Analysis of drug interactions with modified proteins by high-performance affinity chromatography: binding of glibenclamide to normal and glycated human serum albumin

Abstract

High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, K(a), 1.4-1.9 × 10(6)M(-1) at pH 7.4 and 37 °C) and lower affinity sites (K(a), 4.4-7.2 × 10(4)M(-1)). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood.

Figure 1

Structure of glibenclamide. The region within the dashed box shows the core structure of a sulfonylurea drug.

Figure 2

Example of frontal analysis studies for glibenclamide on a normal HSA column at pH 7.4 and 37°C. These results were obtained at 0.5 mL/min and using glibenclamide concentrations of 50, 20, 10, and 5 μM (top-to-bottom). The small initial step changes shown to the left occurred at or near the column void time and probably represent only a small difference in composition and background absorbance of each drug solution versus the application buffer; these small step changes were not included in the data analysis and integration of the much larger frontal analysis curves that are shown to the right.

Figure 3

Data obtained for glibenclamide on a normal HSA column as fit to (a) a one-site binding model generated by using Eq. (1) or (b) a two-site binding model generated by using Eq. (3). The insets in (a) and (b) show the corresponding residual plots. Each point represents the average of four runs, with typical relative standard deviations that ranged from ±0.02% to ±7.9% (average, ±1.9%).

Figure 4

A double-reciprocal plot for frontal analysis experiments that examined the binding of glibenclamide with normal HSA. The data in this plot were the same as utilized in Figure 3. The best-fit line was generated by using the data for 0.5–5 μM glibenclamide and gave a correlation coefficient of 0.996 (n = 7).

Figure 5

Plots prepared according to Eq. (6) that show how the reciprocal of the retention factor for (a) R-warfarin or (b) L-tryptophan changed on normal HSA or glycated HSA columns as the concentration of glibenclamide was varied in the mobile phase. These results are for normal HSA (●) and gHSA2 (▲). Each point in these plots is the mean of four runs, with relative standard deviations in (a) that ranged from ±0.3% to ±2.6 (average, ±2.1%) and in (b) that ranged from ±2.2% to ±15.4 (average, ±7.1%). The correlation coefficients for the normal HSA and gHSA2 plots in (a) were 0.966 (n = 6) and 0.975 (n = 8), respectively, while the correlation coefficients in (b) were 0.970 (n = 7) and 0.993 (n = 8).

Figure 6

Plots prepared according to Eq. (6) that show how the reciprocal of the retention factor for digitoxin on HSA or glycated HSA columns changed as the concentration of glibenclamide was varied in the mobile phase. These results are for normal HSA (●) and gHSA3 (◆). The correlation coefficients for these plots were 0.998 (n = 5) and 0.999 (n = 5), respectively. Each point in these plots is the mean of four runs, with relative standard deviations that ranged from ±0.6% to ±4.6 (average, ±1.8%).