Daniela Schneider1, Dirk Bier2, Andreas Bauer3, Bernd Neumaier4, Marcus Holschbach5. 1. Institute of Neuroscience and Medicine, Molecular Organization of the Brain (INM-2), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany. Electronic address: d.schneider@fz-juelich.de. 2. Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany. Electronic address: d.bier@fz-juelich.de. 3. Institute of Neuroscience and Medicine, Molecular Organization of the Brain (INM-2), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany; Neurological Department, Medical Faculty, Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany. Electronic address: an.bauer@fz-juelich.de. 4. Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany. Electronic address: b.neumaier@fz-juelich.de. 5. Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany. Electronic address: m.holschbach@fz-juelich.de.
Abstract
INTRODUCTION: In vitro metabolism models such as liver microsomes represent an important tool for the development of novel radioligands. Comparability and physiological relevance of in vitro metabolism data critically depend on the careful evaluation and optimization of assay protocols. We therefore investigated the influence of incubation conditions on the microsomal stability of xanthine-derived A1 adenosine receptor (A1AR) ligands which have been developed for positron emission tomography (PET). METHODS: Substrate depletion assays using rat liver microsomes (RLM) were performed for three analogous compounds which differ with regard to the metabolically vulnerable substituent at the xanthine C8 position. Incubation conditions were varied systematically. Additionally, the stability of the cofactor NADPH during incubation was investigated. RESULTS: Microsomal metabolism was strongly influenced by buffer pH, organic solvents and preincubation time. Substrate depletion values varied up to 5-fold depending on incubation matrix composition, however, the rank order of metabolic stability remained unchanged. Prolonged incubation periods led to drastic loss in enzyme activity which could not be prevented by addition of metal chelators or antioxidants. Cofactor NADPH was rapidly oxidized in microsomal matrix, even in the absence of cytochrome P450 substrates. DISCUSSION: In summary, short incubation times, precise pH control and minimal concentrations of organic solvents are mandatory to obtain reliable microsomal stability data. Furthermore, in vitro metabolic stability of the tested A1AR ligands varied largely depending on the particular C8 substituent. Consequently, structural modifications at the xanthine C8 position appear to be a promising strategy for the improvement of A1AR PET radioligands.
INTRODUCTION: In vitro metabolism models such as liver microsomes represent an important tool for the development of novel radioligands. Comparability and physiological relevance of in vitro metabolism data critically depend on the careful evaluation and optimization of assay protocols. We therefore investigated the influence of incubation conditions on the microsomal stability of xanthine-derived A1 adenosine receptor (A1AR) ligands which have been developed for positron emission tomography (PET). METHODS: Substrate depletion assays using rat liver microsomes (RLM) were performed for three analogous compounds which differ with regard to the metabolically vulnerable substituent at the xanthine C8 position. Incubation conditions were varied systematically. Additionally, the stability of the cofactor NADPH during incubation was investigated. RESULTS: Microsomal metabolism was strongly influenced by buffer pH, organic solvents and preincubation time. Substrate depletion values varied up to 5-fold depending on incubation matrix composition, however, the rank order of metabolic stability remained unchanged. Prolonged incubation periods led to drastic loss in enzyme activity which could not be prevented by addition of metal chelators or antioxidants. Cofactor NADPH was rapidly oxidized in microsomal matrix, even in the absence of cytochrome P450 substrates. DISCUSSION: In summary, short incubation times, precise pH control and minimal concentrations of organic solvents are mandatory to obtain reliable microsomal stability data. Furthermore, in vitro metabolic stability of the tested A1AR ligands varied largely depending on the particular C8 substituent. Consequently, structural modifications at the xanthine C8 position appear to be a promising strategy for the improvement of A1AR PET radioligands.