Pharmacology of Targeted Therapy to Brain Tumors

描述(申请人提供)：这项研究的目标是设计改进的非病毒基因载体，并将其应用于大鼠脑肿瘤模型的基因导向酶前体药物治疗(GDEPT)方案。我们要解决的问题是：表达酵母胞嘧啶脱氨酶的优化非病毒GDEPT方案是否会提供比靶向脂质体化疗方案更好的脑肿瘤治疗方案？ 指导这一计划的假设是，多水平靶向的组合将导致肿瘤特异性胞嘧啶脱氨酶基因的强劲表达和改进的肿瘤治疗。这些计划中使用的四个靶向水平是：对流增强扩散(CED)进入脑瘤、生物响应性脂质体基因制剂、CD44受体靶向和肿瘤基质靶向酶。肿瘤定位的胞嘧啶脱氨酶然后将全身给药的前药氟胞嘧啶(FC)转化为活性物质氟尿嘧啶(FU)。这种特殊的方法组合将增强载体进入肿瘤的途径，改善载体通过肿瘤的渗透，增加载体与肿瘤细胞的附着，并改善治疗基因的细胞内递送和旁观者活性。 该计划有四个主要活动：(1)合成新的成分，并将这些成分与质粒DNA组装成小直径、稳定的脂质体，命名为纳米脂粒(NLP)(2)CED进入大脑后，表征控制NLP在细胞中的表现(pH敏感性、细胞结合、基因转移活性、毒性)和在动物中的表现(分布、药代动力学、毒性和基因表达)的因素。(3)构建靶向胞嘧啶脱氨酶的细胞外基质以改善旁观者效应。(4)比较优化后的GDEPT与以CD44为靶向、理化性质与NLP相似的脂质体中氟罗丹酸的治疗效果。氟胞嘧啶和氟甲酸均通过FU发挥治疗作用。肿瘤和血浆中的氟尿嘧啶及其代谢物浓度将被测量。我们将确定与非靶细胞相比，哪种方法在肿瘤中提供了更大的药物浓度x时间分布。 合成化学、分子生物学、药剂学和药理学的结合将使我们能够确定当CED给出两种方案时，非病毒GDEPT是否优于靶向前药治疗脑肿瘤。这些结果将为人脑肿瘤的靶向治疗提供理论依据。

DESCRIPTION (provided by applicant): The objectives of this research are to devise improved non-viral gene carriers and to apply them in a Gene-Directed Enzyme Prodrug Therapy (GDEPT) protocol in a rat brain human tumor model. The question we will address is: will an optimized non-viral GDEPT protocol expressing yeast cytosine deaminase provide better therapy then a targeted liposome chemotherapy protocol in brain tumors? The hypothesis guiding this plan is that a combination of multiple levels of targeting will result in robust tumor-specific cytosine deaminase gene expression and improved tumor therapy. The four levels of targeting employed in these plans are: convective enhanced diffusion (CED) into the brain tumor, bioresponsive liposome gene formulations, CD44 receptor targeting, and a tumor matrix targeted enzyme. Tumor-localized cytosine deaminase will then convert the systemically administered prodrug, fluorocytosine (FC), to fluorouracil (FU), the active agent. This particular combination of approaches will enhance access of the carrier to the tumor, improve percolation of the carrier through the tumor, increase carrier attachment to tumor cells and improve intracellular delivery of the plasmid and bystander activity of the therapeutic gene. There are four major activities in the program: (1) synthesis of novel components and assembly of the components with plasmid DNA into a small diameter, stable liposome designated a nanolipoparticle (NLP)(2) characterization of the factors that control the performance of the NLP in cells (pH sensitivity, cell association, gene transfer activity, toxicity) and in animals (distribution, pharmacokinetics, toxicity and gene expression) after CED into the brain. (3) Construction of an extracellular matrix targeted cytosine deaminase to improve the bystander effect. (4) Comparison of the therapeutic effect of the optimized GDEPT to the effect of fluoroorotic acid delivered in a CD44 targeted liposome of similar physicochemical characteristics as the NLP. Fluorocytosine and fluoroorotic acid both exert their therapeutic effect through FU. The concentration of FU and metabolites will be measured in the tumor and plasma. We will determine which approach provides a greater drug concentration x time profile in the tumor as opposed to non-target cells. The combination of synthetic chemistry, molecular biology, pharmaceutics, and pharmacology will permit us to determine if a non-viral GDEPT is superior to a targeted prodrug for the treatment of brain tumors when both protocols are given by CED. These results will provide a rationale for targeted therapies for human brain tumors.