The (R)-(+)- and (S)-(-)-enantiomers of [¹⁸F]fluspidine have different potential for brain imaging of σ₁ receptors

Objectives: It ist widely accepted that σ₁ receptors represent a novel biological target for the pharmacological treatment of various brain diseases, e.g. depression and neurodegeneration. From our series of σ₁-specific racemic ¹⁸F-fluoroalkylated spirocyclic piperidines we have chosen the superior [¹⁸F]fluspidine for detailed investigation of the (R)-(+)- and (S)-(-)-enantiomers with the aim to identify their individual potential for disease-related neuroimaging studies.

Methods: By semi-preparative chiral HPLC on immobilized amylose-tris-(3,5-dimethylphenyl)-carbamate as stationary phase the racemic tosylate precursor of [¹⁸F]fluspidine was enantioseparated. Automated radiosynthesis of the two enantiomers was accomplished by nucleophilic substitution and biodistribution studies were performed in CD-1 mice (dose: 240-480 kBq). Furthermore brain pharmacokinetics of the two enantiomers was investigated by dynamic PET studies in pigs (dose: 270-420 MBq). Additionally, the highly selective σ₁ receptor agonist SA4503 (5mg/kg) was used in blocking (bolus plus infusion) studies to assess target specificity. SUV values were calculated for 24 MR-defined brain regions. Using a metabolite-corrected plasma input function compartment modelling was applied to estimate kinetic parameters of both enantiomers.

Results: Enantiomerically pure (R)- and (S)-tosylate precursors were obtained with high enantiomeric excess of >98 % and >96 %. (R)- and (S)-[¹⁸F]fluspidine were synthetized within ~70 min with RCY of 35-45% (EOS), RCP of >99%, and A_S of 550 GBq/μmol and 870 GBq/μmol. In mice, both radiotracers readily passed the blood-brain barrier. However, large differences in brain pharmacokinetics of the two enantiomers were found with continuous increase of brain uptake of (R)-[¹⁸F]fluspidine (3.57 %ID/g at 5', 6.01% ID/g at 240' p.i.) in comparison to (S)-[¹⁸F]fluspidine with higher initial brain uptake (4.35 %ID/g at 5' p.i.) and rapid clearance (1.04% ID/g at 240' p.i.). Dynamic PET studies in pigs confirmed these enantiomer-related differences (Fig.).
[Fluspidine kinetics in pig brain]
Under baseline conditions, the initial brain uptake of (S)-[¹⁸F]fluspidine was higher than that of (R)-[¹⁸F]fluspidine (e.g. SUV_{max, Cerebellum} ~3.4 vs. ~2.9; K₁: 0.72 vs. 0.56 ml/g/min). Clearance of (S)-[¹⁸F]fluspidine from brain was fast (SUV_Cerebellum ~1.1 at 95-120' p.i.) whereas the uptake of (R)-[¹⁸F]fluspidine remained close to the initial level (SUV_Cerebellum ~2.5 at 95-120' p.i.). Accordingly the binding potential (k₃/k₄) of (S)-[¹⁸F]fluspidine was much lower (1.7) than that of (R)-[¹⁸F]fluspidine (16.3). In comparison to baseline data, application of σ₁ specific SA4503 reduced the uptake of (S)- and (R)-[¹⁸F]fluspidine in the target region cerebellum by initially 40% and 15% (SUV_max ~2.0 and ~2.5, respectively) and later by ~80% (SUV ~0.2 and ~0.6 at 95-120' p.i., respectively) . Washout kinetics and SUV values determined under blocking conditions indicate both target specificity of the binding as well as minor nonspecific binding of the two radiotracers.

Conclusions: We successfully developed and validated an automated synthesis of the two enantiomers of [¹⁸F]fluspidine. The pharmacokinetics of (S)-[¹⁸F]fluspidine as investigated in two different animal models suggests that this radiotracer is most suitable for upcoming studies of depression-related changes in receptor expression in human brain. The irreversible-like binding behaviour of (R)-[¹⁸F]fluspidine may have advantages for tumor imaging.

Acknowledgements: Supported by DFG (STE 601/10-2, WU 176/7-2) and NIH (T32 EB004822).