Abstract
This study investigates the molecular interactions and physicochemical properties of N(6)-cyclohexyladenosine (CHA), a neuroprotective adenosine A1 receptor agonist, and 8-(p-sulfophenyl)theophylline (8-SPT), a co-administered non-selective adenosine receptor antagonist with limited blood-brain barrier permeability, and 2-hydroxypropyl-β-cyclodextrin (HPβCD), which serves as an excipient. The aim is to systematically evaluate how inclusion complexation with HPβCD affects the solubility and compatibility of drugs in combined formulations, using binding constant analysis to guide the investigation. Two analytical approaches were employed to determine the binding affinities of CHA and 8-SPT: capillary electrophoresis (CE) with UV/Vis absorption detection and nuclear magnetic resonance (NMR) spectroscopy. CE method development utilized two distinct buffers: phosphate buffer (pH 2.5) for protonated CHA and acetate buffer (pH 4.6) for 8-SPT. The detection limits were 2.0 ± 1.0 µg/mL (6 ± 3 µM) at 270 nm for CHA and 2.0 ± 1.0 µg/mL (6 ± 3 µM) at 210 nm for 8-SPT. CE analysis revealed apparent and average binding constants of 136 ± 13 M(-1) for protonated CHA and 121 ± 19 M(-1) for 8-SPT. NMR spectroscopy established the solubility profile of CHA in water and determined an apparent and average binding constant of 308 ± 30 M(-1) for neutral CHA. The relatively small binding constants suggest that 8-SPT does not significantly impact CHA solubility, stability, or availability in combined drug formulations, ensuring sufficient drug availability upon infusion into the bloodstream. These competitive binding constant studies provide valuable guidance for predicting drug solubility and compatibility in combined formulations.