Characterization of drug binding with alpha1-acid glycoprotein in clinical samples using ultrafast affinity extraction

Abstract

Many drugs bind to serum transport proteins, which can affect both drug distribution and activity in the body. α₁-Acid glycoprotein (AGP) is a key transport protein for basic and neutral drugs. Both elevated levels and altered glycosylation patterns of AGP have been seen in clinical conditions such as systemic lupus erythematosus (SLE). This study developed, optimized, and used the method of ultrafast affinity extraction (UAE) to examine whether these changes in AGP are associated with changes in the binding by some drugs to this transport protein. This approach used affinity microcolumns to capture and measure, in serum, the free fractions of several drugs known to bind AGP. These measurements were made with pooled normal control serum and serum samples from individuals with SLE. Immunoaffinity chromatography was used to obtain the content of AGP and HSA in these samples, and CE was used to examine the glycoform pattern for AGP in each serum sample. The free drug fractions measured for normal control serum ranged from 3.5 to 29.1%, in agreement with the results of ultrafiltration, and provided binding constants of ~10⁵-10⁶ M^-1 for the given drugs with AGP at 37⁰C. Analysis of a screening set of SLE serum samples by UAE gave decreased free fractions (relative change, 12-55%) vs normal serum when spiked with the same types and amounts of drugs. These changes were related in some cases to AGP content, with some SLE samples having AGP levels 1.3- to 2.1-fold above the upper end of the normal range. In other cases, the changes in free fractions appeared to be linked to alterations in the glycoforms and binding constants of AGP, with some affinities differing by 1.2- to 1.5-fold vs normal AGP. This approach can be employed with other solute-protein systems and to investigate binding by other drugs or transport proteins directly in clinical samples.

Keywords: Alpha(1)-acid glycoprotein; Drug-protein binding; Human serum albumin; Systemic lupus erythematosus; Ultrafast affinity extraction.

Figure 1.

General scheme for the two-column affinity system that was employed in this study for measuring free drug fractions and studying drug-protein interactions in serum.

Figure 2.

Structures of drugs that were examined in this study for their binding with AGP. The asterisk in some of these structures indicates the location of a chiral center.

Figure 3.

Effect of flow rate on the measured free fraction for (a) imatinib and (b) propranolol in the presence of soluble AGP. In (a) a 3 μL sample containing 1.7 μM imatinib and 20 μM AGP was used, with the latter representing a typical normal serum concentration of AGP [2,7]. In (b) a 5 μL sample containing 0.54 μM propranolol and 20 μM AGP was employed. Both experiments used a 2 mm × 2.1 mm i.d. AGP microcolumn. The error bars represent a range of ± 1 S.D.

Figure 4.

(a) Example of a chromatogram obtained during ultrafast affinity extraction for a 10 μL mixture of 5 μM aprindine and 20 μM AGP injected onto a 5 mm × 2.1 mm i.d. AGP microcolumn at 1.5 mL/min, and (b) effect of valve switching time in a two-column system on the measured free drug fraction for a sample containing 5 μM aprindine, 496 μM HSA, and 20 μM AGP. The error bars in (b) represent a range of ± 1 standard error of the mean (n = 4–5).

Figure 5.

Electropherograms for AGP from SLE serum or pooled normal control serum. The conditions are provided in Section 2.7.

Figure 6.

Glycoform patterns obtained for AGP from individual SLE serum samples versus pooled normal control serum for (a) SLE 1, (b) SLE 2, (c) SLE 3, (d) SLE 4, (e) SLE 5, and (f) SLE 6. The error bars shown in all plots for the migration times and % peak areas represent a range of ± 1 S.D. (n = 3).