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疗法由于存在两个瓶颈限制而相对无效：[1]相对于正常组织，缺乏对癌细胞的药物作用的选择性;以及[2]细胞膜的不渗透性，其将药物摄取限制为简单的小的和疏水的试剂。本文提出了一种新的药物递送方法，其不仅具有同时克服这两种限制的潜力，而且还普遍适用于递送所有类型（例如大或小）的药物以治疗所有类型的癌症（例如乳腺肿瘤、恶性黑色素瘤）。该方法利用所谓的蛋白转导结构域（PTD）肽（例如来自HIV蛋白的达特）的已证实的和无与伦比的跨膜能力，以运送附着的物质穿过膜屏障，实现高效的细胞药物摄取，以及靶向和前药特征，以产生选择性药物作用并终止药物诱导的毒副作用。简言之，该方法由两种组分组成的大复合物组成：[1]靶向组分由与肝素分子连接的特异性靶向部分（例如抗体）组成，[2]药物组分通过可裂解的S-S键将递送的药物偶联至PTD肽而制备。这两种组分可以通过高度阴离子肝素和阳离子PTD之间的电荷-电荷相互作用自动缔合。文献中已证实，PTD与肝素结合将掩盖其跨膜活性;推测是由于抑制了PTD吸附至细胞表面。这种前药特征将避免药物被正常细胞摄取，减轻药物诱导的毒性作用。在通过药物复合物上的抗体靶向肿瘤后，将施用硫酸鱼精蛋白（已知其与肝素的结合强于任何现有PTD肽的临床肝素解毒剂）以暴露肝素抑制并恢复PTD-药物缀合物上PTD的跨膜活性。在鱼精蛋白注射前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物在肿瘤靶点处积聚但最小量保留在循环中的时间），从而可以避免正常组织非特异性摄取PTD-药物缀合物引起的全身毒性。一旦进入肿瘤，药物将通过谷胱甘肽和还原酶活性的胞质水平升高而降解S-S键而释放，诱导原发性肿瘤细胞凋亡。如果使用大蛋白质药物进行递送，则释放的药物将不受多药耐药（MDR）效应的影响，因为蛋白质是细胞膜不可渗透的。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递送细胞不可渗透的蛋白质毒素，在小鼠中观察到几乎完全的肿瘤消退。在这个应用程序中，我们计划进行一个全面的研究，旨在证明这种传输系统的实时可行性和实用性。选择小的常规抗肿瘤剂（多柔比星）和大的亲水性蛋白质药物（白树毒素）来代表可能的抗肿瘤剂的全谱，而选择基于使用VEGF 121的高效血管靶向策略来测试该系统在治疗携带模型CT-26实体瘤的小鼠中的作用。除了该系统对癌症治疗的普遍适用性之外，该系统还可以导致发现和开发新的药物竞技场，这些药物显示出巨大的治疗前景，但由于细胞吸收差或急性毒性作用而被认为是不可用的。在这方面，这个项目的真正价值和影响是重大的，深远的，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.