Potential Clinically Relevant Effects of Sialylation on Human Serum AAG-Drug Interactions Assessed by Isothermal Titration Calorimetry: Insight into Pharmacoglycomics?

Abstract

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10^-5-10^-6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.

Keywords: alpha-1 acid glycoprotein; binding affinity; drugs; isothermal titration calorimetry; plasma protein binding; sialic acid.

Figure 1

Distribution of glycan structures of AAG variants. UPLC–FLD chromatograms with structures of different fluorescently labeled N-glycans in native (top panel) and desialylated AAG (bottom panel) determined and assigned by UPLC–MS/MS using the previously published protocol [34]. The diamond shape on baseline represents the beginning and end of peak integration.

Figure 2

(A) Microcalorimetric titrations for examined drugs with native and desialylated AAG. Top charts show the differential power in $\mathsf{\mu }$W per injectant with subtracted baseline. Bottom charts show dependence of successive enthalpy changes in kJ mol⁻¹ as a function of the AAG–drug molar ratio. (B) Microcalorimetric titrations for examined drugs with native and desialylated AAG. Top charts show the differential power in $\mathsf{\mu }$W per injectant with subtracted baseline. Bottom charts show dependence of successive enthalpy changes in kJ mol⁻¹ as a function of the AAG–drug molar ratio. Enlarged view of injections can be seen in Figure S6. ● experimental; ―calculated.

Figure 3

Free fraction of drugs. Percentage difference of free drug at (A) peak (C_max), and (B) trough therapeutic concentrations (C_trough), depending on the plasma concentrations of AAG$+$s or AAG$-$s. Values are calculated vs. the native form (AAG$+$s) as reference.

Figure 4

Free fraction of drugs. Percentage of free drug at (A) peak (C_max) and (B) trough therapeutic concentrations (C_trough), depending on the plasma concentrations of AAG$+$s with 0.64 mM HSA or AAG$-$s with 0.64 mM HSA.

Figure 5

Free fraction of drugs including HSA concentration. Percentage difference of free drug at (A) peak (C_max) and (B) trough therapeutic concentrations (C_trough), depending on the plasma concentrations of AAG$+$s or AAG$-$s and HSA. Values are calculated vs. the native form (AAG$+$s) as reference.