Thermally Targeted Delivery of DOX

描述(申请人提供)：目前实体肿瘤的治疗受到肿瘤对放射或化疗的固有抵抗力以及全身应用抗肿瘤药物的毒性的限制。我们的长期目标是通过为局部肿瘤开发一种靶向治疗方法来克服这些限制，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性多肽载体，用于化疗阿霉素，它可以抑制培养的癌细胞的增殖。我们的假设是，在全身给药后，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积聚，从而抑制肿瘤生长。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，这些生物聚合物在生理温度(37℃)下可溶于水溶液，但在温度高于40℃时聚集。Tat膜转位序列源自HIV-1 Tat蛋白，已知有助于大货物蛋白的跨细胞膜运输，与ELP连接以促进细胞进入。在ELP的C末端含有一个四肽GFLG连接子。这种溶酶体可降解的多肽连接物将ELP多肽从可与阿霉素偶联的半胱氨酸残基中分离出来。我们的初步体外结果显示，TAT-ELP-Dox结构在MES-SA和MES-SA/Dx5多药耐药子宫肉瘤细胞中与非热响应对照多肽相比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，本研究将针对以下具体目标进行研究：(1)通过测定TAT-ELP-Dox的血药浓度曲线和定量放射自显影技术来测定TAT-ELP-Dox在裸鼠模型正常和肿瘤组织中的血浆动力学及体内分布；(2)通过反复给予TAT-ELP-Dox联合局部热疗来评价TAT-ELP-Dox对有或无局部热疗的裸鼠大腿肿瘤移植瘤的治疗效果。通过局部热疗将化疗多肽载体特异性靶向实体瘤，可提高治疗的特异性和有效性，降低对正常组织的细胞毒作用。这项拟议研究的成功完成将提供比现有/替代技术具有竞争优势的技术，并将提供一种替代或增强目前治疗局部肿瘤的治疗方法的替代手段。目前对实体肿瘤的治疗是有限的，因为只有一小部分给药的药物到达肿瘤部位，而其余的药物分布在全身，当以根除癌细胞所需的剂量使用药物时，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允许药物特异性地输送到肿瘤部位的方法来克服这一限制。这将增加治疗的特异性，并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.