Contribution of Structural Motifs to Heparin Clearance

描述（由申请人提供）：肝素是一种广泛使用的重要药物，特别是对于美国老龄化人群。它是一种基于碳水化合物的抗凝剂，被老年患者用于多种应用，包括血栓性疾病、手术和肾透析。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。肝素由于其主链沿着富含硫酸基团而具有高度可变的结构，并且其化学结构决定其生物学效应。肝素对于老年患者来说难以给药，并且具有充分记录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，手术期间的抗凝作用应在给药停止后迅速消除，而在深静脉血栓形成治疗中，该作用应延长。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仍存在显著需求。我们建议确定不同结构基序对肝素清除率的影响，目的是开发具有不同细胞内化和清除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法来合成具有确定的硫酸化模式、硫酸化密度和长度的放射性标记的肝素结构。将在实验细胞系（Flp-In 293细胞）中测试这些构建体的内化速率，并且将使用生物化学测定来确定它们与肝素清除受体HARE的结合亲和力。还将在人内皮细胞中测试构建体，已知人内皮细胞在体内内化和降解肝素。最后，我们将制备具有不同内化速率的稳定同位素标记的构建体，并在小鼠模型中测试其清除率和抗凝活性。从这些研究中，我们希望阐明控制肝素清除的特定碳水化合物结构，这将是创造针对特定应用和患者的抗凝药物的有力工具。 公共卫生相关性：肝素是手术、肾透析和血栓形成治疗中常用的抗凝剂。该项目旨在阐明不同的肝素结构如何控制肝素从体内清除的速率，这将允许创建更安全且专门针对不同治疗的肝素。

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. PUBLIC HEALTH RELEVANCE: Heparin is a common anticoagulant used during surgery, kidney dialysis and the treatment of thrombosis. This project aims to elucidate how different heparin structures control the rates at which heparin is cleared from the body, which would allow the creation of heparins that are safer and specifically tailored to different treatments.