【作者】 王娜；

【导师】 赵春景； 张景勍；

【作者基本信息】 重庆医科大学 ， 药理学， 2010， 硕士

【摘要】 尿酸酶(Uricase, UOX)在嘌呤代谢途径中有重要的作用,它以分子氧作为受体催化尿酸的分解。大多数哺乳动物体内含有UOX,可将核酸组成单位中嘌呤核苷酸的代谢产物尿酸分解成溶解度比尿酸高的尿囊素;而人体内缺乏UOX,嘌呤分解代谢只能生成尿酸,随后尿酸经肾脏排泄,这就导致人体内血尿酸的浓度高于其他哺乳动物。当尿酸生成速度超过肾脏的排泄能力时,血浆尿酸水平会显著升高,造成高尿酸血症。UOX可以显著降低血浆中的尿酸水平,并可以治疗由高尿酸血症继发引起的痛风和肾脏疾病等。脂质纳米粒作为药物的载体,具有缓释性,靶向性,降低药物毒性等特点。本文制备了UOX脂质纳米粒并对其理化性质进行研究,以期延长UOX在体内的作用时间,提高酶的活性,增加稳定性,降低免疫原性和抗原性。方法:1.采用葡聚糖凝胶G-200层析柱分离脂质纳米粒与游离药物,用考马斯亮兰G-250染色法测定蛋白质的含量,从而测定包封率。2.采用逆向蒸发法制备UOX脂质纳米粒。运用单因素设计,筛选UOX脂质纳米粒的最佳处方。3.通过测定酶的活性,考察UOX脂质纳米粒和UOX的最适温度、最适pH值及米氏常数(Km)。采用荧光分光光度法对含有异硫氰酸荧光素(FITC)的空白脂质体、UOX脂质体以及UOX进行荧光扫描,考察UOX与膜的相互作用。4.通过测定酶的活性,考察UOX脂质纳米粒和UOX的贮存稳定性、热稳定性、酸碱稳定性、抗胰酶水解能力和部分有机化合物对尿酸酶的酶活力的影响。5.分别以UOX脂质纳米粒和UOX作为抗原免疫大鼠,制备抗UOX血清,通过ELISA法进行血清抗UOX抗体效价检测,比较大鼠体内的免疫原性。6.采用次黄嘌呤500 mg/kg和氧嗪酸钾100 mg/kg构建大鼠高尿酸血症动物模型,对UOX脂质纳米粒进行体内初步药效学研究。结果:1.采用葡聚糖凝胶G-200层析柱分离脂质纳米粒与游离药物。UOX的凝胶层析柱平均回收率为98.29%。表明该法操作简便,灵敏度高,重现性好,专属性强,能准确测定UOX脂质纳米粒中UOX的含量及包封率。2.优化工艺制备得到UOX脂质纳米粒的最佳处方为:醚水比为3:1;SPC:CH=1:1;投药量为2 mg。在最佳处方条件下,平均包封率为64.24%,平均粒径为206.73 nm,多分散系数为0.247,Zeta电位为-37.33 mV。3.UOX脂质纳米粒与UOX的最适温度均为40℃,并且UOX脂质纳米粒的活性明显高于游离UOX。在最适温度下,UOX脂质纳米粒的最适pH值为8.0,而游离UOX的最适pH值为8.5,且UOX脂质纳米粒的活性明显高于游离UOX。UOX脂质纳米粒的Km低于游离UOX,说明UOX脂质纳米粒与底物之间的亲和力增加。采用荧光分光光度法对含有异硫氰酸荧光素(FITC)的空白脂质体、UOX脂质体以及UOX进行荧光扫描,结果表明UOX分子与FITC之间存在静电作用和疏水作用,因而UOX脂质纳米粒中UOX与脂质膜有相互作用。4.稳定性实验结果表明,在贮存稳定性、热稳定性、酸碱稳定性、抗胰酶水解能力和部分有机化合物对酶活力的影响方面,UOX脂质纳米粒的稳定性均明显高于UOX。5.免疫原性实验结果表明,以UOX脂质纳米粒和UOX为抗原制备的大鼠抗血清效价分别为1: 500和1: 8000,说明UOX脂质纳米粒能有效降低免疫原性。6.体内初步药效学实验表明,注射UOX脂质纳米粒后,尿酸从高浓度(489.61μmol/L)降到人的正常浓度(240μmol/L)时间低于3 h,然而在注射UOX后从高浓度降到人的正常浓度则多用了6 h。UOX脂质纳米粒抑制尿酸水平高达88.72%。在静脉注射UOX纳米粒后,尿酸水平在12 h时达到最低水平(55.24μmol/L),与此同时,UOX组降到189.94μmol/L,模型组则只降到271.67μmol/L。体内药效学研究表明在大鼠模型上UOX脂质纳米粒能更明显的降低尿酸水平。结论:本文成功制备了UOX脂质纳米粒,其包封率高,提高了酶的活性,增加了稳定性,降低了免疫原性,并在体内可以明显降低血尿酸的水平。更多还原

【Abstract】 Uricase (UOX) plays an important role in the metabolism of purine in vivo, and it can catalyze uric acid into allantoin. Unlike nearly all other mammals, humans lack the enzyme uricase, which catalyzes the oxidation of uric acid to allantoin, a more soluble product that is readily excreted in the urine. The lack of uricase in humans results in plasma uric acid concentrations that are much higher than in most mammals. When less uric acid is excreted than is produced, the plasma urate concentration rises and may cause hyperuricemia. Treatment with uricase can dramatically lower the levels of urate in plasma and has potential for therapy of gout and uric acid nephropathy resulting from hyperuricemia.Liposomes are commonly recognized as a superior drug delivery vehicles which show the advantages of delayed-release, target effects and lower drug toxicity.In this study, lipid vesicles containing uricase were designed and prepared in an attempt that a general strategy to improve its pharmacokinetics characters such as half-life, increase its enzyme activity, stabilize and deliver multimeric enzymes through entrapping them in the lipid vesicles might be affirmed. Meanwhile it can reduce immunogenicity and antigenicity.Method:1. Uricase-containing lipid vesicles were separated from free uricase by gel exclusion chromatography performed with Sephadex G-200 column. And then the protein content was determined by the Coomassie blue method initially reported by Bradford.2. UOXLVs were prepared by reverse-phase evaporation method. Single factor tests were conducted to optimize the reaction conditions.3. Determinating the optimum temperature, optimum pH and the Michaelis constant of UOXLVs and UOX by measuring the enzyme activity. The FITC fluorescence in lipid vesicle suspensions was measured in the presence and absence of uricase using a fluorescence Spectrophotometer to examine a lipid vesicle–uricase interaction.4. Measuring the storage stability, thermal stability, pH stability, stability to proteolytic digestion and effect of various chemical agents on uricase activity of UOXLVs and UOX by measuring the enzyme activity.5. The rat antiserum was prepared by inoculating the rat with UOXLVs and UOX. The antibody against UOX was measured through indirect ELISA using UOXLVs or UOX as coating antigen to analyze the immunogenicity of UOXLVs and UOX in rats.6. Hypoxanthine (at the dose of 500 mg/kg) combined with oxonic acid potassium salt (at the dose of 100 mg/kg) were used to prepare the hyperuricemia rat model.Result:1. Uricase-containing lipid vesicles were separated from free uricase by gel exclusion chromatography performed with Sephadex G-200 column, and the average recovery rate of gel column of UOX is 98.29%. The method is simple, sensitive, and reproducible, which could accurately determinate the contents of UOX and entrapment efficiency in UOXLVs.2. The best formula for UOXLVS by optimize process including: the volume ratio of diethyl ether and buffer was worked out to be 3:1; the molar ratio of SPC and CH is 1:1; and the dosage is 2 mg. In the condition of the optimal formulation, UOX was encapsulated is 64.24%, average particle size is 206.73 nm, the polydispersity index is 0.247, and zeta potential is -37.33 mV.3. The results show that, the optimum temperature of UOXLVs and free uricase were the same as 40℃. The temperature range at which UOXLVs remained a higher activity than that of free uricase. UOXLVs did shift the optimal pH of enzyme activity from 8.5 (for free uricase) to 8.0, and the activity of UOXLVs was significantly higher than free UOX. The Km of UOXLVs is lower than free UOX, which shows the affinity increasing between UOXLVs and the substrate.The FITC fluorescence in lipid vesicle suspensions was measured in the presence and absence of uricase using a fluorescence Spectrophotometer to examine a lipid vesicle–uricase interaction. It was found that the FITC and uricase molecules competitively interacted with the interfacial region of zwitterionic lipid vesicle membranes through electrostatic and hydrophobic interactions.4. Stability experiments showed, in the aspect of its storage stability, thermal stability, pH stability, resistance to trypsin hydrolysis and some organic compounds the effects on the enzyme activity, all the stability of UOXLVs was higher than that of UOX.5. The results of experimental on Immunogenicity showed that the antiserum titer which was based on the antigen of UOXLVs and UOX was separately 1: 500 and 1: 8000, indicating that UOXLVs can reduce immunogenicity effectively.6. Preliminary pharmacodynamic study in vivo show that it took less than 3 h to decrease the uric acid from the high concentration (489.61μmol/L) to the normal uric acid level (240μmol/L in human) after intravenous injection of UOXLVs, while the time was about more than 6 h in the case of free uricase group. UOXLVs suppressed uric acid levels up to 88.72%. The lowest uric acid level (55.24μmol/L) was achieved at 12 h after intravenous injection of UOXLVs (compared with the 189.94μmol/L in the case of the free uricase group and 271.67μmol/L in the model group). In vivo pharmacodynamic studies explicitly suggested that UOXLVs distinctively decreased the uric acid level on the hyperuricemia rat model.Conclusion:The uricase-containing lipid vesicles with high entrapment efficiency were prepared and characterized in terms of their stability and activity. All the results obtained showed that the UOXLVs were advantageous to maintain higher stability and activity than that of free uricase. The immunogenicity of free uricase can be notablely decreased when delivered in the form of UOXLVs. Moreover, in vivo pharmacodynamic studies demonstrated that UOXLVs could decrease the uric acid level obviously and reduce immunogenicity protection at the same time.更多还原