【作者】 安洋；

【导师】 刘继华； 张银龙；

【作者基本信息】 吉林大学 ， 药物分析学， 2025， 博士

【摘要】 血栓性疾病是严重威胁人类生命健康的重大疾病之一,每年约有80%的心血管疾病患者死于血栓形成,已逐渐成为世界疾病负担的主要原因。目前,临床上常用的抗栓药物根据其作用机制和和靶点主要分为抗血小板药物（APAs）、抗凝药物和纤溶药物。其中APAs通过抑制血小板的聚集和活化以预防和减少血栓形成,已经成为血栓治疗的关键药物之一。但是,APAs的长期使用具有导致严重出血并发症的风险,限制了该类药物在临床上的安全应用。因此,在发生危及生命的出血或紧急侵入性手术前,需要及时清除APAs或采取相应的逆转策略减轻出血风险,快速有效地恢复正常的止血功能。然而,目前市场上并没有相关的逆转剂上市。临床上通常采用血小板输注的方式来中和血液中过量的APAs,但是血小板输注的治疗效果有限,且难以把控抑制出血风险与血栓形成间的平衡。因此,本研究从APAs容易引起出血并发症的痛点出发,结合点击化学及主客体化学等分子识别技术设计、合成了两类APAs逆转剂。实验结果证明逆转剂可以有效降低APAs引发的出血风险,为抗栓药物的临床应用提供安全保障。首先,本研究借助点击化学的优势设计合成了两种用于逆转APAs的功能化金纳米平台。由点击化学基团二苯并环辛炔（DBCO）修饰的金纳米颗粒（Au NPs）Au NPs-PEG-DBCO能与叠氮基团修饰的替格瑞洛（N₃-替格瑞洛）特异性结合,而马来酰亚胺（MAL）修饰的金纳米颗粒（Au NPs）Au NPs-PEG-MAL可以与氯吡格雷活性代谢产物（CLP-AM）结合。体外结合实验证实,Au NPs-PEG-DBCO与N₃-替格瑞洛的结合效率为88.0±3.43%,Au NPs-PEG-MAL与CLP-AM的结合效率为45.45±6.89%。表明功能化的纳米颗粒可以在室温条件下与抗血小板药物结合。体内尾尖出血模型实验表明,Au NPs-PEG-DBCO和Au NPs-PEG-MAL可显著缩短N₃-替格瑞洛和氯吡格雷尾尖出血时间,在最长出血时间点的逆转效率分别为43.85%和64.31%;体内急性肝损伤小鼠模型实验同样表明Au NPs-PEG-DBCO和Au NPs-PEG-MAL对N₃-替格瑞洛和CLP-AM的逆转效率分别为68.53%和62.12%,均高于血小板输注的逆转效率19.73%和28.25%。总之,体外血小板结合实验和体内尾尖出血试验以及急性肝损伤实验均验证了金纳米平台的有效性。综上,我们通过“一步法”成功制备了可以有效逆转抗血小板药物替格瑞洛和氯吡格雷的金纳米平台,并对其安全性和有效性进行了全面评估。该平台可通过简单的方法轻松制备,并具有逆转APAs的巨大潜力。其次,本研究针对一线APAs的疏水性结构共性,以一步化学偶联法构建了一种基于β-环糊精（β-CD）的链状分子容器透明质酸-环糊精（HA-CD）,其能够通过主客体相互作用广谱捕获和隔离APAs,降低有效血药浓度。体外血小板聚集实验表明,CLP-AM能够显著抑制ADP诱导的血小板聚集,聚集率从51.4%降低到7.1%。β-CD或HA-CD的引入可以分别将受抑制的血小板聚集率恢复至23.1%和24.7%。同样的,β-CD分别可以将替格瑞洛和阿司匹林抑制的血小板聚集率恢复至15.7%和20.8%,而HA-CD分别可以恢复至17.2%和22.1%。上述结果显示,HA-CD能够与APAs形成竞争性结合,恢复抗血小板药物抑制的血小板活性。此外,在体内实验中,无论是单一或双重抗血小板疗法下的尾尖出血模型和急性肝损伤模型均表明,HA-CD可以显著减少APAs引起的出血时间和出血量。总之,这项工作为安全使用APAs提供了一种简单、经济、广谱的逆转策略。更多还原

【Abstract】 Thrombophilia is one of the major diseases that threaten human life and health.About 80%of cardiovascular disease patients die from thrombosis every year,and it has gradually become a major cause of disease burden in the world.Currently,antithrombotic drugs commonly used in clinical practice are mainly categorized into antiplatelet drugs,anticoagulant drugs and fibrinolytic drugs according to their mechanism of action and targets.Among them,antiplatelet agents（APAs）have become one of the key drugs in thrombosis therapy by inhibiting platelet aggregation and activation to prevent and reduce thrombosis.However,bleeding complications associated with the long-term use of APAs have severely limited the widespread clinical use of this class of drugs.Therefore,in emergency situations,it is essential to rapidly reverse the effect of APAs and restore the hemostatic function of platelets.However,there are currently no specific antiplatelet reversal agents approved for clinical use.Platelet transfusions are commonly used clinically to neutralize excess APAs in the blood,but the therapeutic efficacy of platelet transfusions is limited and it is difficult to control the balance between inhibiting the risk of bleeding and thrombosis.Here,in this study,from the pain point that APAs are prone to cause bleeding complications,we designed and synthesized two types of APAs reversal agents by combining molecular recognition techniques,such as click chemistry and subject-object chemistry,which practically reduced the risk of APAs-induced bleeding,and provided a safety guarantee for the clinical application of antithrombotic drugs.First,we designed and synthesized two functionalized gold nanoplatform to reverse APAs with the advantage of click chemistry in this study.The gold nanoparticles（Au NPs）,modified with dibenzocyclooctyne or maleimide on their surface,are able to neutralize the antiplatelet activity of azido-modified ticagrelor and clopidogrel,respectively.In vitro binding assays confirmed that the binding efficiency of Au NPs-PEG-DBCO with N₃-ticagrelor was 88.0±3.43%,and the binding efficiency of Au NPs-PEG-MAL with CLP-AM was 45.45±6.89%.This indicates that functionalized nanoparticles can bind to antiplatelet drugs at room temperature.In vivo experiments using tail-tip bleeding models showed that Au NPs-PEG-DBCO and Au NPs-PEG-MAL significantly shortened the bleeding time of N₃-ticagrelor and clopidogrel,with reversal efficiencies of 43.85%and 64.31%at the longest bleeding time point,respectively.Similarly,in an acute liver injury mouse model,the reversal efficiencies of Au NPs-PEG-DBCO and Au NPs-PEG-MAL for N₃-ticagrelor and CLP-AM were 68.53%and 62.12%,respectively,both of which were higher than the reversal efficiencies achieved by platelet transfusion（19.73%and 28.25%,respectively）.In summary,our study introduces a gold nanoplatform that can be easily prepared through a“one-step”method and has great potential to be used for the functional reversal of various APAs.Secondly,in this work,a chained molecular container based on cyclodextrins was constructed in a one-step chemical coupling method for the commonality of hydrophobic structures of APAs,which is capable of broad-spectrum trapping and sequestration of APAs through host-guest interactions.In vitro platelet aggregation experiments demonstrated that CLP-AM could significantly inhibit ADP-induced platelet aggregation,reducing the aggregation rate from 51.4%to 7.1%.The introduction ofβ-CD or HA-CD could restore the inhibited platelet aggregation rate to23.1%and 24.7%,respectively.Similarly,β-CD could restore the platelet aggregation rates inhibited by ticagrelor and aspirin to 15.7%and 20.8%,respectively,while HA-CD could restore them to 17.2%and 22.1%,respectively.These results indicate that the molecular container HA-CD can form competitive binding with APAs,restoring the activity of platelets inhibited by antiplatelet drugs.Furthermore,both the in vivo tail bleeding model and acute liver injury model under either single or dual antiplatelet therapy demonstrated that it significantly reduced bleeding.Overall,this work provides a simple,cost-effective,and broad-spectrum reversal strategy for the safe use of APAs.更多还原