LC-MS supported investigations on the metabolism of (+)-[¹⁸F]flubatine, a radiotracer for imaging of α4β2 nicotinic acetylcholine receptors by PET

Objectives: (+)-[¹⁸F]flubatine [1] is currently being investigated for imaging of α4β2 nicotinic acetylcholine receptors (nAChRs) in patients with early Alzheimer’s disease. Accompanying these studies, we performed LC-MS supported in vitro and in vivo investigations to detect and characterize radiometabolites in plasma and urine.
Methods: Liver microsomes from mouse (MLM) or human (HLM) were incubated for 90 or 120 min in PBS or TRIS buffer with 10 µM (+)-, and, for comparison, (-)-flubatine and synthesized references. Further, an anaesthesized pig was infused with (+)-flubatine (67 µg/kg). Plasma and urine samples were taken after 30 and 45 min, respectively. Structures of metabolites in these in vitro and in vivo samples were elucidated by LC-MS.
For comparison MLM and HLM were incubated with 7 MBq (+)-[¹⁸F]flubatine and the radiometabolite patterns were monitored by radio-HPLC. Additionally, plasma (15, 30 min p.i) and urine (90-130 min p.i.) samples of human subjects receiving 283 (259-299) MBq (+)-[18F]flubatine i.v. were investigated.
Results: After microsomal incubations, up to seven metabolites exclusively hydroxylated at the azabicyclic ring system were detected (Fig.1A), among them some which were glucuronidated subsequently. Accordingly, seven hydroxylated metabolites and one glucuronide of (+)-flubatine were found in pig. Radiometabolites of (+)-[¹⁸F]flubatine obtained after HLM and MLM incubations corresponded to those of (+)-flubatine. Plasma (15, 30 min p.i.) and urine (90-130 min p.i.) samples obtained from humans showed 92-99% and 88-99% (n=7) of unchanged tracer, respectively. Two radiometabolites could be detected in both plasma and urine and were finally assigned to 1 and 3 (Fig.1B).
Conclusions: (+)-[¹⁸F]Flubatine showed high metabolic stability in human. The structures of the two radiometabolites found in plasma and urine could be assigned by the presented approach.
Acknowledgements: Supported by the Helmholtz Validation Found.
References: [1] Smits R, et al. (2014) Bioorg Med Chem, 22, 804-12