PTD-Mediated Protein or Drug Delivery for Cancer Therapy

描述(由申请人提供)： 大多数现有的抗癌药物治疗相对无效，因为存在两个瓶颈限制：[1]相对于正常组织，药物作用于癌细胞缺乏选择性；[2)细胞膜的不通透性，限制药物对简单的小而疏水的药物的摄取。这里提出了一种新的药物输送方法，它不仅具有同时克服这两个限制的潜力，而且还普遍适用于用于治疗所有类型的癌症(例如乳腺癌、恶性黑色素瘤)的所有类型的药物(例如大或小)的输送。该方法利用已证实的和无与伦比的蛋白质转导结构域(PTD)多肽(例如来自HIV蛋白质的TAT)的跨膜能力来运送附着的物种通过膜屏障实现高效的细胞药物摄取，以及靶向性和前药特性，从而产生选择性药物作用和终止药物诱导的毒副作用。简而言之，该方法由两个组分组成：[1]靶向组分由与肝素分子相连的特定靶向部分(如抗体)组成，[2]药物组分是通过可切割的S-S键将所传递的药物偶联到PTD多肽上而制成的。这两个组分可以通过高阴离子肝素和阳离子PTD之间的电荷-电荷相互作用自动结合。文献中已经证实，PTD与肝素结合会掩盖其跨膜活性；推测是由于抑制了PTD对细胞表面的吸附。这一前药特征将避免正常细胞摄取药物，减轻药物诱导的毒性效应。在药物复合体上的抗体靶向肿瘤后，将使用一种临床肝素解毒剂硫酸鱼精蛋白，它与肝素的结合强度比任何现有的PTD多肽都要强，以揭示肝素抑制并恢复PTD-药物结合物上PTD的跨膜活性。在注射鱼精蛋白之前，将进行靶向药代动力学研究，以确定鱼精蛋白的最佳给药时间(定义为最大程度的药物复合体在肿瘤靶区积累，但循环中残留最少量的时间)，从而避免正常组织非特异性摄取PTD-药物结合物造成的全身毒性。一旦进入肿瘤，药物将通过细胞内谷胱甘肽和还原酶活性升高而降解S-S连接而释放，诱导原代肿瘤细胞凋亡。如果使用大的蛋白质药物进行传递，释放的药物将不受多药耐药(MDR)效应的影响，因为蛋白质不能渗透到细胞膜。初步研究产生了非常有希望的结果，因为通过使用这种方法传递细胞不渗透蛋白毒素，在小鼠身上观察到几乎完全的肿瘤消退。在这项应用中，我们计划进行一项全面的研究，旨在展示该交付系统的实时可行性和实用性。选择一种小的常规抗肿瘤药物(阿霉素)和一种大的亲水蛋白药物(凝胶)来代表可能的抗肿瘤药物的全谱，而基于血管内皮生长因子121的高效血管靶向策略被选择用于治疗携带CT-26实体瘤的小鼠。除了该系统对癌症治疗的普遍适用性外，该系统还可能导致发现和开发一个新的药物领域，这些药物显示出巨大的治疗前景，但由于细胞摄取不良或急性毒性作用而被认为不可用。在这方面，这个项目的真正价值和影响是重大的、深远的和普遍的。

DESCRIPTION (provided by applicant): Most existing anti-cancer drug therapies are relatively ineffective due to the presence of two bottleneck limitations: [1] the lack of selectivity of drug action towards cancer cells as opposed to normal tissues; and [2] the impermeability of the cell membrane that restricts drug uptake to simply small and hydrophobic agents. Proposed herein is a novel drug delivery approach that not only carries the potential to subdue these two limitations simultaneously but also be applied universally to delivery of drugs of all types (e.g. large or small) for treatment of cancers of all types (e.g. breast tumor, malignant melanoma). The approach utilizes the proven and unmatched trans-membrane ability of the so-called protein transduction domain (PTD) peptides (e.g. TAT from HIV protein) to ferry attached species across the membrane barrier in achieving highly effective cellular drug uptake, as well as the targeting and prodrug features to, yield selective drug actions and abort drug-induced toxic side effects. Briefly, the approach consists of a large complex made of two components: [1] a targeting component consists of a specific targeting moiety (e.g. antibody) linked with a heparin molecule and [2] a drug component is made by coupling, via cleavable S-S bonds, the delivered drug to a PTD peptide. These two components can associate automatically by a charge-charge interaction between the highly anionic heparin and cationic PTD. It has been confirmed in the literature that binding PTD with heparin would mask its trans-membrane activity; presumably due to inhibition of PTD adsorption to the cell surface. This prodrug feature would avoid drug uptake by normal cells, alleviating drug-induced toxic effects. Following tumor targeting by the antibody on the drug complex, protamine sulfate, a clinical heparin antidote that is known to bind heparin stronger than any existing PTD peptide, will be administered to unmask heparin-inhibition and restore the trans-membrane activity of PTD on the PTD-Drug conjugate. A study of the targeting pharmacokinetics will be carried out prior to protamine injection to determine the optimal dosing time of protamine (defined as the time when a maximum degree of the drug complexes has accumulated at the tumor target but a minimum amount is remained in the circulation), so that systemic toxicity caused by non-specific uptake of the PTD-Drug conjugates by normal tissues can be avoided. Once inside the tumor, the drug will be released by degradation of the S-S linkage by elevated cytosolic levels of glutathione and reductase activity, inducing apoptosis to primary tumor cells. If a large protein drug is used for delivery, the released drug will be unaffected by the multidrug resistant (MDR) effect because proteins are impermeable to cell membrane. Preliminary studies yielded extremely promising results, as a nearly complete tumor regression was observed in mice by using this approach in delivering a cell-impermeable protein toxin. In this application, we plan to conduct a full-scale study aimed at demonstrating the real-time feasibility and utility of this delivery system. A small conventional anti-tumor agent (doxorubicin) and a large hydrophilic protein drug (gelonin) are selected to represent the full spectrum of possible anti-tumor agents, whereas a highly effective vascular targeting strategy based on the use of VEGF121 is chosen to test the system in treating mice harboring a model CT-26 solid tumor. Aside from the universal applicability of this system to cancer treatment, the system could lead to discovery and development of a new arena of drugs that show great therapeutic promise but considered unusable due to poor cell uptake or acute toxic effects. To this regard, the true value and impact of this project is significant, far-reaching, and prevalent.