Using capillary electrophoresis/frontal analysis to screen drugs interacting with human serum proteins

Abstract

We have used capillary electrophoresis in the frontal analysis mode (CE/FA) to determine the binding capacity of beta-adrenoceptor blocking drugs to individual serum proteins, serum protein mixtures and human serum. The free drug concentration was directly measured from the height of the frontal peak and used to calculate the bound drug concentration. From the bound drug concentration, the percentage of drug bound to the serum proteins alpha1-acid glycoprotein (AGP) and human serum albumin (HSA) was then determined. In addition to determining the percent of a drug bound to a protein, the drug-protein association constant (Ka) was determined for AGP binding to beta-blockers. The data-estimated association constants were consistent with literature values. The CE/FA studies on the beta-adrenoceptor blocking drugs and the serum proteins indicated that HSA, AGP, high density lipoprotein (HDL), and low density lipoprotein (LDL) were the main contributors to serum binding for this series of compounds. The serum-drug binding data sorted the beta-adrenoceptor blocking drugs into high and low binding categories. The protein mixture (AGP + HSA + HDL + LDL) resulted in dividing the beta-blockers into the same high/low rankings. The protein mixture (AGP + HSA + HDL + LDL) was amenable to automation, did not autoaggregate, and had constant concentrations for the proteins.

Capillary electrophoresis frontal analysis: Principles and applications for the study of drug‐plasma protein binding

“…The other compound (phenol) had a very weak affinity and thus the HSA concentration had to be increased to obtain reliable measurements of the free drug concentration [34]. McDonnell et al [71] studied the binding of 8 b-adrenoceptor blocking drugs and Jia et al [72] the binding of 17 cationic and neutral drugs to HSA and AGP with only minimal alterations of the experimental conditions. Jia et al used pressureassisted CE-FA to shorten analysis times and improve sample throughput.…”

“…The utility and validity of binding studies performed with CE-FA have been demonstrated in several studies using HSA and various cationic ligands such as propranolol and verapamil [36,42,[71][72][73][74]. CE-FA is ideally suited for the measurement of these interactions because the electrophoretic mobilities of the cationic ligands and HSA (negatively charged at physiological pH) are very different; moreover, the interactions between HSA and cationic drugs in general are relatively weak [65,66].…”

“…Previously, we found limits of detection around 1 mM for small drug molecules (0.6 mM for warfarin [34]), thus, with the used CE-FA setup determination of accurate dissociation constants below several mM would probably be difficult. The sensitivity limitations of CE-FA have been pointed out in several studies [14,34,43,71,72]. To the best of our knowledge, other more sensitive detection methods than UV have not been applied in CE-FA studies so far.…”

“…Using verapamil as a ligand Shibukawa et al [36] obtained good accordance between results generated using CE-FA and ultrafiltration. CE-FA studies of AGP binding include work focused on estimation of the degree of binding of cationic drugs [36,71,72] and studies directed at elucidating the effect of AGP structure and glycation on binding [48,78,83].…”

“…Binding to antitrypsin and haptoglobulins has also been probed by CE-FA [71]. Binding of drugs to plasma, serum, and other protein mixtures has received little attention.…”