Use of a column-switching high-performance liquid chromatography method to assess the presence of specific binding of (R)- and (S)-[¹¹C]rolipram and their labeled metabolites to the phosphodiesterase-4 enzyme in rat plasma and tissues

Introduction: To complement recent studies using the high-affinity ¹¹C-labeled phosphodiesterase-4 (PDE4) inhibitor (R)-rolipram and the less active enantiomer (S)-[¹¹C]rolipram for in vivo quantification of PDE4 levels, we evaluated the presence of radiolabeled metabolites and their potential binding to PDE4 in the rat plasma, brain, heart, pancreas, skeletal muscle and brown adipose tissue. Methods: A reverse-phase capture and analytical HPLC column-switch method was used to detect (R)-[¹¹C]rolipram, (S)-[¹¹C]rolipram and their radiolabeled metabolites in rat plasma and tissue extracts. The relative proportion of PDE4-specific binding of the radiotracers and their labeled metabolites was analyzed following co-injections with a saturating dose of unlabeled (R)-rolipram at 45 min post-tracer injection in tissue extracts. Results: Radiolabeled metabolites were found in the plasma (72-75% of total radioactive signal), and in the heart, skeletal muscle, pancreas and brown adipose tissue (44-52%), but not in the brain. In comparison to polar labeled metabolites, the proportion of unchanged (R)-[¹¹C]rolipram was reduced in PDE4-rich organs by co-injection of unlabeled (R)-rolipram. Conversely, no changes were obtained in brown adipose tissue, or with (S)-[¹¹C]rolipram, suggesting that radiolabeled metabolites of (R)-[¹¹C]rolipram display no specific binding to PDE4. Conclusions: Radiolabeled hydrophilic metabolites are unlikely to compete with (R)-[¹¹C]rolipram for PDE4-specific retention. However, due to the high proportion of the radioactive metabolites in the total radioactive signal, any kinetic modeling calculations in the peripheral tissues will need to take into account the presence of labeled metabolites