【作者】 李德馨；

【导师】 吴春福； 王思玲；

【作者基本信息】 沈阳药科大学 ， 药学， 2005， 博士

【摘要】 白内障是世界首位致盲眼病，由白内障导致的视力低下和眼盲是急待解决的医学和社会问题，研究白内障的发病机制和针对病因的抗白内障药物制剂的开发是解决问题的关键。双硫仑（disulfiram，DSF）为二乙基二硫代氨基甲酸（Diethyldithiocarbamate，DDC）的二聚体，DDC及其衍生物是一类具有抗白内障活性的化合物，其中DSF的活性较高，但由于溶解度较低，限制了DSF的应用。本文采用环糊精包合技术，将DSF包合于羟丙基β环糊精（HPβCD）以增加DSF的溶解度，从研究药物的角膜透过性入手，研制成适于眼部给药并符合眼用制剂质量要求的DSF—HPβCD滴眼液，并考察了DSF—HPβCD滴眼液的抗白内障效果以及家兔眼内的药动学过程。之前的研究一般将DDC及其衍生物的抗白内障机理归因于它们的抗氧化作用和对抗机体中过多的自由基。为更进一步从细胞分子水平探讨这个问题，本文首次在人晶体上皮细胞（HLEC）中诱导表达了一氧化氮合酶（inducible nitric oxide synthase，iNOS），发现DDC对iNOS的表达具有抑制作用，从一个崭新的角度解释其抗白内障机理。 首先以相溶解度法和HPLC法研究了DSF与HPβCD形成包合物的特点。其相溶解度曲线符合Higuchi所阐述的An型，包合物的稳定常数为1772.77L·mol^-1（25℃）。包合过程的焓变（△H）为-25.25kJ-mol^-1，虽然包合物形成是熵减少地过程，但是焓效应弥补了熵效应的不利作用，对包合物的形成起支配作用，综合结果是该包合反应的吉布斯自由能△G为负，表明这一反应是一个自发的过程。 分别采用溶液搅拌法、超声波法、直接混合加热法制备DSF—HPβCD包合物，其中溶液搅拌法效果最好，包合物中DSF平均含量为7.606％（w／w）。制得的包合物以差示扫描量热法、红外光谱法和X-射线粉末衍射法鉴定。结果显示包合物的理化性质不同于纯药物及其与HPβCD的物理混合物，包合物以无定型形式存在，且水中的溶解度由0.02％提高到1.34％，完全能够满足制备DSF滴眼剂的要求。 离体角膜透过实验和DSF在角膜匀浆中的代谢研究表明DSF为DDC的前药，应用于眼部时，在透过角膜过程中被代谢成为活性形式DDC，即首次提出这一过程具有酶促反应的特点，米氏常数（K_m）和最大反应速度(V_max)分别为12.05±1.04μmol·L^-1和0.8696±0.0215μmol·min^-1。 离体家兔角膜透过实验研究表明，某些高分子聚合物和渗透促进剂能够显著提高DSF的表观渗透系数，增加DDC的角膜透过量，其中0.1％HPMC和1.0％F₆₈的促渗更多还原

【Abstract】 Cataracts are still the most important cause of blindness in the world. Cataract-induced visual dysfunction and blindness is a significant and increasing global problem. The solutions for this problem are founding out the fundamental mechanism of cataract and delay its formation by drug therapies. Diethyldithiocarbamate （DDC） and its derivates are thus a group of chemical substances. Disulfiram （DSF）, the dimer of DDC, presents good anti-cataract activity. But the use of DSF in ocular treatment was limited by its poor water solubility. In the present studies, DSF was included into hydroxypropyl-8-cyclodextrin to increase its water solubility. Based on in vitro transcorneal transit studies, a DSF-HPβCD based ocular drug delivery system was formulated. The ocular pharmacokinetics of DSF—HPβCD eye drops in rabbit eyes and the pharmacology to selenite-induced cataract rats and hereditary Shumiya cataract rats （SCR） were evaluated. Because of the mechanism of cataract formation was still not clear, the explanation for DSF’s anti-cataract effect was thought to be their anti-oxidation effects and the scavenging ability for free radicals. In a previous study, excessive nitric oxide （NO） production was found to be one reason for cataract. In the present study, for the first time, human lens epithelial cells （HLEC） were found to be able to express inducible nitric oxide synthase （iNOS）. DDC was observed to inhibit iNOS expression in HLEC effectively, which explained the anti-cataract effect of DSF at a new point of view.Interaction between DSF and HPBCD was studied by means of phase solubility method and HPLC assay. The formation of DSF-HPBCD inclusion complex accorded with the A_n type described by Higuchi. The apparent stability constant of the inclusion complex was found to be 1772.77 L ? mol^-1 HPLC assay gave out the enthalpy change（△H ）of the system during the formation of DSF-HPBCD, which is -25.25 kJ·mol^-1 The negative free energy change of this reaction demonstrated the formation of DSF-HPBCD was spontaneous.DSF-HPBCD inclusion complex were prepared using solution mixing method, ultrasonic method and directly mixing and heating. A best outcome was observed by using solution mixing method. DSF-HPBCD was subjected to differential scanning calorimetry analysis（DSC）, infrared spectrometry （IR） and X-ray powder diffraction assay for the identification of inclusion complex formation.Results from in vitro permeability of DSF across isolated rabbit cornea and DSF metabolism study in cornea homogenate demonstrated that, when applied in the eye, DSF was definitely converted to its active form DDC, and the later passing through the cornea and diffusing into the aqueous humor. The metabolism process was of some characters of enzymatic reaction. The K_m and V_max were found to be 12.05 ± 1.04 μmol·L^-1 and 0.8696 ± 0.0215 μmol·min^-1 respectively.更多还原