Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (K a ) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average K a values of 1.3 (± 0.2) × 10 5 M −1 and 3.5 (± 3.0) × 10 2 M −1 for acetohexamide and values of 8.7 (± 0.6) × 10 4 and 8.1 (± 1.7) × 10 3 M −1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with K a values of 1.3 (± 0.1) × 10 5 and 4.3 (± 0.3) × 10 4 M −1 , respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with K a values of 5.5 (± 0.2) × 10 4 and 5.3 (± 0.2) × 10 4 M −1 , respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions.

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used highperformance affinity chromatography to examine the changes in acetohexamide-HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K a ) for the high affinity interactions was in the range of 1.2-2.0 × 10 5 M −1 and increased in moving from normal to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.

The presence of elevated levels of glucose in blood during diabetes can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). This study examined the changes that occur in binding of the sulfonylurea drug tolbutamide to HSA as the level of glycation for this protein was increased. High-performance affinity chromatography was used in this work along with columns containing various preparations of in vitro glycated HSA. It was found in frontal analysis experiments that the binding of tolbutamide with all of the tested preparations of glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The association equilibrium constants (K a ) for tolbutamide at its high affinity sites on glycated HSA were in the range of 0.8-1.2 × 10 5 M −1 and increased by 1.4-fold in going from normal HSA to mildly glycated HSA. It was found through competition studies that tolbutamide was binding at both Sudlow sites I and II on the glycated HSA, in agreement with previous studies. The K a for tolbutamide at Sudlow site II increased by 1.1 to 1.4-fold in going from normal HSA to glycated HSA. At Sudlow site I, the K a for tolbutamide increased by 1.2 to 1.3-fold in going from normal HSA to the glycated HSA samples. This information demonstrates the effects that glycation can have on drug interactions on HSA and should provide a better quantitative understanding of how the protein binding of tolbutamide in serum may be affected for individuals with diabetes.

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.

This report used high-performance affinity microcolumns to examine the changes in binding by sulfonylurea drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple drugs and experimental conditions over hundreds of sample application cycles. Both the overall binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in site-specific binding that occurred between sulfonylurea drugs or as the level of HSA glycation was varied. This method is not limited to clinical samples of glycated HSA but could be adapted for work with other modified proteins of interest in personalized medicine.