基于Massall技术的从亚细胞到整体水平的PEG化吉西他滨药代动力学研究

Gemcitabine is the first-line chemotherapy drug for pancreatic cancer, non-small cell lung cancer, metastatic bladder cancer and metastatic breast cancer. However, there are still some limitations in its clinical application, such as short biological half-life, toxic and side effects, drug resistance and so on. Modification of gemcitabine with PEG at the 4-N site is an effective method to solve the above problems, which could reduce the metabolic inactivation caused by cytidine deaminase and accumulate the drug in tumor tissues through EPR effect. However, PEG modification could lead to changes of the pharmacokinetic behavior of small molecule chemotherapeutic drugs in vivo, especially in DNA-rich organelles in tumor cells. For the PEGylated gemcitabine, there has always been the drug release process after dosing. At least four kinds of pharmaceuticals (PEGylated gemcitabine, released PEG, released gemcitabine and its metabolites) would be present in vivo. The dynamic changes of these substances in vivo are much more complex than small molecule drug delivery system, which would have an important impact on its pharmacology and toxicology. Therefore, it is needed to establish a precise and reliable analytical method to systematically study and explain the pharmacokinetic behavior of PEGylated drugs. Due to the broad continuous mass spectra generated by the large size and charge distributions of PEGylated molecules, this project intends to establish an LC-MS/MS method based on Massall technology to elucidate the release, transfer, metabolism and other dynamic behavior of PEGylated gemcitabine in organelles, cells, plasma and tissues. This study will provide a scientific basis for the design of pegylated drugs and the safety and effectivity evaluation.

吉西他滨是胰腺癌、非小细胞肺癌等的一线治疗药物，但临床应用有局限性：半衰期短，毒副作用大，多种癌症出现耐药性。在4-N位点进行PEG修饰是解决问题的重要方法，既减少脱氨酶导致的代谢失活，又通过EPR效应靶向肿瘤。但PEG修饰会导致体内DMPK行为的改变。给药后到达肿瘤细胞内靶点的过程中一直存在药物的释放、分布、代谢过程，体内会同时存在PEG化吉西他滨、PEG、吉西他滨及代谢物等多种成分，这些物质在体内的动态变化远较小分子给药系统复杂，会对药效、毒性带来重要影响。因此，亟需建立精准可靠的分析方法对PEG化药物的DMPK行为进行系统的研究。针对PEG化药物分子量不唯一且呈正态分布特点，本课题拟建立基于Massall的LC-MS/MS分析方法，从亚细胞到整体水平阐明PEG化吉西他滨在靶细胞、靶组织、系统水平的分布、释放、转移、代谢等动力学行为，为PEG化药物的设计及安全性有效性评价提供理论依据。

化疗药物吉西他滨临床应用存在局限性，PEG修饰能改善其多种缺陷，但是PEG修饰之后，PEG化小分子药物体内的药代动力学行为仍然需要精准的监控，这对于PEG化药物的开发是十分重要的。本项目以PEG化吉西他滨为研究对象, 建立了基于数据非依赖性采集技术（Massall）的能够分析PEG化吉西他滨的体内多形态成分（PEG化吉西他滨、释放的游离吉西他滨和吉西他滨代谢产物dFdU）的UPLC-MS/MS分析方法, 并将此方法应用于从细胞水平到整体动物水平多尺度PEG化吉西他滨的纳米药代动力学研究中。结果表明，相对于普通注射制剂，PEG修饰可延长药物血浆半衰期，同时血浆和组织中较低的游离吉西他滨浓度表明毒副作用降低。通过量化PEG化吉西他滨在体内不同状态、不同部位的经时浓度变化，明确其相对于吉西他滨给药后的分布和代谢差异，为PEG化小分子药物安全性评价提供有效证据。同时，在原有研究计划基础上，本课题建立了基于源内裂解技术（In-source CID）的液相色谱串联质谱技术，并应用于药用高分子辅料PEG-PLA的药代动力学研究，为科学设计新型高分子药用辅料及其相关安全性评价奠定基础。在本基金的资助下，已发表SCI论文7篇（均已标注基金号），申报专利1项，培养硕士研究生3人。