小分子化合物“金丝桃素”与“依文思蓝”的坏死亲和性机理研究及其在癌症诊疗中的应用

We have previously demonstrated that a combination of vascular disrupting agent and 131I labeled necrosis-avid agent could effectively realize a targeted therapeutic and diagnostic or theragnostic strategy for handling solid tumors. However, mechanisms of necrosis-affinity for those small molecular natural or synthetic compounds, such as Hypericin (Hyp) and Evans blue (EB), are still unknown, and the applicability of such a steategy for specific tumor types still needs to be verified. These doubts may impair the clinical translation of this method. Our preliminary results indicated that albumin could bind Hyp and EB, which facilitated their mass accumulation in necrosis. In living cells, the agents seemed to highly accumulate in lysosomes and be cleared subsequently. But in dead cells they retained and moreover, they distributed in the area with a high concentration of fetal bovine serum (FBS). So, it is hypothesized that the agents with bound proteins are processed in lysosomes and cleared by living cells; but they are extracted from the blood to the dead cells with higher protein levels in the cytoplasm due to massive proteins resulting from the diffused enzymatic activity by the broken lysosomes. To prove this hypothesis, experiments will be conducted to trace the in vivo and in vitro dynamics of the agents and their protein-binding profiles in living cells, and to verify possible mechanisms of necrosis-affinity as mentioned above. In addition, one of the best agents will be chosen for combining the vascular disrupting agent and labeling with I-131 to explore the theragnostic potentials in animal tumor models such as osteosarcoma and liver cancers. This study will provide theoretical foundation and efficacy evaluation for further clinical development of this necrosis targeted anticancer theragnostic strategy.

本课题组已利用“肿瘤血管破坏药物+131I坏死亲和物”初步实现了针对实体瘤的诊断治疗一体化策略。但小分子坏死亲和物如金丝桃素（Hyp） 和依文思蓝（EB）的作用机理不明，且适用的实体瘤类型也没有验证，影响该成果的临床转化。本项目前期研究发现白蛋白可与Hyp、EB结合、且在坏死部位浓聚；二者进入活细胞先形成亮点（疑为溶酶体）、随后被活细胞清除，进入死细胞则始终弥散分布于胞浆；环境内蛋白浓度越高越易吸引二者分布。因此提出假设：二者结合血浆蛋白入胞，活细胞状态下溶酶体分解结合蛋白、二者随之被清除；死细胞内溶酶体崩解弥散，大量未及分解的白蛋白将Hyp或EB从低白蛋白浓度的血浆“萃取”到高白蛋白浓度的坏死细胞内。本研究将对Hyp、EB在活细胞的动力学过程和在死细胞的坏死亲和机理进行深入研究，并优选其中之一用于各部位骨肉瘤、各不同来源肝肿瘤的诊断、治疗，为该策略推向临床应用提供理论依据和实际效果评价。

本课题组已利用“肿瘤血管破坏药物”联合“131I坏死亲和物”、初步实现了广谱作用于各类实体瘤的“梯次双靶诊断治疗一体化新策略”。但小分子坏死亲和物如金丝桃素（Hyp） 和依文思蓝（EB）的作用机理不明，适用范围有待优化、各项减毒增效措施还需进一步完善，这些都关系到该类坏死亲和物及其前期成果的临床转化。本项目比较了Hyp和EB的坏死亲和性的差异；分析其血液中的载体蛋白成分、进入细胞的方式、亚细胞分布、降解途径等问题，并针对前人报导的坏死亲和机理（细胞膜磷脂结合、DNA嵌合、脂蛋白依懒性）一一进行了研究与判定。本项目发现Hyp与EB不但靶向部位有区别、而且入胞模式也有差异：EB不进入活细胞。因此推广到临床应用中，认为EB的同位素标记物更适合做坏死（如心梗、脑梗）的诊断、而Hyp由于其脂溶性及肝脏代谢的特点，更适合作为治疗性核素的靶向药物。本项目否定了前人提出的Hyp坏死亲和性为DNA嵌合、细胞膜磷脂结合、脂蛋白依赖性入胞的机理，观测到其坏死亲和性更依赖于蛋白的浓度、而非单一的某种蛋白成分。另外，本研究发现Hyp血浆中主要与白蛋白结合，并以此开发了新的131I-Hyp制剂，可更好的降低核素对正常组织的一过性副作用。最后，本研究贴近临床需求，积极改进“梯次双靶”，解决了针对“大型肿瘤”、“不影响效果的前提下局部给药、降低给药剂量”、“第一靶药物专利过期”等问题，尝试了“梯次双靶抗实体瘤疗法”与“高强度聚焦超声”联用、介入下的“梯次双靶抗实体瘤疗法”、制剂学等各项减毒增效措施，并着眼未来，开发CA4P替代性药物及其监控方法，使该方法推向临床更加完善。