Biomarkers for 12-lipoxygenase inhibition as a therapeutic intervention for heparin-induced thrombocytopenia and thrombosis (HIT/T)

该项目的目标是开发用于评估VLX-1005的高度敏感和可重复性的生物标志物 治疗肝素引起的血小板减少和血栓形成(HIT/T)。VLX-1005，一种12-LOX抑制剂 有效抑制免疫介导的血栓形成，包括HIT/T，具有治疗HIT/T患者的潜力。 HIT/T是一种获得性危及生命的血小板减少和血栓并发症，发生在患者身上 接触普通肝素(UFH)或低分子肝素(LMWH)：使用最广泛 世界上最大的抗凝血剂。在中国，每年约有1200万患者接触UFH和LMWH 仅美国每年就有大约24,000个开发中的HIT/T。HIT/T经常导致异常的血小板活化和 聚集导致a)血小板衍生的微粒释放，b)增加凝血酶生成导致 导致静脉肢体坏疽的灾难性动脉和静脉血栓形成，c)深静脉血栓形成， 和d)肺栓塞和死亡。目前对HIT/T的治疗仅限于阿加曲班(一种直接凝血酶 抑制物)。不幸的是，这带来了严重出血的重大风险，限制了其有效性。近期 有证据表明，酶12-脂氧合酶(12-LOX)及其代谢产物12-LOX- 羟基二十碳四烯酸(12-HETE)是HIT/T潜在病理的关键因素，包括 促进血小板反应性、血栓形成/稳定性以及血管闭塞。VLX-1005选择性和 有效地抑制12-LOX，阻止异常的血小板激活和血栓形成，并已在许多 动物模型，包括肠系膜小动脉损伤和激光诱导的提睾肌小动脉血栓形成。而当 治疗HIT/T的VLX-1005的开发进展迅速，具有高度的 评估VLX-1005的敏感生物标志物，因为它经常干预和预防发病率和死亡率 与疾病一起观察。为此，我们建议2旨在解决这一对临床试验准备情况的担忧。 在目标1中，将评估几个凝血状态和血小板激活的生物标志物，包括 凝血酶-抗凝血酶(TAT)酶联免疫吸附试验、12-HETE试验、光传输聚集法(LTA)和 HIT/T小鼠模型的血液阻抗聚集法(WBIA)这些生物标志物将作为一个指标 VLX-1005对凝血和血小板激活的影响。由于VLX-1005尚未显示出效果 凝血或纤维蛋白形成，我们预计VLX-1005不会改变患者的凝血条件， 然而，由于减少的血小板激活环境可能导致凝血酶激活减少，这些 生物标志物将告知VLX-1005是否正在改变上游凝血。目标2将采取生物标志物优化 并将其应用于体外人体血液样本，以确定生物标志物的有效性，以报告 VLX-1005对血液有影响。生物标志物分析对于计划中的VLX临床试验是必不可少的- 1005用于治疗HIT/T。

The goal of this project is to develop highly sensitive and reproducible biomarkers for assessment of VLX-1005 treatment of heparin-induced thrombocytopenia and thrombosis (HIT/T). VLX-1005, a 12-LOX inhibitor that potently inhibits immune-mediated thrombosis including HIT/T, has the potential to treat HIT/T in patients. HIT/T is an acquired life-threatening thrombocytopenic and thrombotic complications that occur in patients exposed to unfractionated heparin (UFH) or low molecular weight heparin (LMWH): the most widely used anticoagulants in the world. Approximately 12 million patients are exposed to UFH and LMWH annually in the US alone with ~24,000 developing HIT/T every year. HIT/T often results in aberrant platelet activation and aggregation leading to a) platelet-derived microparticle release, b) increased thrombin generation leading to catastrophic arterial and venous thrombosis that results in venous limb gangrene, c) deep venous thrombosis, and d) pulmonary embolism and death. Current treatment of HIT/T is limited to argatroban (a direct thrombin inhibitor). Unfortunately, this carries a significant risk of severe bleeding limited its effectiveness. Recent evidence has established that the enzyme 12- lipoxygenase (12-LOX), and its metabolic product, 12- hydroxyeicosatetraenoic acid (12-HETE) are key contributors to the underlying pathology of HIT/T including promoting platelet reactivity, thrombus formation/stability as well as vessel occlusion. VLX-1005 selectively and potently inhibits 12-LOX, halts aberrant platelet activation and thrombosis and has shown efficacy in numerous animal models, including mesenteric arteriole injury and laser-induced cremaster arteriole thrombosis. While the development of VLX-1005 for treatment of HIT/T is rapidly progressing, it will be important to have highly sensitive biomarkers for assessment of VLX-1005 as it intervenes and prevents morbidity and mortality often observed with the disease. To this end, we propose 2 Aims to address this concern for clinical trial readiness. In Aim 1, several biomarkers for coagulation state and platelet activation will be assessed including the thrombin-antithrombin (TAT) ELISA assay, 12-HETE assay, light transmission aggregometry (LTA), and whole blood impedance aggregometry (WBIA) in mouse models of HIT/T. These biomarkers will serve as an indicator of VLX-1005 effects on both coagulation and platelet activation. As VLX-1005 has not been shown to effect coagulation or fibrin formation, we do not expect VLX-1005 to alter coagulation conditions in the patient, however since a decreased platelet activation environment may result in decreased thrombin activation, these biomarker will inform if VLX-1005 is altering upstream coagulation. Aim 2 will take the biomarker optimization from Aim 1 and apply it to human blood samples ex vivo to determine the efficacy of the biomarkers to report VLX-1005 effects in the blood. The biomarker assay profile is essential for the planned clinical trials with VLX- 1005 for treatment of patients with HIT/T.