LC-MS Supported Investigation Of The In Vitro And In Vivo Metabolism Of [¹⁸F]Flubatine – A New Radiotracer For Imaging Of α4β2 Nicotinic Acetylcholine Receptors

Imaging of brain α4β2 nicotinic acetylcholine receptors (nAChRs), a subtype involved in learning and memory processes, can potentially help to predict prognosis of dementia in neurodegenerative disorders like Alzheimer´s disease (AD). Two [¹⁸F]flubatine enantiomers [¹⁸F]-1 (derivatives of homoepibatidine) are promising radiotracers for neuroimaging of α4β2-nAChRs [1] and are currently investigated in clinical studies. Therefore, their metabolism was investigated by two different approaches. Initially, (+)- and (−)-1, were incubated with liver microsomes from mouse or human to produce phase I metabolites. After precipitation with acetonitrile, LC-MS/MS studies of the supernatants were performed to compare metabolic stabilities and for structural elucidation of metabolites. Thereafter, (+)-[¹⁸F]-1 (~280 MBq) was injected into mice. Liver, plasma and urine samples were taken, precipitated and investigated by radio-HPLC. In order to conclude about chemical identities of the radiometabolites of (+)-[¹⁸F]-1, we applied identical chromatographic conditions to both LC-MS and radio-HPLC. LC-MS revealed that both flubatine enantiomers retained a high level of metabolic stability. Furthermore, they were metabolized to higher extent by mouse than by human microsomes. In direct comparison, (+)-1 proved to be more stable than (−)-1. A series of metabolites resulting from oxidation was detected (M0-M6). Presence of each metabolite was species and enantiomer dependent. MS/MS examinations revealed the azabicyclic ring system being exclusively affected by oxidation, namely C- and N-hydroxylation. Using 3-hydroxy flubatine as reference, we identified the radiolabelled derivative (Mc) as one of the metabolites. Correlation with radio-HPLC supports the existence of further radiometabolites resulting from oxidation beside others (Fig. 1). We conclude, that our combined approach offers a useful tool for assignment and finally identification of radiotracer metabolites.

[1] Brust, P. et al. Preclinical Aspects of Nicotinic Acetylcholine Receptor Imaging. In PET and SPECT of Neurobiological Systems; Dierckx, R. A.; Otte, A.; Vries, E. F. de; van Waarde, A.; Luiten, P. G., Eds.; Springer Berlin Heidelberg, 2014; pp 465-512.