LC-MS supported Studies on the in vitro Metabolism of both Enantiomers of Flubatine and the in vivo Metabolism of (+)-[18F]Flubatine – a Positron Emission Tomography Radioligand for Imaging α4β2-Nicotinic Acetylcholine Receptors

Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer’s disease, a detailed examination of the metabolism in vitro and in vivo is required. (+)- and (–)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH. Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS. Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, that was identified as the main metabolic pathway in this assay. Further, after injection of 280 MBq (+)-[18F]flubatine (specific acitivity > 350 GBq/µmol) into a CD-1 mouse, samples were prepared from liver, plasma, and urine after 30 min and investigated by HPLC with radioactivity detection. For structure elucidation of the radiometabolites of (+)-[18F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation we assigned 3 of 4 main in vivo radiometabolites to products exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.