Contribution of Structural Motifs to Heparin Clearance

说明(申请人提供)：肝素是一种广泛使用和重要的药物，特别是对美国老龄化的人口。它是一种以碳水化合物为基础的抗凝剂，被老年患者用于许多应用，包括血栓性疾病、手术和肾脏透析。该项目的长期目标是创造比现有药物更安全、更有效、更适合不同应用的合成肝素药物。由于其骨架上含有丰富的硫酸盐基团，肝素具有高度可变的结构，其化学结构决定了其生物效应。肝素对老年患者来说很难给药，而且有很好的文献记载的出血副作用和污染问题。此外，不同的肝素应用需要不同的药物清除率；例如，术中的抗凝作用应在给药停止后迅速消除，而在深静脉血栓治疗中，抗凝作用应延长。尽管市场上有几种肝素药物，但对具有同源结构和受控清除率的肝素的需求很大。我们建议确定不同的结构基序对肝素清除速率的影响，目的是开发具有不同细胞内化和清除速率的结构定义的肝素类似物。为了实现这一目标，我们将使用一种独特的化学酶方法来合成具有特定硫化模式、硫化密度和长度的放射性标记肝素结构。这些结构的内化率将在实验细胞系(FLP-in 293细胞)中进行测试，并将使用生化分析来确定它们与肝素清除受体HARE的结合亲和力。这些构建物还将在人体内皮细胞中进行测试，众所周知，内皮细胞在体内可以内化和降解肝素。最后，我们将制备稳定的具有不同内化率的同位素标记的构建体，并在小鼠模型中测试它们的清除和抗凝活性。通过这些研究，我们希望阐明控制肝素清除的特定碳水化合物结构，这将是开发针对特定应用和患者量身定做的抗凝药物的有力工具。

DESCRIPTION (provided by applicant): Heparin is a widely-used and important drug, particularly for the aging US population. It is a carbohydrate-based anticoagulant that used by elderly patients for many applications, including thrombotic disorders, in surgery, and during kidney dialysis. The long-term goal of this project is to create synthetic heparin drugs that are safer, more effective, and better tailored to different applications than the currently-available drugs. Heparin has a highly-variable structure due to the abundance of sulfate groups along its backbone, and its chemical structure determines its biological effect. Heparin is difficult to dose for elderly patients, and has well- documented bleeding side effects and contamination issues. In addition, different heparin applications require different rates of drug clearance; for example, the anticoagulant effect during surgery should be eliminated quickly after administration stops, while in deep vein thrombosis treatment the effect should be prolonged. Although there are several heparin drugs on the market, there is a significant need for heparins with homologous structures and controlled rates of clearance. We propose to determine the effects of different structural motifs on the rate of heparin clearance with the aim of developing structurally-defined heparin analogs with varied cellular internalization and clearance rates. To achieve this goal, we will use a unique chemoenzymatic method to synthesize radioactively-labeled heparin constructs having defined sulfation patterns, sulfation densities and lengths. The internalization rates of these constructs will be tested in an experimental cell line (Flp-In 293 cells), and their binding affinities to the heparin clearance receptor, HARE, will be determined using biochemical assays. The constructs will also be tested in human endothelial cells, which are known to internalize and degrade heparin in vivo. Lastly, we will prepare stable isotopically-labeled constructs having different internalization rates and test their clearance and anticoagulant activity in a mouse model. From these studies, we hope to elucidate the specific carbohydrate structures that control heparin clearance, which will be a powerful tool in creating anticoagulant drugs that are tailored to specific applications and patients.