Characterization Of Drug Interactions With Serum Proteins by Using High-Performance Affinity Chromatography

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding

Characterization Of Drug Interactions With Serum Proteins by Using High-Performance Affinity Chromatography

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding

Pharmaceutical And Biomedical Applications Of Affinity Chromatography: Recent Trends And Developments

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered

Pharmaceutical And Biomedical Applications Of Affinity Chromatography: Recent Trends And Developments

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered

Affinity Chromatography in Environmental Analysis and Drug-Protein Interaction Studies

This dissertation will examine the use of novel affinity sorbents to extract emerging contaminants from water. These contaminants include carbamazepine, an anti-epileptic drug which is resistant to natural degradation in the environmental and to drinking water treatment procedures. This drug has been found in fish, drinking water, estuarine and coastal waters, and river sediment and has been used as a general marker of contaminants in wastewater. Carbamazepine was one of the most commonly detected compounds in surface-water and groundwater samples in a recent reconnaissance study of untreated drinking water sources in the U.S. Besides using this drug as a representative contaminant for testing albumin-based extraction methods, other sections of this dissertation will include a discussion of the combination of on-line immunoextraction using anti-carbamazepine antibodies with RPLC/MS. Research will be presented involving the use of this method with molecularly imprinted polymers (MIPs) to extract emerging contaminants from water. Other studies in this dissertation will include the use of serum protein columns to not only retain drugs but to provide chiral separations. This approach will be used to examine the retention of some chiral drugs by the serum protein α1-acid glycoprotein. Another part of this dissertation will include a discussion of how chromatographic theory can be used to describe the binding and extraction behavior of albumin columns when used to retain emerging contaminants. In addition, it will be shown how the same types of protein columns can be used to examine the kinetics of drug-protein interactions. Possible future directions for this work will also be discussed. Adviser: David S. Hag

Environmental Analysis By On-Line Immunoextraction And Reversed-Phase Liquid Chromatography: Optimization Of The Immunoextraction/RPLC Interface

The use of antibodies in HPLC columns for on-line immunoextraction combined with reversed-phase liquid chromatography (RPLC) is of growing interest in environmental and agricultural analysis. This technique is typically performed by using a small RPLC precolumn to capture and concentrate analytes as they elute from the immunoextraction column; however, there is little information on the conditions required for optimizing this interface. This study examined the behavior of this interface by using 2,4-dichlorophenoxyacetic acid (2,4-D) and related herbicides as model analytes. It was found that analyte dissociation from immunoextraction columns followed first-order decay and that the elution of these analytes through the immunoextraction/RPLC interface gave an exponentiallymodified Gaussian profile. Computer simulations were used to see how analyte elution through the interface changed with different dissociation and retention conditions. Several guidelines were developed from this work that could be used for developing and optimizing on-line immunoextraction/RPLC systems for other chemicals of environmental or agricultural interest

KINETIC STUDIES OF DRUG-PROTEIN INTERACTIONS BY USING PEAK PROFILING AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: EXAMINATION OF MULTI-SITE INTERACTIONS OF DRUGS WITH HUMAN SERUM ALBUMIN COLUMNS

Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (± 0.2) s-1 and 0.67 (± 0.04) s-1 at pH 7.4 and 37 °C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins

Pharmaceutical and biomedical applications of affinity chromatography: Recent trends and developments

a b s t r a c t Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered