The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.
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) × 105 M−1, which agreed with a previously reported value of 1.0 (± 0.1) × 105 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.
No abstract
Several approaches were compared for the entrapment of proteins within hydrazide-activated silica for use in affinity microcolumns and high performance affinity chromatography. Human serum albumin (HSA) and concanavalin A (Con A) were used as model proteins for this work. Items considered in this study included the role played by the solution volume, amount of added protein, and use of slurry vs. on-column entrapment on the levels of solute retention and extent of protein immobilization that could be obtained by means of entrapment. The levels of retention and protein immobilization were evaluated by injecting warfarin or 4-methylumbellipheryl α-Dmannopyranoside as solutes with known binding properties for HSA or Con A. Altering both the solution volume and amount of added protein led to an increase of up to 17-fold in the extent of protein immobilization for HSA in slurry-based entrapment; on-column entrapment provided an additional 3.6-fold increase in protein content vs. the optimized slurry method. Similar general trends were seen for Con A. The protein contents obtained by entrapment for HSA or Con A (i.e., up to ~87 and 46 mg/g silica, respectively) were comparable to or higher than levels reported for the covalent immobilization of these proteins onto silica. The retention of warfarin on the entrapped HSA was at least 1.7-fold higher than has been obtained under comparable support and mobile phase conditions when using covalent immobilization. These results indicated that entrapment can be an attractive alternative to covalent immobilization for proteins such as HSA and Con A, with this approach serving as a potential means for obtaining good solute binding and retention in work with affinity microcolumns or related microscale devices.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.