High‐throughput analysis of drug dissociation from serum proteins using affinity silica monoliths

Abstract

A noncompetitive peak decay method was used with 1 mm × 4.6 mm i.d. silica monoliths to measure the dissociation rate constants (kd) for various drugs with human serum albumin (HSA) and α1-acid glycoprotein (AGP). Flow rates up to 9 mL/min were used in these experiments, resulting in analysis times of only 20-30 s. Using a silica monolith containing immobilized HSA, dissociation rate constants were measured for amitriptyline, carboplatin, cisplatin, chloramphenicol, nortriptyline, quinidine, and verapamil, giving values that ranged from 0.37 s−1 to 0.78 s−1. Similar work with an immobilized AGP silica monolith gave kd values for amitriptyline, nortriptyline, and lidocaine of 0.39 s−1 to 0.73 s−1. These kd values showed good agreement with values determined for drugs with similar structures and/or affinities for HSA or AGP. It was found that a kd of up to roughly 0.80 s−1 could be measured by this approach. This information made it possible to obtain a better understanding of the advantages and possible limitations of the noncompetitive peak decay method and in the use of affinity silica monoliths for the high-throughput analysis of drug-protein dissociation.

Determination of Rate Constants and Equilibrium Constants for Solution-Phase Drug–Protein Interactions by Ultrafast Affinity Extraction

“…9,15,40,43 The association rate constants for tolbutamide, acetohexamide, and racemic verapamil gave k a values of 6.4 (±2.4) × 10 4 M –1 s –1 , 1.1 (±0.3) × 10 5 M –1 s –1 , and 5.4 (±1.5) × 10 3 M –1 s –1 , respectively, which were comparable to the results calculated from previously reported k d and K a or nK a ′ values for these systems. 15,17,19−21 Gliclazide and chlorpromazine gave k a values of 4.7 (±0.5) × 10 4 M –1 s –1 and 2.1 (±0.3) × 10 5 M –1 s –1 , which agreed with the range of values that have been reported for drugs with comparable affinities and dissociation rates for HSA. 15,17,41,42 …”

“…15,17,19−21 Gliclazide and chlorpromazine gave k a values of 4.7 (±0.5) × 10 4 M –1 s –1 and 2.1 (±0.3) × 10 5 M –1 s –1 , which agreed with the range of values that have been reported for drugs with comparable affinities and dissociation rates for HSA. 15,17,41,42 …”

Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography

“…(1) and the ultrafast affinity extraction data. The k d values for quinidine and diazepam with HSA were 0.58 (± 0.02) and 0.63 (± 0.05) s −1 , respectively, which had absolute differences of only 0.05–0.19 s −1 from values in the literature [22,45]. The relative precisions of these k d values were ± 3.4 to 7.9%.…”

Analytical methods for kinetic studies of biological interactions: A review

“…This is illustrated in Fig. 8 for racemic warfarin that had been applied to immobilized HSA in a small silica monolith column [142]. Other drugs (e.g., diazepam, imipramine, acetohexamide, tolbutamide, amitriptyline, quinidine, verapamil, amitriptyline, lidocaine, and nortriptyline) and binding agents (e.g., alpha 1 -acid glycoprotein) have also been studied with this method [142,143].…”

“…8 for racemic warfarin that had been applied to immobilized HSA in a small silica monolith column [142]. Other drugs (e.g., diazepam, imipramine, acetohexamide, tolbutamide, amitriptyline, quinidine, verapamil, amitriptyline, lidocaine, and nortriptyline) and binding agents (e.g., alpha 1 -acid glycoprotein) have also been studied with this method [142,143]. The peak decay method has further been employed to study the dissociation rates of various targets from immobilized antibodies during the selection of elution conditions for immunoaffinity chromatography [144].…”