Tumor drug delivery systems based on polymalic acid

描述（由申请人提供）：将研究一种基于绒泡菌天然衍生的聚苹果酸（PMLA）的新型纳米级药物递送系统（药物尺寸20-30 nm）。该系统初步数据显示，pmla基药物（Polycefin）对脑癌具有显著的抗肿瘤和抗血管生成活性，且无毒性。聚烯烃具有一种独特的靶向多聚体肿瘤相关蛋白的药物载体的能力，这在其他递送系统中是不可行的。用含有Morpholino反义寡核苷酸到血管肿瘤蛋白laminin-8链上的Polycefin进行有限次治疗后，移植的人脑肿瘤大鼠的存活率显著提高（p<0.0004），肿瘤血管密度降低75% （p<0.001）。初步数据显示聚苯乙烯可穿透血肿瘤屏障（BTB），并在脑和乳腺肿瘤中特异性积累。在本研究中，我们将在体外和体内研究不同Polycefin变体的结构与肿瘤治疗效果之间的关系，特别是结合前药（反义寡核苷酸）和肿瘤靶向部分的数量和特异性。为了改善肿瘤传递和抗癌药物的疗效，将研究聚苯乙烯变异体。该项目将专注于治疗脑胶质瘤和浸润性乳腺癌的第二代偶联物，靶向表皮生长因子受体（EGFR），该受体在这些肿瘤中过表达。此外，我们之前的目标，laminin-8，将被使用。最终目标是通过详细的临床前评估，从这些基于聚合物的偶联物中为每种肿瘤类型选择一种先导载体药物化合物，这将为进一步的临床应用奠定基础。为实现这一目标，我们提出：药物载体的结构优化。这包括(a)通过与亮氨酸乙基酯交换当前载体中的缬氨酸来增强疏水性，从而增加膜透性，从而改善内体破坏活性；(b)用Alexa Fluor 680交换现有的荧光素报告蛋白，以获得更好的体外和体内成像。目标2。优化单个PMLA载体上反义寡核苷酸的数量和相对丰度，增加待抑制分子靶标的数量。体外测定靶蛋白的抑制作用及药物对肿瘤细胞增殖、侵袭和凋亡的影响，选择抑制作用最强的药物。目标2 b。优化PMLA载体上靶向单克隆抗体的细胞数量和相对丰度，促进BTB穿透，提高肿瘤靶向特异性。体外测定靶蛋白抑制作用及药物对肿瘤细胞增殖、侵袭和凋亡的影响。目标3。那些特性。测定载体系统本身（含/不含反义寡核苷酸和单克隆抗体）和先导载体-药物化合物的药物毒性和半衰期。处理次数的优化。目标4。评估先导化合物对动物存活、肿瘤大小、血管性、细胞凋亡及其分子靶点EGFR和层粘连蛋白-8在异种移植人类肿瘤中的表达的影响。

DESCRIPTION (provided by applicant): A new nanoscale drug delivery system (drug size 20-30 nm) based on naturally derived poly malic acid (PMLA) from Physarum polycephalum will be studied. Preliminary data using this system show significant anti-tumor and anti-angiogenic activity of PMLA-based drug (Polycefin) against brain cancer with no toxicity. Polycefin has a unique ability of targeting multimeric tumor-associated proteins with one drug vehicle, which is not feasible with other delivery systems. Rats with xenografted human brain tumors after a limited number of treatments with Polycefin bearing Morpholino antisense oligonucleotides to chains of vascular tumor protein, laminin-8 showed significantly increased survival (p<0.0004) and 75% decrease in tumor vascularity (p<0.001). Preliminary data show that Polycefin penetrates blood-tumor barrier (BTB) and accumulates specifically in brain and breast tumors. In this proposal, the relationship between tumor treatment efficacy and the structure of different Polycefin variants, in particular, the number and specificity of conjugated prodrugs (antisense oligonucleotides) and tumor-targeting moieties will be evaluated in vitro and in vivo. Polycefin variants will be studied in order to improve tumor delivery and the efficacy of anti-cancer drug(s). The project will focus on second-generation conjugates to treat brain gliomas and invasive breast cancer, targeting epidermal growth factor receptor (EGFR), which is overexpressed in these tumors. Additionally, our previous target, laminin-8, will be used. The ultimate goal is to select a lead vehicle-drug compound for each tumor type from these polymer-based conjugates by detailed preclinical evaluation, which would set grounds for further clinical use. To accomplish this goal, we propose: Aim 1. Structural optimization of the drug vehicle. This includes (a) improvement of endosomal disruption activity through enhanced hydrophobicity by exchange of valine in the present carrier with leucine ethylester thereby increasing membrane permeation; (b) exchanging the existing fluorescein reporter by Alexa Fluor 680 for superior imaging in vitro and in vivo. Aim 2a. Optimization of the number and relative abundance of antisense oligonucleotides on one PMLA carrier to increase the number of molecular targets to be inhibited. In vitro determination of target protein inhibition and the drug effects on tumor cell proliferation, invasion and apoptosis, to select the strongest inhibiting drug. Aim 2b. Optimization of the number and relative abundance of cell targeting monoclonal antibodies on PMLA carrier for facilitating BTB penetration and increasing tumor targeting specificity. In vitro determination of target protein inhibition and the drug effects on tumor cell proliferation, invasion and apoptosis. Aim 3. Pro-drug characterization. Determination of drug toxicity and half-life of the carrier system itself (with/without antisense oligonucleotides and monoclonal antibodies) and of the lead carrier-drug compounds. Optimization of the treatment number. Aim 4. Evaluation of the effects of lead compounds on animal survival, tumor size, vascularity, apoptosis, and expression of their molecular targets, EGFR and laminin-8, in xenotransplanted human tumors.