UOMVLs体外活性、代谢动力学及抗高尿酸血症的研究

发布时间：2018-06-08 10:26

本文选题：尿酸酶多囊脂质体 + 活性　；参考：《重庆医科大学》2014年博士论文

【摘要】：本文构建的尿酸酶多囊脂质体(lipid-based multivesicular carrier loaded with uricase, UOMVLs),是一种酶类药物的新型制剂,为了考察其临床应用价值,有必要对其进行药理学方面的评价。本文考察了UOMVLs的酶代谢动力学、药物代谢动力学、药效学,还考察了UOMVLs的免疫原性、溶血性和血管刺激性。结果显示UOMVLs能克服尿酸酶在体内活性低,半衰期较短,易诱发机体免疫反应等缺点。UOMVLs可以在体内保持较长时间的活性,有效地降低高尿酸血症大鼠体内的尿酸水平,而无溶血性和刺激性,为UOMVLs的临床应用价值提供了理论基础。尿酸氧化酶(uricase, UOX)在嘌呤代谢途径中起着十分重要的作用,它能催化底物尿酸,使其代谢分解生成尿囊素。大多数哺乳动物的体内都含有尿酸酶,可以将嘌吟核苷酸代谢产物尿酸分解,但是在人体内却缺乏有生物活性的尿酸酶,尿酸只能通过排泄的方式去除。由于尿酸及其盐类在水中的溶解度非常低,当血液中积累过多就会导致高尿酸血症,继而导致痛风综合症,肾结石等。尿酸酶可以通过将尿酸氧化成溶解度更高的尿囊素来降低患者体内的尿酸浓度,从而消除痛风石沉积、肾结石等症状。多囊脂质体(multivescular liposomes, MVLs)主要用于递送亲水性药物,具有缓释性,降低药物毒性等作用。本文主要研究内容包括以下几部分：第一部分,采用复乳法构建了尿酸酶多囊脂质体,并对其生化特性进行了考察,包括包封率、粒径分布、Zeta电位、形态结构、UOMVLs和游离尿酸酶活性达最大时的温度和pH。构建的尿酸酶多囊脂质体为非同心圆囊泡结构,其包封率为(63.75±3.65)%,粒径大小为(22.5±1.70)μm, Zeta电位为(-41.81±6.59) mV, UOMVLs和游离尿酸酶活性达最大时的温度为40℃,pH为8.0。第二部分考察了高温、低温、酸碱度、胰蛋白酶对UOMVLs和游离尿酸酶活性的影响,并对UOMVLs和游离尿酸酶活性在模拟血浆中的活性变化进行了考察。结果显示上述因素对UOMVLs的影响均小于游离酶。第三部分考察了UOMVLs的相关机制。采用荧光分光光度法对UOMVLs和游离尿酸酶活性提高的机制进行考察,结果显示尿酸酶的结构在高温下易发生改变,而UOMVLs中尿酸酶通过与脂质膜作用增强了尿酸酶的热稳定性；结果还显示经多囊脂质体包裹后的尿酸酶活性增加。通过对UOMVLs在pH7.4条件下形态学改变的观察,推测UOMVLs释放的主要方式是胞裂外排和直接裂解。第四部分考察了UOMVLs的代谢动力学,包括酶学代谢动力学、体内静脉注射UOMVLs的代谢动力学和体内皮下注射UOMVLs的代谢动力学。酶学代谢动力学结果为：UOMVLs和游离尿酸酶的米氏常数Km值分别为(12.29±3.5)和(14.27±3.1)说明相对于游离尿酸酶,UOMVLs对底物尿酸的亲和力较好。UOMVLs的体内代谢动力学考察中,将所得的大鼠体内血药浓度使用DAS 2.1.1药动学程序软件进行统计分析,得到了静脉注射UOMVLs的主要药代动力参数为：AUC(0-72h)为(446.27±70.60) U/L-h, MRT(0-72h)为(4.17±0.31)h,Cmax为(73.04±6.35) U/L, Tmax为(1.00±0.00)h；静脉注射游离尿酸酶的主要药代动力参数为：AUC(0-72h)为(27.80±10.36)U/L·h,MRT(0-72h)为(0.91±0.20)h：,Cmax为(13.91±6.03)U/L,Tmax为(0.67±0.29)h,生物等效性分析结果显示UOMVLs和游离尿酸酶生物不等效,UOMVLs经静脉注射生物利用度为1605.3%。皮下注射UOMVLs的主要药代动力参数为：AUC(0-72h)为(179.13±17.76) U/L·h,MRT(0-72h)为(4.37±0.52) h, Cmax为(60.33±6.42) U/L,Tmax为(4.00±0.88)h；皮下注射游离尿酸酶的主要药代动力参数为AUC(0-72h)为(87.61±10.54)U/L·h, MRT(0-72h)为(2.26±0.03)h, Cmax为(38.26±6.03) U/L,Tmax为(1.00±0.00)h,生物等效性分析结果显示UOMVLs和游离尿酸酶生物不等效,UOMVLs经皮下注射生物利用度为204.5%。第五部分考察了静脉注射和皮下注射给药后UOMVLs在高尿酸血症的大鼠体内的药效学。UOMVLs较游离尿酸酶表现出了非常明显的降尿酸优势。高尿酸血症大鼠通过静脉注射UOMVLs后,体内尿酸水平达到大鼠正常尿酸水平所需时间为8.96 h,所用时间仅为游离尿酸酶组的0.2倍。皮下注射UOMVLs也表现出了明显的降尿酸效果。第六部分考察了UOMVLs的免疫原性。用ELISA法对样品进行测定,结果显示给大鼠静脉连续给药6周后,UOMVLs组大鼠体内产生的抗体量明显低于游离尿酸酶；抗体效价结果也显示UOMVLs能有效地降低尿酸酶的免疫原性。第七部分考察了UOMVLs的溶血性和血管刺激性。结果表明,UOMVLs溶血率很低,几乎无浴血性；刺激性实验结果显示UOMVLs无刺激性。溶血性和血管刺激性结果表明UOMVLs可用于注射给药。
[Abstract]:The lipid-based multivesicular carrier loaded with uricase (UOMVLs) is a new preparation of the enzyme drug. In order to investigate its clinical application value, it is necessary to evaluate its pharmacology. In this paper, the enzyme metabolism kinetics, pharmacokinetics, pharmacodynamics, pharmacodynamics, and pharmacodynamics of UOMVLs are also investigated. The immunogenicity, hemolysis and vascular irritation of UOMVLs were investigated. The results showed that UOMVLs could overcome the disadvantages of low activity of uricase in vivo, short half life and easy to induce the immune response of the body..UOMVLs could keep the activity in the body for a long time and effectively reduce the level of uric acid in the hyperuricemia rats, without hemolysis and stimulation. It provides a theoretical basis for the clinical application of UOMVLs. Uricase (UOX) plays a very important role in the metabolic pathway of purine. It can catalyze the substrate uric acid and decompose it into allantoin. Most mammals have urate in vivo, which can decompose uric acid by the metabolites of purinyin nucleotides. It is in the human body that there is a lack of bioactive uricase, and uric acid can only be removed by excretion. Because the solubility of uric acid and its salts is very low in water, hyperuricemia can lead to hyperuricemia in the blood, resulting in gout syndrome, kidney stones and so on. Uricase can be oxidized to higher solubility by uricase. Allantoin to reduce the concentration of uric acid in the patient's body, thus eliminating the symptoms of gout deposits and kidney stones. Multivescular liposomes (MVLs) is mainly used to deliver hydrophilic drugs, which have the effect of slow release and lower drug toxicity. The main contents of this paper include the following parts: the first part, using the compound emulsion method The biochemical characteristics of urate polycystic liposomes were investigated, including the encapsulation efficiency, the particle size distribution, the Zeta potential, the morphological structure, the temperature of the UOMVLs and the free urate activity at the maximum temperature and the pH. constructed by the urate polycystic liposome as a non concentric circular vesicle structure, the encapsulation efficiency was (63.75 + 3.65)%, the size of the particle was (22.5 + 1.70) m, Z The ETA potential was (-41.81 + 6.59) mV, UOMVLs and free urate activity reached the maximum temperature of 40, and pH was 8.0. second to investigate the effects of high temperature, low temperature, pH, trypsin on the activity of UOMVLs and free urate, and the activity of UOMVLs and free urate activity in simulated plasma was investigated. The results showed that the activity of the enzyme in the simulated plasma was changed. The effect of these factors on UOMVLs was less than that of free enzyme. Third part of the mechanism of UOMVLs was investigated. The mechanism of UOMVLs and free urate activity was investigated by fluorescence spectrophotometry. The results showed that the structure of urate was easily changed at high temperature, and urate in UOMVLs enhanced uric acid through the action of lipid membrane. The thermal stability of the enzyme showed that the activity of urate after polycystic liposome was increased. By observing the morphological changes of UOMVLs under pH7.4 conditions, it was suggested that the main modes of release of UOMVLs were the fissure discharge and direct lysis. The fourth part investigated the metabolic kinetics of UOMVLs, including enzyme metabolism kinetics, and intravenous infusion in vivo. The metabolic kinetics of UOMVLs and the metabolic kinetics of UOMVLs were injected subcutaneously in the body. The enzyme metabolic kinetics results were that the mite constant Km value of UOMVLs and free uric acid enzyme was (12.29 + 3.5) and (14.27 + 3.1), respectively, relative to the free urate, and UOMVLs was in the metabolic kinetics of the affinity and force of the substrate uric acid in vivo, The blood drug concentration in the rat was analyzed by DAS 2.1.1 pharmacokinetic program software, and the main pharmacokinetic parameters of UOMVLs were obtained: AUC (0-72h) was (446.27 + 70.60) U/L-h, MRT (0-72h) was (4.17 + 0.31) h, Cmax was (73.04 + 6.35) U/L, Tmax was (1 + 0), and the main intravenous injection of free urate The pharmacokinetic parameters were as follows: AUC (0-72h) was (27.80 + 10.36) U/L / h, MRT (0-72h) was (0.91 + 0.20) h:, Cmax was (13.91 + 6.03) U/L, Tmax was (0.67 + 0.29) H. Bioequivalence analysis showed that UOMVLs and free urate organisms were not equivalent. AUC (0-72h) is (179.13 + 17.76) U/L. H, MRT (0-72h) is (4.37 + 0.52) h, Cmax is (60.33 + 6.42) U/L, Tmax is (4 + 0.88) h, and the main pharmacokinetic parameter of subcutaneous injection of free uric acid enzyme is AUC (87.61 + 10.54) The results of sex analysis showed that UOMVLs and free uricase were not equivalent, and the bioavailability of UOMVLs by subcutaneous injection was 204.5%. fifth. The pharmacodynamic.UOMVLs of UOMVLs in hyperuricemia rats after intravenous and subcutaneous injection showed a very obvious lower uricase advantage than that of free uric acid enzyme. After intravenous injection of UOMVLs, the time required for the uric acid level in the body to reach the normal uric acid level of the rat was 8.96 h, and the time used was only 0.2 times that of the free urate group. The subcutaneous injection of UOMVLs also showed an obvious effect of reducing uric acid. The sixth part examined the immunogenicity of UOMVLs. The samples were measured by ELISA, and the results showed that After 6 weeks of continuous intravenous administration, the amount of antibody produced in the UOMVLs group was significantly lower than that of the free urate, and the antibody titer results also showed that UOMVLs could effectively reduce the immunogenicity of urate. The seventh part examined the hemolysis and vascular irritation of UOMVLs. The results showed that the hemolysis rate of UOMVLs was very low, and the stimulation was almost no bath blood. The results of sexual experiments showed that UOMVLs was not irritant. Hemolysis and vascular irritation showed that UOMVLs could be used for injection.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

【相似文献】