The free, or non-bound, form of many drugs and hormones in blood or serum is of great interest since this is believed to represent the active form of these compounds, making it the ideal analytical tool for patient diagnosis or treatment. But there is currently no general, fast approach for measuring free solute levels within the body. Recent work with L-thyroxine and R/S-warfarin has demonstrated that free solute measurements are possible by using millisecond-scale affinity extractions and on-line chromatographic immunoassays. This proposal seeks to further develop and refine this approach for its use with other analytes. The first part of this project will consider several new applications for these assays (e.g., therapeutic monitoring and pharmacological studies). Alternative assay formats will also be examined, such as 1) the use of LC/MS or near-infrared fluorescent labels for detection, 2) the use of monolithic supports in affinity extraction columns, 3) the use of aptamers as ligands I for these columns, and 4) the development of HPLC displacement or immunometric assays for quantitating the extracted drugs and hormones. The second part of this project will use computer simulations and model systems to optimize these methods. Specific items to be examined will include: 1) the degree of extraction that is needed for free drug/hormone analysis by affinity extraction, 2) the effect on these assays when multiple binding agents for a solute are in a sample, and 3) the affinities and dissociation kinetics that are needed for ligands used in such assays. The last section will examine the interactions of new analytes with their binding agents in serum or plasma to aid in the development of the free solute assays. This will involve the use of several affinity methods based on HPLC, CE or LC/MS to 1) identify' the agents in serum or plasma that interact with each new candidate for free fraction measurements, 2) determine the equilibrium constants for these interactions, and 3) estimate the rare constants for these processes (e.g., the rare of analyte release from its binding agents). The result of this work should be the creation of new, more rapid tools for determining free drug or hormone levels, providing a better means for studying how such compounds behave and interact within our bodies.
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