拮抗CXCR4/Axl双靶标的多肽共递送系统及其克服白血病耐药的功能研究

It has been a big challenge for the treatment of acute myeloid leukemia (AML). Although the existing chemotherapy regimens lead to a relative high complete remission rate, high percentage of relapse still occurs in patients who have received the chemotherapeutic treatment; moreover, the patients will develop drug resistance to the chemotherapeutics. It has been recognized that AML cells would upregulate some proteins expression when the cells are attacked by chemotherapeutics, these proteins reduce the sensitivity of the AML cells and guide the cells home to the bone marrow where they obtain survival signals to form miniature residue diseases, which are the major reasons for the relapse and drug resistance after the complete remission. These processes are regulated by multiple mechanisms, in which CXCR4/CXCL12 and Axl/GAS6 are two crucial axes, the former plays roles in the migration and adhesion of AML cells to bone marrow matrix cells, while the later enhances the ability of anti-apoptosis for AML cells. Therefore CXCR4 and Axl are two important targeted proteins on the cells membrane. Nevertheless, the research of targeting the two proteins at the same time by peptide antagonists has not been touched yet. . This project aims to (1) develop co-delivery system of two antagonistic peptides targeting CXCR4 and Axl by investigating key physicochemical factors in the assembly of micelles; (2) study the synergetic effect of the two antagonistic peptides loaded in micelles in multiple AML cell lines and AML animal models by using cell and molecular biological technologies; and (3) study the efficacy of dual-peptide co-delivery system in the combination with typical chemotherapeutics applied in AML treatment in AML animal models. In the previous work we have developed effective antagonistic peptide E5 targeting CXCR4 and a peptide targeting Axl with high binding efficiency in AML cells. Meanwhile a micelles system loading E5 has been developed and demonstrated effective in AML cells as well as breast cancer cells. All of these provided solid basement for this project. This research is of important implications to develop novel strategies in AML treatments.

急性髓系白血病（AML）的治疗面临严峻挑战；虽然现行化疗方案可获得较高完全缓解率，但AML细胞会上调某些蛋白以降低其对药物的敏感性和向骨髓归巢，导致日后的复发与耐药。CXCR4/CXCL12和Axl/GAS6是调控此过程的重要生物轴，前者引导AML细胞向骨髓趋化和粘附，后者促进细胞抗凋亡能力，CXCR4和Axl是导致AML复发和产生耐药的重要靶蛋白。迄今同时拮抗上述两种蛋白并用于AML治疗的研究尚未见报道。本项目拟将前期研发的靶向CXCR4和Axl的两种拮抗多肽整合到磷脂胶束中，通过研究关键理化因素的影响规律实现双肽共递送系统构建，并在AML细胞株和AML小鼠模型上研究该递送系统及其与化疗药联用的治疗功效与协同拮抗机制，增强AML细胞对化疗药物的敏感性；并利用胶束的保护提高多肽的体内稳定性。此研究对于建立难治性AML的生物治疗新方法，进一步认识AML耐药机制和提供药物研发靶点具有重要意义。

本项目聚焦化学合成拮抗多肽及应用于急性髓细胞白血病（AML）治疗的研究，以AML细胞表达的耐药相关蛋白CXCR4、Axl、CD123和MUC-1为靶标，设计新型多肽序列，并建立相应的纳米药物递送系统，在难治性AML动物模型上研究拮抗多肽及其纳米胶束递送系统的药效与这作用机制。项目执行期间，新设计合成了针对CD123的拮抗多肽和针对MUC-1的靶向多肽；同时，对前期开发的CXCR4和Axl拮抗多肽在应用剂型方面做了进一步优化研究，在此基础上，建立了适合于多肽与化疗药物共递送的纳米胶束系统，以提高多肽在生理环境中的稳定性及其生物功能。.项目完成了CXCR4拮抗多肽E5及其纳米胶束剂型的药效与分子机制研究，论文发表在纳米科技综合性权威期刊Small上，所申请的PCT专利获美国和欧洲授权。建立了多肽与化药共递送系统，将E5和阿霉素组装到磷脂胶束上，该双药递送系统同时发挥了CXCR4拮抗功能、特异性识别和靶向递送功能、以及化疗药细胞毒作用，在耐药性白血病细胞株表现出优越的抗肿瘤活性，在难治性AML小鼠模型上获得显著治疗功效。论文发表在药学领域高影响力期刊Pharmacological Research。新设计合成了CD123拮抗多肽PO-6及其纳米胶束递送系统，在难治性AML小鼠模型上完成了药效学和分子机制研究，并揭示了纳米递送系统的关键作用，研究论文发表在血液肿瘤学领域高影响力期刊Journal of Hematology & Oncology和材料科学综合性高影响力期刊ACS Applied Materials Interface，并获国家发明专利授权1项，申请PCT专利1项。此外，设计合成了靶向MUC-1的多肽序列，已提交国家发明专利申请。建立了用于Axl拮抗多肽及其与化药共递送的纳米胶束递送系统，准备申请国家发明专利。上述多肽序列及其纳米胶束剂型技术具有重要的临床转化应用价值，有望为难治性AML提供新的治疗药物及新的治疗策略。.项目部分研究成果分别在Wiley出版社MaterialsViews中国的官方微博、“化学与材料科学”公众号、和中国医学科学院北京协和医院网站/公众号、中国医学科学院基础医学研究所网站展示和推介。生物技术网和生物通等网站对MaterialsViews中国上的新闻报道进行了转载。