Cong Bi

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.

Analytical methods for kinetic studies of biological interactions: A review

The rates at which biological interactions occur can provide important information concerning the mechanism and behavior of these processes in living systems. This review discusses several analytical methods that can be used to examine the kinetics of biological interactions. These techniques include common or traditional methods such as stopped-flow analysis and surface plasmon resonance spectroscopy, as well as alternative methods based on affinity chromatography and capillary electrophoresis. The general principles and theory behind these approaches are examined, and it is shown how each technique can be utilized to provide information on the kinetics of biological interactions. Examples of applications are also given for each method. In addition, a discussion is provided on the relative advantages or potential limitations of each technique regarding its use in kinetic studies.

Studies of metabolite–protein interactions: A review

The study of metabolomics can provide valuable information about biochemical pathways and processes at the molecular level. There have been many reports that have examined the structure, identity and concentrations of metabolites in biological systems. However, the binding of metabolites with proteins is also of growing interest. This review examines past reports that have looked at the binding of various types of metabolites with proteins. An overview of the techniques that have been used to characterize and study metabolite-protein binding is first provided. This is followed by examples of studies that have investigated the binding of hormones, fatty acids, drugs or other xenobiotics, and their metabolites with transport proteins and receptors. These examples include reports that have considered the structure of the resulting solute-protein complexes, the nature of the binding sites, the strength of these interactions, the variations in these interactions with solute structure, and the kinetics of these reactions. The possible effects of metabolic diseases on these processes, including the impact of alterations in the structure and function of proteins, are also considered.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum.

Dual-Target Binding Ligands with Modulated Pharmacokinetics for Endoradiotherapy of Prostate Cancer

Prostate-specific membrane antigen (PSMA)–targeted radiotherapy of prostate cancer (PCa) has emerged recently as a promising approach to the treatment of disseminated disease. A small number of ligands have been evaluated in patients, and although early tumor response is encouraging, relapse rate is high and these compounds localize to the parotid, salivary, and lacrimal glands as well as to the kidney, leading to dose-limiting toxicities and adverse events affecting quality of life. We envision that dual-target binding ligands displaying high affinity for PSMA and appropriate affinity for human serum albumin (HSA) may demonstrate a higher therapeutic index and be suitable for treatment of PCa by targeted α-therapy. Methods: Six novel urea-based ligands with varying affinities for PSMA and HSA were synthesized, labeled with 131I, and evaluated by in vitro binding and uptake assays in LNCaP cells. Four compounds were advanced for further evaluation in a preclinical model of PCa. The compounds were compared with MIP-1095, a PSMA ligand currently in clinical evaluation. Results: The compounds demonstrated affinity for PSMA on the order of 4–40 nM and affinity for HSA in the range of 1–53 μM. Compounds with relatively high affinity for HSA (≤2 μM) showed high and sustained blood-pool activity and reduced uptake in the kidneys. 131I-RPS-027, with a 50% inhibitory concentration (PSMA) of 15 nM and a dissociation constant (HSA) of 11.2 μM, cleared from the blood over the course of 48 h and showed good tumor uptake (10 percentage injected dose per gram) and retention and a greater than 5-fold decrease in kidney uptake relative to MIP-1095. The tumor-to-kidney ratio of 131I-RPS-027 was greater than 3:1 at 24 h after injection, increasing to 7:1 by 72 h. Conclusion: RPS-027 shows dual targeting to PSMA and albumin, resulting in a high tumor uptake, highly favorable tissue distribution, and promising therapeutic profile in a preclinical model of prostate cancer. In comparison to existing ligands proposed for targeted therapy of prostate cancer, RPS-027 has tumor-to-tissue ratios that predict a significant reduction in side effects during therapy. Using iodine/radioiodine as a surrogate for the radiohalogen 211At, we therefore propose dual-target binding ligands such as RPS-027 as next-generation radiopharmaceuticals for targeted α-therapy using 211At.

Development of enhanced capacity affinity microcolumns by using a hybrid of protein cross-linking/modification and immobilization.

A hybrid method was examined for increasing the binding capacity and activity of protein-based affinity columns by using a combination of protein cross-linking/modification and covalent immobilization. Various applications of this approach in the study of drug-protein interactions and in use with affinity microcolumns were considered. Human serum albumin (HSA) was utilized as a model protein for this work. Bismaleimidohexane (BMH, a homobifunctional maleimide) was used to modify and/or cross-link HSA through the single free sulfhydryl group that is present on this protein. Up to a 75-113% increase in protein content was obtained when comparing affinity supports that were prepared with BMH versus reference supports that were made by using only covalent immobilization. Several drugs that are known to bind HSA (e.g., warfarin, verapamil and carbamazepine) were further found to have a significant increase in retention on HSA microcolumns that were treated with BMH (i.e., a 70-100% increase in protein-based retention). These BMH-treated HSA microcolumns were used in chiral separations and in ultrafast affinity extraction to measure free drug fractions in drug/protein mixtures, with the latter method giving association equilibrium constants that had good agreement with literature values. In addition, it was found that the reversible binding of HSA with ethacrynic acid, an agent that can combine irreversibly with the free sulfhydryl group on this protein, could be examined by using the BMH-treated HSA microcolumns. The same hybrid immobilization method could be extended to other proteins or alternative applications that may require protein-based affinity columns with enhanced binding capacities and activities.

High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples.

Kinetic analysis of drug-protein interactions by affinity chromatography.

Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body.

Chromatographic studies of drug interactions with alpha1-acid glycoprotein by ultrafast affinity extraction and peak profiling ☆

Interactions with serum proteins such as alpha1-acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 104-106M-1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s-1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest.