Receptor properties (number and affinity of receptors) have wide-ranging implications. Due to lack of methods for studying receptors in vivo, little is known about the role of receptor pathophysiology in diseases, regulatory processes, drug efficacy and toxicology. In vitro methods for studying receptors cannot determine the quantity present in the body because only an unknown fraction of the receptors can be sampled due to the heterogeneously dispersed location in the body. The physical and biochemical integrity of the receptor tissue may also be influenced by in vitro preparations and introduce experimental artifacts. There are no methods available for studying receptor properties in vivo to overcome these problems. Endogenous regulatory proteins and hormones, and their equivalent recombinant biopharmaceuticals, are often primarily eliminated via binding to specific receptors through which they exert their action. The pharmacokinetics (PK) of such substances depend on their receptor binding. Previous attempts to use PK analysis to determine the in vivo receptor properties have been unsuccessful. Overall Study Hypothesis: it is possible to characterize the in vivo properties of receptors by PK using tracers. Objective: to determine the merit of an entirely new in vivo PK approach, the Tracer Interaction Method, TIM, that uses a highly sensitive, novel tracer methodology. Erythropoietin receptors (EPO-R) will be used in the study because EPO is eliminated via a receptor-mediated process.
Aim 1 : To analyze EPO-R properties in vivo in adult sheep by TIM and correlate the results to PK parameters obtained by other methods and in vitro measurements.
Aim 2 : To analyze EPO-R properties in vivo in sheep by TIM and correlate the results to a pharmacokinetic/pharmacodynamic (PK/PD) analysis using reticulocytes and hemoglobin as primary PD outcome variables. Methods: Controlled phlebotomies will be done in sheep to create various degrees of anemia sufficient to result in acute increases in endogenous EPO plasma levels and induce changes in the EPO-R pool. TIM will be applied at different stages of receptor induction. Receptor parameters from TIM will be correlated to PK parameters determined by a regular tracer kinetic method and by a system analysis-based approach (DDA). Using metabolic activity, FACS flow cytometry and binding analysis, in vitro measurements of EPO-R on erythroid progenitor cells from bone marrow aspirates will be correlated to in vivo receptor parameters derived by TIM. By filling a void in receptor analysis TIM will potentially have a high impact in basic medical research with direct bearing on development of more efficacious and specifically acting drugs, improved understanding of the mechanism of drugs, hormonal regulation and optimized drug treatment.
Showing the most recent 10 out of 15 publications
