仿刺参糖胺聚糖吸收和代谢特征与体内抗凝血活性分子机制研究

Holothurian glycosaminoglycan (HGAG) is a sulfated polysaccharide isolated from marine organisms and its chemical structure with backbone of chondroitin sulfate and sulfated fucose branches is unique. Studies indicated that HGAG has potent anticoagulant and antithrombotic activities. HGAG via oral administration possessed obvious activities, less bleeding tendency and wider therapeutic window than heparin, which could meet the clinical demand for the treatment of thrombotic diseases. However, the structure and molecular weight of HGAG with low oral bioavailability was heterogeneous, so that its trace analysis in vivo was much difficult. Therefore, the undefined absorption and transport mechanism as well as metabolic pathways of HGAG resulted in unclear anticoagulant molecular mechanism in vivo. So that the research and development of HGAG as well as its clinical use is limited seriously. In this study, Apostichopus japonicus holothurian glycosaminoglycan (AHG) will be studied through polysaccharide, disaccharide and monosaccharide analysis. The sensitive analytical methods for the determination of AHG will be established. The experimental models and methods on animal level, organ level, cellular level and molecular level will be employed to elucidate the absorption and metabolism characters. The active form of AHG in vivo will be revealed and its anticoagulant molecular mechanism will be clarified. The results would provide very important scientific basis for further research and development.

海参糖胺聚糖（HGAG）是一类海洋来源的结构独特的硫酸酯化多糖，主链具有硫酸软骨素类骨架，并带有硫酸岩藻糖支链，具有显著的抗凝血及抗血栓活性，与肝素相比，具有口服有效、出血风险低、治疗窗宽的特点，符合血栓性疾病临床治疗需求。但HGAG结构和分子量不均一，口服生物利用度低，体内微量分析困难，其吸收转运机制和代谢途径尚不明确，体内抗凝血活性分子机制不清楚，严重影响其进一步研究开发和临床应用。本研究拟以仿刺参糖胺聚糖（AHG）为研究对象，基于其结构特点，通过多糖整体、主链二糖和单糖组成分析，建立灵敏的微量定性定量多角度分析方法，应用动物水平、器官水平、细胞水平和分子水平的多层次实验模型和研究方法，明确AHG的吸收代谢特征，揭示其体内活性形式，阐明体内抗凝血活性分子机制，为AHG的进一步研究和开发提供重要科学依据。

海参糖胺聚糖（HGAG）是一类海洋来源的岩藻糖基化硫酸软骨素，具有显著的抗凝血及抗血栓活性，口服有效、出血风险低、治疗窗宽，符合血栓性疾病临床治疗需求。但HGAG结构和分子量不均一，体内微量分析困难，其吸收转运特征和机制尚不明确，体内抗凝血活性分子机制不清楚，严重影响其进一步研究开发和临床应用。本研究以仿刺参糖胺聚糖（AHG）为研究对象，制备了不同分子量的解聚AHG（DAHGs）和脱硫酸酯基衍生物（dsAHG）、脱岩藻糖支链衍生物（dfAHG）、羧基还原衍生物（reAHG），应用多种方法对其结构进行了表征。建立了AHG及DAHGs的高效液相色谱结合柱后衍生定量分析方法，并基于主链二糖分析策略建立了生物样品中DAHGs的LC-MS/MS定量分析方法，为研究其吸收转运特征和体内药物动力学过程提供了方法学基础。首次在细胞、组织器官和动物水平阐明了AHG及DAHGs的吸收和转运特征和机制，揭示了AHG及DAHGs的吸收转运涉及膜动转运机制，证实其可在肠道被吸收进入体循环，但生物利用度低（<2%），体内活性形式可能是原型药物。硫酸酯基、岩藻糖支链和羧基是AHG抑制FIXa-FVIIIa-Ca2+-磷脂内源性酶复合物、FIIa、FXa的必需基团。DAHG-3和DAHG-2在体内外对FXII无激活作用，安全性高。通过建立PK-PD结合模型，阐明了大鼠体内DAHGs抗凝血作用的“时间-浓度-效应”之间的关系，发现DAHGs在体内抑制凝血因子效应强度与其药物浓度呈现良好的相关性，其原型形式可通过抑制FIXa-FVIIIa-Ca2+-磷脂内源性酶复合物、FIIa、FXa的活性发挥抗凝血作用。本研究为DAHGs作为口服抗凝血药物进一步研究和开发提供了重要的科学依据。