Influence of incubation conditions on microsomal metabolism of xanthine-derived A

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 A 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. 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. 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 A

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