【作者】 金圣煊；

【导师】 毕殿洲； 邓意辉；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2005， 博士

【摘要】 高效抗逆转录病毒疗法（HAART）能抑制HIV复制，明显降低了感染人群的发病率和死亡率，但仍存在药物难以到达的病毒贮库、无法彻底清除HIV、长期应用易产生多药耐药毒株等诸多问题。因此继续寻找高效低毒抗HIV药物，以及针对HIV贮库开发新的给药系统是当前国际上极为关注的热门领域。本文以齐多夫定（AZT）为原料药，进行亲脂性前药改性，将药物有效地包封于脂质体中，利用脂质体的被动靶向特点，可将药物导入巨噬细胞这—HIV的重要贮库；利用右旋糖酐硫酸酯（DS）能够与HIV外膜蛋白gp120特异性结合的特点，首次合成了DS的脂肪酸衍生物，用于脂质体表面修饰，制备主动靶向脂质体，可将药物传递至HIV感染的细胞，阻断HIV感染细胞与正常细胞的融合作用；在此基础上，用聚乙二醇（PEG）衍生物包衣，构建立体稳定主动靶向脂质体，使之既保持脂质体的长循环特点，又具主动寻靶能力，可将药物传递至HIV感染的深层部位，如脑等。具体研究内容如下：合成了双重前药——齐多夫定肉豆蔻酸酯（AZT-M），并测定有关理化性质。AZT-M为白色粉末，熔点为56℃，不溶于水，易溶于氯仿等非极性有机溶剂，在正辛醇—pH7.4磷酸盐缓冲液中的表观油水分配系数logP_app为5.30（AZT为0.07）。采用改良乙醇注入法结合微射流技术整粒，制备AZT-M普通脂质体（AZT-ML）。较为系统地考察了微射流仪匀化压力和循环次数对脂质体粒径的影响，并对磷脂种类和用量、药脂比、胆固醇用量和水化介质的种类等处方因素进行筛选，得到优化工艺和处方，制得平均粒径在100nm以下，包封率在98％以上的AZT-ML；选择合适的冻干工艺和保护剂，制得AZT-ML冻干制剂，4℃避光贮存稳定性良好。首次合成肉豆蔻酰化DS（myristoyl DS，DSM）和棕榈酰化DS（palmitoyl DS，DSP），考察了DSM和DSP的加入方式、用量对AZT-ML的粒径、Zeta电位和包衣效率等的影响，结果表明内加法优于外加法，内加法可使DSM和DSP的包衣效率达到90％以上；随着DSM或DSP浓度的提高，AZT-ML的粒径逐渐增大，当浓度过高（2mg／mL）时，会造成脂质体聚集；DSM或DSP表面修饰后，AZT-ML Zeta电位下降；DSM或DSP表面修饰能在一定程度上抑制Triton X-100诱发的钙黄绿素泄漏，提高了脂质体的膜稳定性。采用外加法，用聚乙二醇单甲醚（2000）胆固醇琥珀酸酯（CHS-PEG）对DSM-AZT-ML进行包衣，制备立体稳定主动靶向脂质体（PEG-DSM-AZT-ML），PEG包衣后可增加脂质体的物理稳定性。为了预测AZT-M在体内的降解趋势以及评价脂质体包裹对AZT-M稳定性的影响，比较脂质体包裹前后AZT-M在不同pH缓冲液、不同种动物血浆以及大鼠组织匀浆中的降解情况。结果表明，AZT-M在pH 4.0～9.0缓冲液中的降解机理为专属碱催化和水催化降解，随着介质pH的增大、温度的升高，降解速率加快，在pH 4.0～7.0降解缓慢：AZT-M在混悬液和脂质体中的降解趋势一致，在pH 4.0～7.4时降解速率基本接近，当pH 9.0、温度为80℃时，AZT-M在脂质体中的降解速率明显低于其在混悬液中的降解速率。AZT-M在血浆和组织匀浆中的降解过程符合伪一级动力学规律，血浆和组织匀浆中的酶能显著加速AZT-M的降解，且存在明显的种间差异，AZT-M在血浆中的降解速率依次为小鼠＞大鼠（?）兔。经脂质体包裹后，明显提高了AZT-M的稳定性，药物在血浆和组织匀浆中的半衰期顺序依次为PEG-DSM-AZT-ML＞DSM-AZT-ML≈AZT-ML＞AZT-M。以AZT为指标成分，评价大鼠静脉注射AZT溶液剂和AZT-M脂质体后药动学及组织分布情况。药动学研究结果表明，AZT-ML、DSM-AZT-ML和PEG-DSM-AZT-ML的AUC_0-∞分别为AZT的1.6、1.9和2.3倍；总体清除率(CL_tot)则由AZT的16.6±2.3 mL／min分别下降为10.8±2.2 mL／min、8.6±1.4 mL／min和7.1±1.1 mL／min，稳态分布体积(V_ss)由AZT的1.2±0.3 L分别下降为0.8±0.2 L、0.8±0.1 L和0.7±0.2 L；PEG-DSM-AZT-ML还能显著延长药物的末端消除半衰期(t_1／2)和平均滞留时间（MRT）。组织分布研究结果表明，AZT-ML给药后可被动靶向网状内皮系统，如60 min时，在脾、肝和肺中的药物浓度分别是AZT溶液剂的6.9、4.7和4．3倍；DSM-AZT-ML给药后在肺中的分布量较高，5 min、60 min和240 min时的药物浓度分别是AZT溶液剂的1.6、9.3和3.6倍；AZT-M脂质体给药后，在脑中的AZT浓度均有了显著提高，且以PEG-DSM-AZT-ML作用最为明显，如给药后60 min时AZT-ML、DSM-AZT-ML和PEG-DSM-AZT-ML在脑中的浓度分别是AZT溶液剂的2.2、2.0和6.1倍。通过台盼蓝染色法测定AZT-M脂质体的细胞毒性，结果发现经脂质体包裹的AZT-M毒性明显降低，其对MT-4细胞的毒性大小依次为PEG-DSM-AZT-ML＜DSM-AZT-ML＜AZT-ML＜AZT-M。以SF₃₃引起的细胞病变为指标，初步评价AZT-M脂质体的抗HIV-1活性，结果发现AZT-M及其脂质体组在所考察的3个浓度中，对SF₃₃感染的MT-4细胞的保护作用均与阳性药AZT相当。更多还原

【Abstract】 Highly active antiretroviral therapy （HAART） has led to a profound decrease in morbidity and mortality in infected people by suppressing HIV replication. However, the eradication of virus does not seen attainable with the present strategies of interventions which is due to two major obstacles: if resistant mutantions appear the virus will escape further treatment, and latent virus reservoirs exist which cannot be reached with the current treatment regimens. Therefore, the search for new promising agents against HIV from various sources and the development of new drug carrier systems which allow drugs targeting HIV reservoirs have become hot topics in the world.In this paper, zidovudine myristate （AZT-M）, a potential double-barrelelled prodrug to AZT and myristic acid, was synthesized, and then encapsulated in three types of liposomes to fulfill targeted drug delivery, namely, （1） conventional liposomes, which could deliver AZT-M to cells of the mononuclear phagocyte system （MPS）, an important reservoir of HIV; （2） active-targeting liposomes, by the modification with dextran sulfate derivatives （DS, which binds HIV-1 envelope glycoprotein gp120 with high affinity）, which might specifically conjugate with HIV-infected cells and inhibit syncytium formation; （3） sterically stabilized active-targeting liposomes, by coating with poly （ethylene glycol） （PEG） polymer, which might be useful in targeting HIV infected deep tissue such as brain.This research work was granted by the National Natural Science Foundation of China with a key project number of 30371694.The contents in detail are as follows:After the synthesis, the physicochemical properties of AZT-M were investigated. AZT-M, a white powder with mp 56℃, is unsoluble in water, but very soluble in non-polar solvents such as chloroform. logP_app （n-octanol/phosphate buffer） for AZT-M was 5.30 at pH 7.4, larger than that of AZT （0.07）. The very high lipophilicity might be helpful for AZT-M to be encapsulated in liposomes.The conventional liposomes containing AZT-M （AZT-ML） were prepared using a modified ethanol injection method followed by homogenization and filtration. The effects of homogenization pressure and number of cycles of Microfluidize on particle size distribution were investigated, and the factors in the formulations such as species of phospholipids and solvents, ratio of drug and lipid, and amount of cholesterol were tested. By optimizing the formulation and technique, AZT-ML with the mean particle size below 100 ran and the entrapment efficiency （EN） above 98% could be obtained. To improve the long term stability, AZT-ML were freeze-dried by the presence of trehalose, an excellent lyoprotectant. The mean particle size and EN of AZT-ML before and after lyophilization were almost the same. No significant physical instability or chemical degradation was observed in the lyophilized AZT-ML power after the storage of 12 months at 4℃.The novel derivatives of dextran sulfate, myristoyl dextran sulfate （DSM） and palmitoyl dextran sulfate （DSP）, were synthesized. The effects of DSM/DSP concentration and the sequence of the addition on the characteristics of modified AZT-ML were investigated. Results showed that above 90％of the coating efficiency was acquired when polymer solution was added to the lipid solution before liposome formation, which was much higher than that when DSM/DSP solution was added to pre-formed AZT-ML. The particle size of modified AZT-ML was increased with the increasing concentration of polymer, suggesting the formation of coating layer on the surface of the liposoms. However, if the concentration of polymer was too high （2 mg/mL）, aggregation of the liposomes may occur. The Zeta potential of modified AZT-ML was decreased as a result of the adsorption of DSM/DSP with negative charge. The effect of DSM/DSP on membrane permeability was studied by measuring the extent of calcein leakage from liposomes with the treatment of Triton X-100. Results indicated that DSM/DSP could improve the stability of liposomal bilayer membranes to some extent.To prolong the in vivo circulation time of DSM-AZTML, monomethoxy polyethyleneglycol succinyl cholesterol （CHS-PEG） was mixed with the pre-formed liposomes. The physical stability of PEG-DSM-AZT-ML was also increased with the increasing concentration of CHS-PEG..Stability of AZT-M in suspension and liposomes was investigated at various pH buffers or in the presence of animal plasma and tissue homogenates. The degradation of AZT-M in both suspension and liposomes followed a pseudo first-order reaction. Little degradation of AZT-M occurred at pH 4.0～7.0. When pH and temperature of the aqueous were raised, degradation rate of AZT-M increased accordingly. There was no significant difference between the degradation rate of AZT-M in suspension and liposomes at pH 4.0～7.4. However, at pH 9.0 and 80℃, the degradation rate of AZT-M in liposomes was slower than that in suspension. AZT-M underwent rapid degradation by esterases in plasma and tissues. The degradation rates of AZT-M among mice, rat and rabbit plasma were significantly different （mice＞rat＞＞rabbit）, which suggested that there might exist species-dependent metabolism for AZT-M. Liposomes can protect the loaded AZT-M from esterases degradation in plasma and tissue homogenates. The half-lives of AZT-M in different liposomal formulations were longer than that in suspension （PEG-DSM-AZT-ML＞DSM-AZT-ML≈AZT-ML＞AZT-M）.The pharmacokinetic profiles and tissue distribution of AZT after i.v. administration of different formulations of liposomal AZT-M in rats compared with AZT solution were investigated. AUC_0-∞ of AZT in AZT-ML, DSM-AZT-ML and PEG-DSM-AZT-ML were 1.6, 1.9 and 2.3-fold higher than that of AZT solution, respectively. Total body clearance (CL_tot) decreased from 16.6±2.3 mL/min （AZT solution） to 10.8±2.2 mL/min （AZT-ML）, 8.6±1.4 mL/min （DSM-AZT-ML） and 7.1±1.1 mL/min （PEG-DSM-AZT-ML）, respectively. Steady distribution volume (V_ss) declined from 1.2±0.3 L （AZT solution） to 0.8±0.2 L （AZT-ML）, 0.8±0.1 L （DSM-AZT-ML） and 0.7±0.2 L （PEG-DSM-AZT-ML）, respectively. Moreover, terminal half life (t_1/2)and mean resident time （MRT） of AZT in PEG-DSM-AZT-ML were significantly prolonged compared with those of AZT solution.Tissue distribution studies confirmed that AZT-ML were rapidly accumulated in organs of RES. The AZT levels 1 h after injection of AZT-ML were 6.9, 4.7 and 4.3 times as great as those of AZT solution in spleen, liver and lung respectively. Higher concentrations of AZT were observed in lung after dosing with DSM-AZT-ML than with other preparations. Compared with AZT solution, liposomal AZT-M led to significantly higher brain concentrations of AZT. The AZT concentrations in brain 1 h after administration of AZT-ML, DSM-AZT-ML and PEG-DSM-AZT-ML were 2.2, 2.0 and 6.1-fold than those of AZT solution respectively.Cytotoxicity of different formulations of free or liposomal AZT-M against MT-4 cells was tested by the trypan blue exclusion method for viability determination. Liposomal AZT-M significantly decreased the cytotoxicity compared with free AZT-M （PEG-DSM-AZT-ML＜DSM-AZT-ML＜AZT-ML＜AZT-M）. Measurements of the Anti-HIV activity were based on the inhibition assay of SF33 virus-induced-CPE （Cytopathic effect）. Results showed that the anti-HIV potency of free or liposomal AZT-M was comparable with that of AZT at the tested concentrations.更多还原