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的生物标志物 治疗肝素诱导的血小板减少症和血栓形成（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，有可能治疗患者的HIT/T。 HIT/T是一种获得性的危及生命的血小板减少和血栓形成并发症， 暴露于普通肝素（UFH）或低分子量肝素（LMWH）：最广泛使用的 世界上最好的抗凝血剂。每年约有1200万患者暴露于UFH和LMWH， 仅美国每年就有约24，000人发展HIT/T。HIT/T通常导致异常的血小板活化， 聚集导致a）血小板衍生的微粒释放，B）增加的凝血酶生成导致 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HIT/T的治疗仅限于阿加曲班（一种直接凝血酶 抑制剂）。不幸的是，这带来了严重出血的重大风险，限制了其有效性。最近 有证据表明，12-脂氧合酶（12-LOX）及其代谢产物12- 羟基二十碳四烯酸（12-HETE）是HIT/T潜在病理学的关键因素，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 有效抑制12-LOX，阻止异常血小板活化和血栓形成，并在许多疾病中显示出疗效。 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 治疗HIT/T的VLX-1005的开发正在迅速进展，重要的是要高度重视 用于评估VLX-1005的敏感生物标志物，因为它干预和预防发病率和死亡率， 观察疾病。为此，我们提出了2个目标，以解决临床试验准备的这一问题。 在目标1中，将评估凝血状态和血小板活化的几种生物标志物，包括 凝血酶-抗凝血酶（TAT）ELISA测定、12-HETE测定、光透射聚集测定（LTA）和全 在HIT/T小鼠模型中的血液阻抗聚集测定法（WBIA）。这些生物标志物将作为一个指标， VLX-1005对凝血和血小板活化的影响。由于VLX-1005尚未显示出效果， 凝血或纤维蛋白形成，我们不期望VLX-1005改变患者的凝血条件， 然而，由于血小板活化环境减少可能导致凝血酶活化减少，因此这些 生物标志物将告知VLX-1005是否改变上游凝血。目标2将采取生物标志物优化 并将其应用于离体人血液样品，以确定要报告的生物标志物的功效 VLX-1005对血液的影响生物标志物测定特征对于计划的VLX临床试验至关重要- 1005用于治疗HIT/T患者。

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.