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.
