Pharmacology of Targeted Therapy to Brain Tumors

描述（由申请人提供）：本研究的目的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药治疗（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的非病毒GDEPT方案是否比靶向脂质体化疗方案在脑肿瘤中提供更好的治疗？ 指导该计划的假设是，多个靶向水平的组合将导致稳健的肿瘤特异性胞嘧啶脱氨酶基因表达和改善的肿瘤治疗。这些计划中采用的四个水平的靶向是：进入脑肿瘤的对流增强扩散（CED）、生物响应脂质体基因制剂、CD44受体靶向和肿瘤基质靶向酶。然后，肿瘤定位的胞嘧啶脱氨酶将全身给药的前药氟胞嘧啶（FC）转化为活性药物氟尿嘧啶（FU）。这种方法的特定组合将增强载体对肿瘤的接近，改善载体通过肿瘤的渗透，增加载体对肿瘤细胞的附着，并改善质粒的细胞内递送和治疗基因的旁观者活性。 该方案有四项主要活动：（1）合成新组分并将组分与质粒DNA组装成小直径的稳定脂质体，称为纳米脂质体颗粒（NLP）（2）表征控制NLP在细胞中的性能的因素（pH敏感性、细胞结合、基因转移活性、毒性）和动物中在CED进入脑后，这些细胞的分布（分布、药代动力学、毒性和基因表达）。(3)细胞外基质靶向胞嘧啶脱氨酶提高旁观者效应的构建。(4)优化的GDEPT的治疗效果与在具有与NLP相似的物理化学特征的CD44靶向脂质体中递送的氟乳清酸的效果的比较。氟胞嘧啶和氟乳清酸均通过FU发挥治疗作用。将测量肿瘤和血浆中FU和代谢物的浓度。我们将确定哪种方法在肿瘤中提供比非靶细胞更大的药物浓度x时间曲线。 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两种方案都由CED给出时，非病毒GDEPT是否优于用于治疗脑肿瘤的靶向前药上级。这些结果将为人类脑肿瘤的靶向治疗提供理论基础。

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.