Tumor drug delivery systems based on polymalic acid

描述（由申请人提供）：将研究一种基于来自多头绒泡菌的天然聚苹果酸（PMLA）的新型纳米级药物递送系统（药物尺寸20-30 nm）。使用该系统的初步数据显示基于PMLA的药物（Polycefin）对脑癌具有显著的抗肿瘤和抗血管生成活性，并且没有毒性。Polycefin具有用一种药物载体靶向多聚体肿瘤相关蛋白的独特能力，这是其他递送系统不可行的。在用携带针对血管肿瘤蛋白质层粘连蛋白-8链的吗啉代反义寡核苷酸的Polycefin进行有限次数的治疗后，患有异种移植人脑肿瘤的大鼠显示出显著增加的存活率（p<0.0004）和75%的肿瘤血管减少（p<0.001）。初步数据显示，Polycefin可穿透血液肿瘤屏障（BTB），并在脑和乳腺肿瘤中特异性蓄积。在该提案中，将在体外和体内评价肿瘤治疗功效与不同头孢菌素变体的结构之间的关系，特别是缀合前药（反义寡核苷酸）和肿瘤靶向部分的数量和特异性。将研究头孢菌素变体，以改善肿瘤递送和抗癌药物的疗效。该项目将专注于治疗脑胶质瘤和浸润性乳腺癌的第二代缀合物，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，我们以前的目标，层粘连蛋白-8，将被使用。最终目标是通过详细的临床前评价从这些基于聚合物的缀合物中选择用于每种肿瘤类型的先导载体-药物化合物，这将为进一步的临床使用奠定基础。为了实现这一目标，我们提出：目标1。药物载体的结构优化。这包括（a）通过用亮氨酸乙酯交换本发明载体中的缬氨酸来增强疏水性，从而提高膜渗透性，从而改善内体破坏活性;（B）通过Alexa Fluor 680交换现有的荧光素报道分子，用于体外和体内的上级成像。目标2a。优化一个PMLA载体上反义寡核苷酸的数量和相对丰度，以增加待抑制的分子靶标的数量。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，筛选出抑制作用最强的药物。目标2b。优化PMLA载体上细胞靶向单克隆抗体的数量和相对丰度，以促进BTB渗透并增加肿瘤靶向特异性。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：前药表征。测定药物毒性和载体系统本身（含/不含反义寡核苷酸和单克隆抗体）和主要载体药物化合物的半衰期。优化治疗次数。目标4。在异种移植的人类肿瘤中，评价先导化合物对动物存活率、肿瘤大小、血管分布、细胞凋亡及其分子靶点EGFR和层粘连蛋白-8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.