Determination of Hydroxycamptothecin Affinities to Albumin and Membranes by Steady-State Fluorescence Anisotropy Measurements

Abstract

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.

Determination of the Protein-Binding Properties of Camptothecins by Means of Optical Spectroscopy Methods

“…, where F f and F b are the fractions of ligands free and bound to albumin, and L represents the total concentration of HSA [7,12,13]. Because…”

“…The F b fraction can be determined for all examined concentration L by uorescence anisotropy measurements. The procedure was described previously [12]. Finally, by regression 1/F b on 1/L, the association constant can be calculated.…”

“…Using method described in [12], the double-reciprocal plots were drawn (C and D) and the association constants of drugs binding to albumin were calculated (Table III). The uorescence anisotropy of 9-amino-CPT was measured at pH 3 because of low uorescence of this compound at pH 7.4 [15].…”

Fluorescence Spectroscopy in Camptothecins Studies

“…DMPC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Procedure of liposomes preparing was the same as described in previous papers [8,9]. For steady state fluorescence anisotropy measurements, the concentration of camptothecins in final samples was equal 2 µM.…”

“…On the basis of fluorescence anisotropy measurements in dependence on concentration of lipids forming the model membranes the association (binding) constant of drugs to membranes were determined. The association constant, which is a quantitative measure of affinity of drug to membranes, is determined by formulae Procedure of determining the association constant on the basis of fluorescence anisotropy measurements was in details described in previous papers [9,12].…”