title: Fluorescence and analytical ultracentrifugation analyses of the interaction of the tyrosine kinase inhibitor, tyrphostin AG 1478-mesylate, with albumin.
abstract: 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, AG 1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG 1478, 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. AG 1478-mesylate binds to albumin with an apparent single-site affinity (K(d)) of 120 microM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG 1478-mesylate for albumin (K(d)=4-6 microM), and we propose that the AG 1478-mesylate-Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG 1478-mesylate ligand alone. A fluorenylmethoxycarbonyl-sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG 1478, 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.
authors.affiliation: Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, P.O. Royal Melbourne Hospital, Parkville, Vic. 3050, Australia.
authors.forename: Andrew H A
authors.initials: AH
authors.lastname: Clayton
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
authors.forename: Janet
authors.initials: J
authors.lastname: Weinstock
authors.forename: Julie
authors.initials: J
authors.lastname: Rothacker
authors.forename: Keith G
authors.initials: KG
authors.lastname: Watson
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
mesh_terms: D011485:Protein Binding; D011799:Quinazolines; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D020032:Tyrphostins / Q000737:chemistry*; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D011799:Quinazolines; D012709:Serum Albumin; D020032:Tyrphostins; C101044:RTKI cpd