Thermally Targeted Delivery of DOX

描述（由申请人提供）：目前实体瘤的治疗受限于肿瘤对放疗或化疗的固有耐药性以及全身使用抗肿瘤药物的毒性。我们的长期目标是通过开发针对局部肿瘤的靶向治疗方法来克服这些局限性，从而提高治疗的特异性和有效性，并降低正常组织中的细胞毒性。我们已经开发了一种热响应多肽载体化疗阿霉素抑制癌细胞的增殖培养。我们的假设是，在系统给药后，这些基因工程多肽可以通过局部热疗靶向肿瘤部位。这将导致药物在肿瘤中的积累，随后抑制肿瘤生长。所设计的多肽的氨基酸序列是基于弹性蛋白样多肽（ELP）生物聚合物，它在生理温度（37℃）下可溶于水溶液，但当温度升高到40℃以上时聚集。Tat膜易位序列来源于HIV-1 Tat蛋白，已知可促进大货物蛋白跨细胞膜的递送，与ELP结合以促进细胞进入。ELP的c端包含一个四肽GFLG连接体。这种溶酶体可降解肽连接物将ELP多肽从可偶联到阿霉素的半胱氨酸残基中分离出来。我们的初步体外实验结果表明，与非热反应性对照肽相比，tas - elp - dox构建体在MES-SA和MES-SA/Dx5多药耐药子宫肉瘤细胞中具有非常显著的作用。为了解决Tat-ELP-Dox可以热靶向肿瘤组织的假设，将解决以下具体目标：(1)通过测定血浆浓度曲线和定量放射自显像法测定组织浓度，测定Tat- ELP-Dox在胸腺模型大鼠正常和肿瘤组织中的血浆动力学和体内分布；(2)通过反复给药联合局部热疗，评价Tat-ELP-Dox对胸腺大鼠大腿肿瘤异种移植物局部热疗的治疗效果。提出的化疗多肽载体通过局部热疗特异性靶向实体瘤，可以提高治疗的特异性和有效性，降低正常组织中的细胞毒性。该研究的成功完成将提供比现有/替代技术更具竞争优势的技术，并将提供一种有效替代或增强局部肿瘤治疗的替代方法。目前对实体瘤的治疗是有限的，因为只有一小部分给药剂量到达肿瘤部位，而其余的药物分布在全身，当药物以根除癌细胞所需的剂量使用时，会对正常组织产生不良的副作用。我们的长期目标是通过开发一种方法来克服这一限制，使药物能够特异性地递送到肿瘤部位。这将增加治疗的特异性，减少对正常组织的毒性。

DESCRIPTION (provided by applicant): Current treatment of solid tumors is limited by inherent tumor resistance to radiation or chemotherapy and by toxicity from systemic administration of antineoplastic agents. Our long-term goal is to overcome these limitations by developing for localized tumors a targeted therapeutic approach that increases the specificity and efficacy of the therapy and reduces the cytotoxicity in normal tissues. We have developed a thermally responsive polypeptide carrier for the chemotherapeutic doxorubicin which inhibits proliferation of cancer cells in culture. Our hypothesis is that after systemic administration these genetically engineered polypeptides can be targeted to the tumor site by applying local hyperthermia. This will result in accumulation of the agent in the tumor with subsequent inhibition of tumor growth. The amino acid sequence of the designed polypeptides is based on elastin-like polypeptide (ELP) biopolymers which are soluble in aqueous solution at physiological temperature (37 ¿C), but aggregate when the temperature is raised above 40 ¿C. The Tat membrane translocating sequence, derived from the HIV-1 Tat protein, known to facilitate delivery of large cargo proteins across cell membranes, is conjugated to the ELP to facilitate cell entry. A tetrapeptide GFLG linker was included at the C-terminus of ELP. This lysosomally degradable peptide linker separates the ELP polypeptide from a cysteine residue that can be coupled to doxorubicin. Our preliminary in vitro results demonstrate a very significant effect of the Tat-ELP-Dox construct in MES-SA and MES-SA/Dx5 multidrug resistant uterine sarcoma cell cells when compared to a non-thermally responsive control peptide. In order to address the hypothesis that Tat-ELP-Dox can be thermally targeted to tumor tissue, the following specific aims will be addressed: (1) Measure the plasma kinetics and in vivo distribution of Tat- ELP-Dox in normal and neoplastic tissue in an athymic rat model by determining the plasma concentration curve and determination of the tissue concentrations with quantitative autoradiography, and (2) Evaluate the therapeutic efficacy of Tat-ELP-Dox in the treatment of neoplastic xenografts in the thigh of athymic rats with and without localized hyperthermia through repeated administration of the agent coupled with local hyperthermia. Specific targeting of the proposed chemotherapeutic polypeptide carrier to solid tumors by local hyperthermia would increase specificity and efficacy of treatment and reduce the cytotoxicity in normal tissues. The successful completion of the proposed research will provide technology which has competitive advantage over existing/alternate technologies and it would provide an alternative mean to effectively substitute or augment present therapy for treatment of localized tumors. Current treatment of solid tumors is limited as only a small fraction of the administered dose of drug reaches the tumor site, while the rest of the drug is distributed throughout the body causing undesirable side effects to normal tissue when drugs are used in the doses required to eradicate cancer cells. Our long term goal is to overcome this limitation by developing an approach that allows the drug to be delivered specifically to the tumor site. This will increase the specificity of the therapy and reduce the toxicity in normal tissues.