RECEPTOR BINDING DETERMINES ERYTHROPOIETIN DISPOSITION

受体结合决定促红​​细胞生成素的分布

• 批准号: 6030919
• 负责人: Donald M Mock
• 金额: $ 11.79万
• 依托单位: ARKANSAS CHILDREN'S HOSP (LITTLE ROCK)
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-10 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6030919
• 关键词: anemia; biomarker; biotin; bone marrow; erythropoietin; growth factor receptors; hematopoietic stem cells; human tissue; newborn animals; pharmacokinetics; receptor binding; recombinant proteins; sheep

The many red cell transfusions administered to preterm infants as treatment for anemia are expensive and place the recipients at risk for blood-borne infections and other transfusion related complications. The long-term goal of this research project is to develop effective strategies for optimal administration of recombinant human erythropoietin (r-HuEPO) to treat anemia in preterm infants- although other patient populations are likely to benefit as well. Achieving this goal is particularly important and timely because r-HuEPO remains an evolving clinical therapy for this group without a clear consensus of how, when, and for whom it should be used. This proposal seeks funding to develop sensitive and accurate tracer methodologies for EPO as a first step towards later mechanistic pharmacokinetic (PK) and pharmacodynamic (PD) studies in human subjects that will test the following hypothesis: The disposition and the efficacy of r-HuEPO are determined primarily by the number and affinity of EPO receptors in the body; this pool of receptors is finite and saturable. In order to conduct the mechanistic human studies, we must be able to measure tracer quantities of labeled r-HuEPO in plasma to avoid saturating the hypothesized receptor mediated mechanism. Radioactively labeled tracer r-HuEPO could theoretically serve this purpose. However, radiation exposure of experimental subjects (e.g., bone marrow transplant patients and premature infants) is not acceptable. Use of biotinylation biotechnology offers a safe alternative that is sufficiently sensitive for tracer PK studies. Our specific aims are as follows: 1) To biotinylate r-HuEPO. Biotinylation will be accomplished by carbohydrate moiety oxidation followed by biotinylation using biotin hydrazide. 2) To develop assays for specifically measuring BioEPO in tracer amounts. These assays will be developed in a collaboration that combines sensitive, specific assays for EPO (Dr. Widness) and cutting edge detection systems for biotinylated molecules (Dr. Mock). 3) To determine whether biotinylated r-HuEPO (BioEPO) has retained biologic activity and PK behavior identical to native r-HUEPO. Molecular structure and biological activity will be assessed in vitro by antibody binding studies, receptor binding studies using human bone marrow, and growth stimulation studies using hematopoietic precursor cells. Pharmacokinetic studies will be conducted in lambs. The technology developed here will be used for later mechanistic studies of human PK and PD of EPO that will be the subject of subsequent proposals. The knowledge gained from these studies will permit optimal r-HuEPO use. In addition, the biotinylation approach potentially offers a new way to study the mechanism(s) of removal and action of other cytokines.

对早产儿给药的许多红细胞输血 贫血的治疗很昂贵，并使接收者面临 血传播感染和其他与输血有关的并发症。 这 该研究项目的长期目标是发展有效 重组人的最佳管理策略 促红细胞生成素（R-Huepo）治疗早产儿的贫血 - 尽管 其他患者人群也可能受益。 实现这一目标 目标尤其重要，并且及时，因为R-Huepo仍然是 该组不断发展的临床疗法，没有明确共识 如何，何时以及应该使用它。该建议寻求资金 为EPO开发敏感和准确的示踪方法 迈向后来的机械药代动力学（PK）和 人类受试者的药效学（PD）研究将测试 遵循假设：R-Huepo的性格和功效是 主要由EPO受体的数量和亲和力确定 身体;这种受体池是有限且可饱和的。 为了 进行机械性人类研究，我们必须能够测量示踪剂 等离子体中标记的R-Huepo的数量，以避免饱和 假设的受体介导的机制。 放射性标记的示踪剂 R-Huepo理论上可以实现此目的。 但是，辐射 暴露实验受试者（例如，骨髓移植患者 和早产婴儿）是不可接受的。使用生物素化 生物技术提供了一种安全的替代方案 用于示踪剂PK研究。 我们的具体目的如下：1） 生物素基r-Huepo。 生物素化将通过碳水化合物完成 部分氧化，然后使用氢酰胺生物素化。 2） 开发用于特异性测量示踪剂量的生物epo的测定。 这些测定将在合作的合作中开发 敏感的EPO（Dr. widness）和尖端的特定分析 生物素化分子的检测系统（Mock博士）。 3）到 确定生物素化的R-Huepo（BioePo）是否保留了生物学 活性和PK行为与天然R-Huepo相同。 分子 结构和生物学活性将在体外通过抗体评估 结合研究，使用人骨髓的受体结合研究， 使用造血前体细胞的生长刺激研究。 药代动力学研究将在羔羊中进行。 技术 此处开发的将用于人类PK的以后机械研究 和EPO的PD，这将是随后的提案的主题。 这 从这些研究中获得的知识将允许最佳的R-Huepo使用。 此外，生物素化方法可能提供了一种新的方法 研究其他细胞因子的去除和作用的机制。