The free, or non-bound, fraction of many drugs and hormones in blood or serum is of great interest to clinical and pharmaceutical chemists because this fraction is believed to represent the active form of many such agents. As a result, it is the free fraction of these drugs and hormones that should ideally be measured as a tool for patient diagnosis and treatment. However, there is currently no general and fast approach for measuring the free fractions of drugs and hormones in clinical samples. The overall goals of this proposal are 1) to obtain a better understanding of drug and hormone interactions with their binding agents in blood or serum and 2) to use this information to develop fast, reliable free drug and hormone assays. The underlying hypothesis of this project is that a new class of improved free drug and hormone assays can be created based on the techniques of ultrafast affinity extraction and chromatographic immunoassays, as recently demonstrated for compounds such as warfarin, thyroxine, phenytoin and carbamazepine. Future studies will build on these previous efforts by considering new analytes and approaches that can be employed in such work. Particular emphasis will be given to high-throughput methods and techniques that can be adapted for the detection of a broad range of analytes. Advances to be explored in the field of binding studies will include the development of rapid affinity methods for examining both the extent and rate of drug or hormone interactions with serum agents, and the use of affinity microcolumns as tools in personalized medicine to study changes in drug interactions with variants of serum proteins. Improved tools to be created for free drug and hormone measurements will include ultra-high capacity affinity supports for use in fast drug or hormone extractions, chromatographic immunoassays with on-column detection, multi-dimensional systems for free drug/hormone assays, the use of serum protein columns to carry out free drug/hormone assays, and improved analytical platforms for improving the flexibility and sample throughput when using chromatographic immunoassays with near infrared fluorescent labels. An immediate impact of this work will be a better understanding of how drugs and hormones interact with serum binding agents and are transported in the circulatory system. In addition, the new techniques that will be created for free drug/hormone measurements should greatly improve the ability of clinicians and pharmaceutical chemists to study and examine the biological effects of drugs and hormones in the body. This capability should lead to better protocols in clinical testing and in personalized medicine for the treatment and diagnosis of patients suffering from a variety of disorders. The various affinity-based approaches developed in this project for examining solute-ligand binding, rapidly extracting analytes from biological samples, and measuring trace levels of such analytes should also be valuable in many areas of biochemical research, including proteomics, metabolomics and the characterization or screening of drug candidates.

Public Health Relevance

This work will provide a better understanding of how drugs and hormones interact with serum binding agents and will result in faster, more convenient techniques for free drug and hormone measurements in clinical and pharmaceutical samples. The tools created in this work for binding studies and affinity-based separations should also be useful for biomedical research in areas such as proteomics, metabolomics and the screening of drug candidates.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
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Edmonds, Charles G
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University of Nebraska Lincoln
Schools of Arts and Sciences
United States
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Zheng, Xiwei; Li, Zhao; Beeram, Sandya et al. (2014) Analysis of biomolecular interactions using affinity microcolumns: a review. J Chromatogr B Analyt Technol Biomed Life Sci 968:49-63
Zheng, Xiwei; Li, Zhao; Podariu, Maria I et al. (2014) Determination of rate constants and equilibrium constants for solution-phase drug-protein interactions by ultrafast affinity extraction. Anal Chem 86:6454-60
Zheng, Xiwei; Matsuda, Ryan; Hage, David S (2014) Analysis of free drug fractions by ultrafast affinity extraction: interactions of sulfonylurea drugs with normal or glycated human serum albumin. J Chromatogr A 1371:82-9
Sobansky, Matthew R; Hage, David S (2014) Analysis of drug interactions with very low density lipoprotein by high-performance affinity chromatography. Anal Bioanal Chem 406:6203-11
Pfaunmiller, Erika L; Anguizola, Jeanethe A; Milanuk, Mitchell L et al. (2014) Development of microcolumn-based one-site immunometric assays for protein biomarkers. J Chromatogr A 1366:92-100
Moser, Annette C; Willicott, Corey W; Hage, David S (2014) Clinical applications of capillary electrophoresis based immunoassays. Electrophoresis 35:937-55
Anguizola, Jeanethe; Joseph, K S; Barnaby, Omar S et al. (2013) Development of affinity microcolumns for drug-protein binding studies in personalized medicine: interactions of sulfonylurea drugs with in vivo glycated human serum albumin. Anal Chem 85:4453-60
Queralt, Anna Vallverdu; Fragou, Domniki; Panoutsopoulou, Kalliope et al. (2013) Bioanalysis young investigator: award 2013. Bioanalysis 5:1341-5
Anguizola, Jeanethe; Matsuda, Ryan; Barnaby, Omar S et al. (2013) Review: Glycation of human serum albumin. Clin Chim Acta 425:64-76
Jackson, Abby J; Anguizola, Jeanethe; Pfaunmiller, Erika L et al. (2013) Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin. Anal Bioanal Chem 405:5833-41

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