【摘要】：肝片吸蟲(chóng)病是十分常見(jiàn)的人畜共患寄生蟲(chóng)病,普遍存在于牛、羊等反芻動(dòng)物。在我國(guó)各省區(qū)呈區(qū)域性流行,一年四季均可發(fā)生,特別是夏秋季節(jié)非常普遍。牛、羊的感染率最高,羊的感染率可達(dá)30%～50%,甚至部分地區(qū)全部感染,牛的感染率有30%～60%,部分地區(qū)90%以上。肝片吸蟲(chóng)病會(huì)致使動(dòng)物發(fā)育停滯,體重消減,耕牛生產(chǎn)力低下,乳牛產(chǎn)奶量減少,毛和肉產(chǎn)量減少并且品質(zhì)降低,給畜牧業(yè)造成巨大的經(jīng)濟(jì)虧損。硝碘酚腈(Nitroxynil)是20世紀(jì)60年代末研制的高效抗肝片吸蟲(chóng)藥,對(duì)肝片吸蟲(chóng)和大片吸蟲(chóng)的成蟲(chóng)期均都具有很高的療效,然而藥物與血漿蛋白具有高度結(jié)合力(結(jié)合率97%),使游離硝碘酚腈在組織內(nèi)集聚量很少,以致在推薦劑量下,肝實(shí)質(zhì)中的藥物濃度不足以殺滅未成熟的幼蟲(chóng)。在臨床中肌內(nèi)注射與皮下注射為首選方式,然而在給藥部位發(fā)生炎性腫脹,動(dòng)物出現(xiàn)搖頭、走路搖晃、呼吸急促甚至死亡等現(xiàn)象。脂質(zhì)體作為藥物的載體系統(tǒng),將藥物包封于脂質(zhì)雙分子層中,可以提高藥物在機(jī)體內(nèi)的靶向性,增強(qiáng)治療效果,降低毒副作用,已經(jīng)在醫(yī)學(xué)臨床中得到廣泛的應(yīng)用。脂質(zhì)體雙分子層生物膜主要由磷脂和膽固醇組成,未經(jīng)修飾的脂質(zhì)體經(jīng)靜脈給藥進(jìn)入動(dòng)物機(jī)體中,被巨噬細(xì)胞作為“侵入者”吞噬而產(chǎn)生天然靶向性,主要由肝和脾中的吞噬細(xì)胞(網(wǎng)狀內(nèi)皮細(xì)胞)吞噬,因此脂質(zhì)體是用于治療肝寄生蟲(chóng)病、利什曼原蟲(chóng)病等肝臟內(nèi)寄生蟲(chóng)病的理想藥物載體。本試驗(yàn)使用脂質(zhì)體包封技術(shù)制備硝碘酚腈脂質(zhì)體,旨在提高其有效生物利用度、延長(zhǎng)作用時(shí)間、降低藥物毒性作用,并探究其臨床使用意義。1硝碘酚腈脂質(zhì)體的制備方法：采用薄膜超聲法制備硝碘酚腈脂質(zhì)體,以磷脂-膽固醇質(zhì)量比(A)、藥脂比(B)、超聲時(shí)間(C)、水化溫度(D)四個(gè)因素進(jìn)行正交試驗(yàn)設(shè)計(jì)L9(34),以包封率為指標(biāo),篩選最佳工藝。結(jié)果：經(jīng)試驗(yàn)數(shù)據(jù)分析,各因素對(duì)工藝的影響程度：BADC.最佳工藝處方為：磷脂-膽固醇質(zhì)量比為2：1,藥脂比為3：1,超聲時(shí)間6min,水化溫度40℃。經(jīng)重復(fù)性驗(yàn)證該處方包封率為50.05±1.62%。結(jié)論：本試驗(yàn)篩選出的處方及工藝所制備的硝碘酚腈脂質(zhì)體包封率較好、重現(xiàn)性好。2硝碘酚腈脂質(zhì)體的質(zhì)量評(píng)價(jià)2.1HPLC測(cè)定脂質(zhì)體中硝碘酚腈含量方法的建立方法：色譜條件：流動(dòng)相：乙腈-0.01 mol/L磷酸鹽pH6.5(25:75, V/V);色譜柱：Phenomenex-C18 (4.6 mm x 250 mm,5μm);流速：1.5 mL/min;柱溫：40℃；檢測(cè)波長(zhǎng)：271 nm；進(jìn)樣量：10μL。結(jié)果：經(jīng)方法學(xué)驗(yàn)證,該色譜條件適合硝碘酚腈含量測(cè)定,出峰時(shí)間合適,峰形對(duì)稱(chēng),與其他雜質(zhì)峰分離度良好,重現(xiàn)性與精密度均符合要求。標(biāo)準(zhǔn)曲線為y=25250x-173662,R2=0.9999,線性范圍：10-400μg/mL。2.2粒徑的測(cè)定方法：使用Zetasizer Nano分析儀動(dòng)態(tài)光散射法測(cè)定脂質(zhì)體粒徑大小。結(jié)果：硝碘酚腈脂質(zhì)體粒徑于200-300nnm呈正態(tài)分布。2.3包封率測(cè)定方法：以葡聚糖凝膠柱內(nèi)徑與填充高度比(A),加樣量(B),流速(C)三個(gè)因素設(shè)計(jì)正交試驗(yàn),篩選葡聚糖凝膠柱分離硝碘酚腈脂質(zhì)體的最佳條件,收集脂質(zhì)體流出液,使用HPLC測(cè)定硝碘酚腈含量,計(jì)算包封率。結(jié)果：使用100-300μm的葡聚糖凝膠G-50作為層析基質(zhì),葡聚糖凝膠柱內(nèi)徑與填充高度比：2：7,加樣量：1.0mL,流速：1mL-min-1。洗脫曲線顯示,脂質(zhì)體洗脫時(shí)間為4-15min。收集流出液用10%Trixton-100溶液破乳,用HPLC測(cè)定硝碘酚腈含量。結(jié)論：本試驗(yàn)方法制得脂質(zhì)體粒徑分布較均勻,使用葡聚糖凝膠柱分離硝碘酚腈脂質(zhì)體出峰時(shí)間合適,可以有效分離硝碘酚腈脂質(zhì)體與游離硝碘酚腈。3硝碘酚腈脂質(zhì)體在家兔體內(nèi)藥動(dòng)學(xué)試驗(yàn)方法：選擇健康家兔2組,比較耳緣靜脈注射相同劑量(8.8mg/kg)的硝碘酚腈注射液與硝碘酚腈脂質(zhì)體,分別于Smin, 10min,15min、30min、45min、1h、1.5h、2h、4h、 8h、12h、24h、36h采血,使用上述HPLC方法測(cè)定含量,用3P97軟件分析藥動(dòng)學(xué)模型以及藥動(dòng)學(xué)參數(shù)。結(jié)果：HPLC繪制血液中硝碘酚腈標(biāo)準(zhǔn)曲線為：y=27547x-31146,R2=0.9995。線性范圍均為6.25-400μg/mL。硝碘酚腈注射液和硝碘酚腈脂質(zhì)體耳緣靜脈注射后的血藥濃度-時(shí)f間分布均符合—級(jí)吸收二室模型。硝碘酚腈注射液在家兔體內(nèi)主要?jiǎng)恿W(xué)參數(shù)為：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)。硝碘酚腈脂質(zhì)體在家兔體內(nèi)主要?jiǎng)恿W(xué)參數(shù)：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)。硝碘酚腈脂質(zhì)體消除半衰期延長(zhǎng)(T1/2β)、中央室分布容積(Vc)增大,與硝碘酚腈注射液比較有極顯著差異(p0.01)。結(jié)論：硝碘酚腈脂質(zhì)體在家兔體內(nèi)消除半衰期顯著延長(zhǎng)、中央室分布容積增大,生物利用度升高,揭示其具有緩釋作用。4硝碘酚腈脂質(zhì)體的肝臟靶向性初步研究選擇健康KM小鼠2組,尾靜脈注射相同劑量的硝碘酚腈注射液與硝碘酚腈脂質(zhì)體,分別于15min.30min、45min、1h、2h、4h、8h、12h、24h解剖小鼠,采集肝臟組織,使用上述HPLC分析方法進(jìn)行含量測(cè)定。結(jié)果HPLC繪制肝臟組織標(biāo)準(zhǔn)曲線為：y=26156x+36621,R2=0.9996。線性范圍為6.25～400μg/mL。硝碘酚腈注射液與硝碘酚腈脂質(zhì)體肝臟中AUC注=87.00(μg/mL)*h, AUC脂=197.29(μg/mL*h,肝臟相對(duì)攝取率re=2.27。結(jié)論：硝碘酚腈脂質(zhì)體中肝臟re=2.27,AUC 注與AUC脂差異極顯著,表明具有良好的靶向性�？偨Y(jié)：本課題篩選的硝碘酚腈脂質(zhì)體制備工藝合理,所制備出的脂質(zhì)體具有較好的藥動(dòng)學(xué)特征及肝臟靶向性,揭示其可能有效的改善硝碘酚腈的副作用與生物利用度。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S859.795

【參考文獻(xiàn)】

中國(guó)期刊全文數(shù)據(jù)庫(kù) 前10條

1 張曉靜;張頻;;紫杉醇新劑型的開(kāi)發(fā)及臨床研究進(jìn)展[J];癌癥進(jìn)展;2007年01期

2 周紅蕾;李春玲;王貴平;侯加法;;脂質(zhì)體作為疫苗免疫佐劑的應(yīng)用研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2006年02期

3 李晶;;動(dòng)物寄生蟲(chóng)病免疫預(yù)防的研究進(jìn)展[J];山東畜牧獸醫(yī);2014年12期

4 葛紅霞;;淺談牛羊肝片吸蟲(chóng)病的防治措施[J];農(nóng)村實(shí)用科技信息;2014年04期

5 謝鳳云;張欽凱;王百川;潘慶杰;;肝片吸蟲(chóng)的特性及肝片吸蟲(chóng)病的預(yù)防與治療[J];畜牧與飼料科學(xué);2014年01期

6 武鑫朋;孫李妍;慕宏杰;楊紹梅;魏俊花;周繡棣;陳大全;;Box-Behnken效應(yīng)面法優(yōu)化牛蒡苷pH敏感脂質(zhì)體處方[J];齊魯藥事;2011年11期

7 張愛(ài)軍;封新;馬小亞;車(chē)曉俠;閆志勇;;辣椒素脂質(zhì)體凝膠劑的制備與釋放度的測(cè)定[J];西北藥學(xué)雜志;2011年04期

8 范云鵬;王德云;胡元亮;劉家國(guó);高煥;王遠(yuǎn)壘;郭利偉;;正交試驗(yàn)優(yōu)選黃芪多糖脂質(zhì)體的制備工藝[J];中草藥;2011年03期

9 錢(qián)德興,梁秀峰,向定輝;硝碘酚腈、丙硫咪唑驅(qū)除奶山羊消化道蠕蟲(chóng)的效果[J];中國(guó)獸醫(yī)科技;1991年07期

10 高曉黎,季興梅;葡聚糖凝膠柱色譜法測(cè)定脂質(zhì)體包封率的條件篩選[J];中國(guó)藥學(xué)雜志;2003年07期

中國(guó)碩士學(xué)位論文全文數(shù)據(jù)庫(kù) 前1條

1 祝玉祥;紫杉醇磁性脂質(zhì)體制備及動(dòng)物實(shí)驗(yàn)研究[D];東南大學(xué);2006年

，

本文編號(hào)：2143304