本文選題：尿酸酶多囊脂質(zhì)體 + 活性��； 參考：《重慶醫(yī)科大學(xué)》2014年博士論文

【摘要】：本文構(gòu)建的尿酸酶多囊脂質(zhì)體(lipid-based multivesicular carrier loaded with uricase, UOMVLs),是一種酶類藥物的新型制劑,為了考察其臨床應(yīng)用價(jià)值,有必要對(duì)其進(jìn)行藥理學(xué)方面的評(píng)價(jià)。本文考察了UOMVLs的酶代謝動(dòng)力學(xué)、藥物代謝動(dòng)力學(xué)、藥效學(xué),還考察了UOMVLs的免疫原性、溶血性和血管刺激性。結(jié)果顯示UOMVLs能克服尿酸酶在體內(nèi)活性低,半衰期較短,易誘發(fā)機(jī)體免疫反應(yīng)等缺點(diǎn)。UOMVLs可以在體內(nèi)保持較長時(shí)間的活性,有效地降低高尿酸血癥大鼠體內(nèi)的尿酸水平,而無溶血性和刺激性,為UOMVLs的臨床應(yīng)用價(jià)值提供了理論基礎(chǔ)。尿酸氧化酶(uricase, UOX)在嘌呤代謝途徑中起著十分重要的作用,它能催化底物尿酸,使其代謝分解生成尿囊素。大多數(shù)哺乳動(dòng)物的體內(nèi)都含有尿酸酶,可以將嘌吟核苷酸代謝產(chǎn)物尿酸分解,但是在人體內(nèi)卻缺乏有生物活性的尿酸酶,尿酸只能通過排泄的方式去除。由于尿酸及其鹽類在水中的溶解度非常低,當(dāng)血液中積累過多就會(huì)導(dǎo)致高尿酸血癥,繼而導(dǎo)致痛風(fēng)綜合癥,腎結(jié)石等。尿酸酶可以通過將尿酸氧化成溶解度更高的尿囊素來降低患者體內(nèi)的尿酸濃度,從而消除痛風(fēng)石沉積、腎結(jié)石等癥狀。多囊脂質(zhì)體(multivescular liposomes, MVLs)主要用于遞送親水性藥物,具有緩釋性,降低藥物毒性等作用。本文主要研究內(nèi)容包括以下幾部分：第一部分,采用復(fù)乳法構(gòu)建了尿酸酶多囊脂質(zhì)體,并對(duì)其生化特性進(jìn)行了考察,包括包封率、粒徑分布、Zeta電位、形態(tài)結(jié)構(gòu)、UOMVLs和游離尿酸酶活性達(dá)最大時(shí)的溫度和pH。構(gòu)建的尿酸酶多囊脂質(zhì)體為非同心圓囊泡結(jié)構(gòu),其包封率為(63.75±3.65)%,粒徑大小為(22.5±1.70)μm, Zeta電位為(-41.81±6.59) mV, UOMVLs和游離尿酸酶活性達(dá)最大時(shí)的溫度為40℃,pH為8.0。第二部分考察了高溫、低溫、酸堿度、胰蛋白酶對(duì)UOMVLs和游離尿酸酶活性的影響,并對(duì)UOMVLs和游離尿酸酶活性在模擬血漿中的活性變化進(jìn)行了考察。結(jié)果顯示上述因素對(duì)UOMVLs的影響均小于游離酶。第三部分考察了UOMVLs的相關(guān)機(jī)制。采用熒光分光光度法對(duì)UOMVLs和游離尿酸酶活性提高的機(jī)制進(jìn)行考察,結(jié)果顯示尿酸酶的結(jié)構(gòu)在高溫下易發(fā)生改變,而UOMVLs中尿酸酶通過與脂質(zhì)膜作用增強(qiáng)了尿酸酶的熱穩(wěn)定性；結(jié)果還顯示經(jīng)多囊脂質(zhì)體包裹后的尿酸酶活性增加。通過對(duì)UOMVLs在pH7.4條件下形態(tài)學(xué)改變的觀察,推測UOMVLs釋放的主要方式是胞裂外排和直接裂解。第四部分考察了UOMVLs的代謝動(dòng)力學(xué),包括酶學(xué)代謝動(dòng)力學(xué)、體內(nèi)靜脈注射UOMVLs的代謝動(dòng)力學(xué)和體內(nèi)皮下注射UOMVLs的代謝動(dòng)力學(xué)。酶學(xué)代謝動(dòng)力學(xué)結(jié)果為：UOMVLs和游離尿酸酶的米氏常數(shù)Km值分別為(12.29±3.5)和(14.27±3.1)說明相對(duì)于游離尿酸酶,UOMVLs對(duì)底物尿酸的親和力較好。UOMVLs的體內(nèi)代謝動(dòng)力學(xué)考察中,將所得的大鼠體內(nèi)血藥濃度使用DAS 2.1.1藥動(dòng)學(xué)程序軟件進(jìn)行統(tǒng)計(jì)分析,得到了靜脈注射UOMVLs的主要藥代動(dòng)力參數(shù)為：AUC(0-72h)為(446.27±70.60) U/L-h, MRT(0-72h)為(4.17±0.31)h,Cmax為(73.04±6.35) U/L, Tmax為(1.00±0.00)h；靜脈注射游離尿酸酶的主要藥代動(dòng)力參數(shù)為：AUC(0-72h)為(27.80±10.36)U/L·h,MRT(0-72h)為(0.91±0.20)h：,Cmax為(13.91±6.03)U/L,Tmax為(0.67±0.29)h,生物等效性分析結(jié)果顯示UOMVLs和游離尿酸酶生物不等效,UOMVLs經(jīng)靜脈注射生物利用度為1605.3%。皮下注射UOMVLs的主要藥代動(dòng)力參數(shù)為：AUC(0-72h)為(179.13±17.76) U/L·h,MRT(0-72h)為(4.37±0.52) h, Cmax為(60.33±6.42) U/L,Tmax為(4.00±0.88)h；皮下注射游離尿酸酶的主要藥代動(dòng)力參數(shù)為AUC(0-72h)為(87.61±10.54)U/L·h, MRT(0-72h)為(2.26±0.03)h, Cmax為(38.26±6.03) U/L,Tmax為(1.00±0.00)h,生物等效性分析結(jié)果顯示UOMVLs和游離尿酸酶生物不等效,UOMVLs經(jīng)皮下注射生物利用度為204.5%。第五部分考察了靜脈注射和皮下注射給藥后UOMVLs在高尿酸血癥的大鼠體內(nèi)的藥效學(xué)。UOMVLs較游離尿酸酶表現(xiàn)出了非常明顯的降尿酸優(yōu)勢。高尿酸血癥大鼠通過靜脈注射UOMVLs后,體內(nèi)尿酸水平達(dá)到大鼠正常尿酸水平所需時(shí)間為8.96 h,所用時(shí)間僅為游離尿酸酶組的0.2倍。皮下注射UOMVLs也表現(xiàn)出了明顯的降尿酸效果。第六部分考察了UOMVLs的免疫原性。用ELISA法對(duì)樣品進(jìn)行測定,結(jié)果顯示給大鼠靜脈連續(xù)給藥6周后,UOMVLs組大鼠體內(nèi)產(chǎn)生的抗體量明顯低于游離尿酸酶；抗體效價(jià)結(jié)果也顯示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。結(jié)果表明,UOMVLs溶血率很低,幾乎無浴血性；刺激性實(shí)驗(yàn)結(jié)果顯示UOMVLs無刺激性。溶血性和血管刺激性結(jié)果表明UOMVLs可用于注射給藥。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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