of the liposome of nitodipholonitrile prepared by this test was better, and the quality of the liposome of.2 was reproducible, and the quality of the liposome of.2 was reproducible. Evaluation of the method for the determination of nitriodiolitrile content in liposomes by 2.1HPLC: chromatographic conditions: mobile phase: acetonitrile -0.01 mol/L phosphate pH6.5 (25:75, V/V); chromatographic column: Phenomenex-C18 (4.6 mm x 250 mm, 5 mu m); flow rate: 1.5 mL/min; column temperature: 40 C; detection wavelength: 271: 10 micron results: methodological test The chromatographic condition is suitable for the determination of nitramiol nitrile content, the peak time is suitable, the peak shape is symmetrical, the separation degree is good with other impurity peaks, the reproducibility and precision meet the requirements. The standard curve is y=25250x-173662, R2=0.9999, and the linear range: 10-400 mu g/mL.2.2 particle size determination method: using the Zetasizer Nano analyzer dynamic light scattering method Results: the particle size of nitro iodide liposomes was determined by the positive distribution of.2.3 encapsulation efficiency in 200-300nnm: the optimum conditions for the separation of the liposomes from the dextran gel column were screened by three factors such as the internal diameter of the Sephadex column and the height of filling (A), the amount of addition (B) and the velocity of velocity (C). The content of NO3 iodide phenolitrile was measured by HPLC and the encapsulation efficiency was calculated by using HPLC. Results: the internal diameter of the dextran gel column was compared with the filling height using the 100-300 - M dextran gel, 2:7, 1.0mL, velocity: 1mL-min-1. elution curve showed that the elution time of liposome was 4-15min. collection of 10%Trixton-1 from the effluent. 00 solution demulsification and HPLC determination of nitriodiphenolitrile content. Conclusion: this method is made to make the particle size distribution of liposomes is more uniform and the time of separating nitro iodonitrile liposomes with glucan gel column is suitable. It can effectively separate the pharmacokinetics test method of nitodiolitrile liposomes and free nitodiiodolonitrile.3 nitro iodolonitrile liposomes in rabbits. 2 groups of healthy rabbits were selected to compare the nitodiiodolonitrile injection and nitnoiodolonitrile liposomes with the same dose (8.8mg/kg) in the auricular vein. Smin, 10min, 15min, 30min, 45min, 1H, 1.5h, 2h, 4h, 8h, 12h, blood sampling, and pharmacokinetic parameters were analyzed using the above-mentioned method. The standard curve of C to draw blood nitnoiodolonitrile is y=27547x-31146, and the linear range of R2=0.9995. is 6.25-400 mu g/mL. nitnol acetonitrile injection and niodolonitrile liposomes after the auricular vein injection, and the distribution of F is conformed to the level absorption two compartment model. The main kinetic parameters of nitramonitrile injection solution in rabbits are A=1 4.44 + 1.25 mu g/mL, a=3.10 + 0.37/h, B=15.50 + 2.88 mu g/mL, beta =0.031 + 0.0028/h, V (c) =0.29 + 0.00083 L), Tl/2a= 0.22 + 35.76. 8 + 1.22 g/mL, beta =0.0155 + 0.002/h, V (c) =0.4 + 0.025 (mg/kg) / (pg/mL), Tl/2 alpha = 0.38 + 0.011h, T1/2 beta = 44.83 + 0.29h. Significant difference (P0.01). Conclusion: the half-life of nitliodiolitrile liposomes in the rabbit was prolonged significantly, the volume of the central ventricular distribution was increased, the bioavailability was increased, and the liver targeting of the.4 nitliodiolitrile liposome was preliminarily studied in 2 groups of the healthy KM mice and the same dose of nitliodioliodide injection was injected into the tail vein. 15min.30min, 45min, 1H, 2h, 4h, 8h, 12h, 24h of mice were dissected with nitliodiolitrile liposomes to collect liver tissues and use the above HPLC analysis method to determine the content. Results HPLC drew the standard curve of liver tissue: y=26156x+36621, R2=0.9996. linear range was 6.25 to 400 micronnoolitrile injection and nitliodiolitrile liposomes. AUC injection of =87.00 (mu g/mL) *h, AUC fat =197.29 (mu g/mL*h, liver relative uptake rate re=2.27.), re=2.27. conclusion: the liver re=2.27 in nitliodiolitrile liposomes, AUC injection and AUC lipid are very significant, indicating that it has good targeting. The pharmacokinetic characteristics and liver targeting ability of the nitrite were found to be effective in improving the side effects and bioavailability of nitnocyanolitrile.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.795
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