Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction

Abstract

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha₁-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.

Keywords: Affinity microcolumn; Alpha(1)-acid glycoprotein; Drug-protein interactions; High performance affinity chromatography; Human serum albumin; Ultrafast affinity extraction.

Figure 1

The general basis for ultrafast affinity extraction.

Figure 2

(a) The Schiff base method for the immobilization of HSA and (b) the hydrazide method for the immobilization of AGP.

Figure 3

The typical designs for systems used in ultrafast affinity extraction based on (a) a single-column or (b) a two-column system.

Figure 4

General approaches for the separation and analysis of free drug or solutes fractions by using ultrafast affinity extraction using (a) a single-column system or (b) a two-column system.

Figure 5

Effect of injection flow rate on the column residence time (dashed line) and apparent free drug fractions (solid line), as illustrated for 10 μL injections of samples containing 1 μM propranolol and 20 μM soluble AGP onto a 2 mm × 2.1 mm i.d. AGP microcolumn at pH 7.4 and 37 ºC. Reproduced with permission from Ref. [54].

Figure 6

Effect of changing the valve switching time in ultrafast affinity extraction when using a two-column system. These effects are illustrated for (a) the recovery of carbamazepine at 0.5 mL/min on a 10 mm × 2.1 mm i.d. AGP column that was placed on-line with a 5 mm × 2.1 mm i.d. AGP column at various times following the injection of a 3 μL sample mixture of 15 μM carbamazepine/20 μM AGP/481 μM HSA onto the first column at 3.0 mL/min, and (b) the apparent free drug fractions that were measured by using the same chromatographic conditions and sample when using a two-column system. All of the times show represent the elapsed interval after sample injection. The error bars represent ± 1 standard error of the mean (n = 3) and all the measurements were made at pH 7.4 and 37 ºC. Reproduced with permission from Ref. [53].

Figure 7

Typical chromatograms obtained from the application of 185 μM tolbutamide (therapeutic concentration) in the absence or presence of 526 μM HSA (physiological concentration) onto a single 5 mm × 2.1 mm i.d. HSA microcolumn at 2.25 mL/min. Reproduced with permission from Ref. [50].

Figure 8

Analysis of the interactions by verapamil with soluble AGP by using ultrafast affinity extraction and Eqs. (1) and (2). The samples contained 10 μM verapamil and 20 μM AGP. The error bars represent a range of ± 1 S.D. These results are based on data from Ref. [54].