Exploration of the impact of stereochemistry on the identification of the novel translocator protein PET imaging agent [(18)F]GE-180

Introduction: The tricyclic indole compound, [(18)F]GE-180 has been previously identified as a promising positron emission tomography (PET) imaging agent of the translocator protein (TSPO) with the potential to aid in the diagnosis, prognosis and therapy monitoring of degenerative neuroinflammatory conditions such as multiple sclerosis. [(18)F]GE-180 was first identified and evaluated as a racemate, but subsequent evaluations of the resolved enantiomers have shown that the S-enantiomer has a higher affinity for TSPO and an improved in vivo biodistribution performance, in terms of higher uptake in specific brain regions and good clearance (as described previously). Here we describe the additional biological evaluations carried out to confirm the improved performance of the S-enantiomer and including experiments which have demonstrated the stability of the chiral centre to chemical and biological factors.

Materials and methods: GE-180 and the corresponding radiolabelling precursor were separated into single enantiomers using semi-preparative chiral supercritical fluid chromatography (SFC). A detailed comparison of the individual enantiomers and the racemate was carried out in a number of biological studies. TSPO binding affinity was assessed using a radioligand binding assay. Incubation with rat hepatic S9 fractions was used to monitor metabolic stability. In vivo biodistribution studies up to 60 min post injection (PI) in naïve rats were carried out to monitor uptake and clearance. Achiral and chiral in vivo metabolite detection methods were developed to assess the presence of metabolite/s in plasma and brain samples, with the chiral method also determining potential racemisation at the chiral centre.

Results: Evaluation of the chiral stability of the two enantiomers to metabolism by rat S9 fractions, showed no racemisation of enantiomers. There were notable differences in the biodistribution between the racemate and the R- and S-enantiomers. All compounds had similar initial brain uptake between 0.99 and 1.01% injected dose (id) at 2 min PI, with S-[(18)F]GE-180 showing significantly greater retention than the R-enantiomer at 10 and 30 min PI (P<0.05). S-[(18)F]GE-180 uptake to the TSPO-expressing olfactory bulbs was 0.45% id (SD ± 0.17) at 30 min PI in comparison to RS-[(18)F]GE-180 or R-[(18)F]GE-180 levels of 0.41% id ± 0.09 and 0.23% id ± 0.02 respectively, at the same timepoint (P > 0.05). The signal-to-noise ratio (ratio olfactory bulb to striata binding) were similar for both RS-[(18)F]GE-180 and S-[(18)F]GE-180 (3.2 and 3.4 respectively). Initial uptake to the lungs (an organ with high TSPO expression) was more than 3-fold greater with S-[(18)F]GE-180 than R-[(18)F]GE-180, and significantly higher at 10 and 30 min PI (P < 0.05). Furthermore lung uptake of S-[(18)F]GE-180 at 2 and 10 min PI was also significant when compared to the racemate (P < 0.05). The majority of the radioactivity in the rat brain following administration of RS-[(18)F]GE-180 or S-[(18)F]GE-180 was due to the presence of the parent compound (91% ± 1.5 and 94% ± 2.0 of total radioactivity at 60 min PI respectively). In contrast at 60 min PI for the plasma samples, the parent compounds accounted for only 28% ± 1.2 and 21% ± 4.6 of total radioactivity for RS-[(18)F]GE-180 and S-[(18)F]GE-180 respectively. Chiral assessment confirmed that the S-enantiomer was chirally stable in vivo, with no stereochemical conversion in brain and plasma samples up to 60 min PI.

Conclusions: Developing racemic radiotracers, as for racemic therapeutics, is a considerable challenge due to differences of the enantiomers in pharmacokinetics, efficacy and potential toxicity. We have shown that the enantiomers of the promising racemic PET ligand [(18)F]GE-180 do not share identical performance, with S-[(18)F]GE-180 demonstrating higher TSPO affinity, higher brain uptake and better retention to the high TSPO-expressing lungs. Furthermore, S-[(18)F]GE-180 has also been shown to be enantiomerically stable in vivo, with no observed conversation of the eutomer to the distomer. As a single enantiomer, S-[(18)F]GE-180 retains the beneficial characteristics of the racemate and is a promising imaging agent for imaging neuroinflammation in vivo.