Fluorescence and analytical ultracentrifugation analyses of the interaction of the tyrosine kinase inhibitor, tyrphostin AG1478-mesylate, with albumin

• Published in: Analytical biochemistry 2005-07, Vol.342 (2), p.292-299
• Main Authors: Clayton, Andrew H.A., Perugini, Matthew A., Weinstock, Janet, Rothacker, Julie, Watson, Keith G., Burgess, Antony W., Nice, Edouard C.
• Format: Article
• Language: English
• Subjects: AG1478; Analytical ultracentrifugation; Drug binding; Fluorescence; Tyrosine kinase inhibitor; Tyrphostin
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2005.04.002

Quantifying the interaction of drugs with carrier proteins in plasma is of importance for understanding effective drug delivery to disease-affected tissues. In this study, we employed analytical ultracentrifugation and steady-state fluorescence spectroscopy to characterize the interaction of a potential new anticancer drug, AG1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG1478, an epidermal growth factor receptor kinase inhibitor, sediments in 0.1%(w/v) NSA as a complex with a sedimentation coefficient of 3.8 S. This is consistent with the size of human serum albumin. This interaction was quantitated by meniscus depletion sedimentation and fluorescence titration analyses. AG1478-mesylate binds to albumin with an apparent single-site affinity ( K d) of 120 μM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG1478-mesylate for albumin ( K d = 4–6 μM), and we propose that the AG1478-mesylate–Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG1478-mesylate ligand alone. A fluorenylmethoxycarbonyl–sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG1478, which was designed to have improved serum-binding properties, was shown by fluorescence analysis to bind with approximately 100-fold greater affinity than the parent compound. This has significant implications in the effective delivery of therapeutic agents in vivo.