Radiosynthesis and evaluation of (R)- and (S)-¹⁸F-OF-NB1 for imaging the GluN2B subunits of the NMDA receptor in non-human primates

Objectives: The GluN2B subunit-containing NMDA receptor is an attractive therapeutic target for a plethora of neuropathologies, including Alzheimer’s disease, ischemic brain injury, and schizophrenia. Despite vigorous efforts, no suitable PET probe for imaging the GluN2B subunits has been successfully translated into humans. In a prior work, we reported on racemic (±)-¹⁸F-OF-NB1 as a promising radioligand in rodents and its imaging potential in postmortem brain tissues of amyotrophic lateral sclerosis patients (1). We report herein on the in vivo characterization of (R)- and (S)-¹⁸F-OF-NB1 in rhesus monkeys. Methods: Racemic (±)-¹⁸F-OF-NB1 was prepared as previously described (1). Chiral separation of the racemic mixture afforded the enantiopure (R)- and (S)-forms. PET scans were conducted in rhesus monkeys for up to 180 min on a Focus 220 scanner. Blocking studies with the GluN1/2B ligand, Co-101,244 (0.25 mg/kg), and the sigma-1 ligand, FTC-146 (0.125 and 0.027 mg/kg), were conducted to assess in vivo binding specificity and selectivity. Arterial blood sampling coupled with metabolite analysis by HPLC was used to measure the plasma input function. Regional brain time-activity curves (TACs) were produced and fitted with the one-tissue (1T) and two-tissue (2T) compartment models and multilinear analysis-1 (MA1) method to calculate the corresponding regional volumes of distribution (V_T, mL/cm³).

Results: The (S)- and (R)-enantiomers of ¹⁸F-OF-NB1 were successfully prepared in >99% radiochemical and enantiomeric purity with an average molar activity of 59 ± 16 GBq/µmol (n=6) at the end of synthesis. Chirality of the tracer influenced the metabolism and in vivo kinetics considerably. The parent fractions of (S)- and (R)-forms remaining in the plasma at 30 min post-injection were 32% and 57%, respectively. Plasma free fraction was ~15% for both tracers. Although the two enantiomers displayed a similar widespread distribution across the brain, (S)-¹⁸F-OF-NB1 showed a faster tissue clearance than the (R)-enantiomer. Tracer uptake was higher in the cortex, putamen, and cerebellum, moderate in the hippocampus and thalamus, and lowest in the centrum semiovale. Both the 1TC model and MA1 method fitted the TACs well and offered reliable V_T estimates, ranging from 20 mL/cm³ in the centrum semiovale to 39 mL/cm³ in the cingulate cortex for (S)-¹⁸F-OF-NB1. Regional V_T values of (R)-¹⁸F-OF-NB1 were higher than those of the (S)-enantiomer. For (S)-¹⁸F-OF-NB1, receptor occupancy induced by the GluN2B ligand Co-101244 was 81% and 88% in two separate monkeys (96% for (R)-¹⁸F-OF-NB1). Both high (0.125 mg/kg) and low (0.027 mg/kg) doses of the sigma-1 receptor ligand FTC-146 induced similar reduction in specific binding of 48% and 49%, respectively, in two animals imaged with (S)-¹⁸F-OF-NB1. However, a sequential blockade with Co-101244 (0.25 mg/kg) followed by FTC-146 (0.125 mg/kg) did not reveal additional reduction in binding (81% for the single GluN2B blockade and 82% for the sequential blockade). Non-specific volume of distribution (V_ND) of (S)-¹⁸F-OF-NB1, estimated from the GluN2B blocking studies, was 9.3 in Monkey #1 and 7.3 in Monkey #2. Regional binding potential (BP_ND), calculated as BP_ND= (V_T- V_ND)/V_ND, ranged from 2.31 (occipital cortex) to 4.3 (cingulate cortex) for (S)-¹⁸F-OF-NB1.

Conclusions: We have successfully synthesized and evaluated (S)- and (R)-¹⁸F-OF-NB1 as PET probes for imaging the GluN2B subunits of the NMDA receptor in non-human primates. Both appeared to bind specifically to GluN2B in vivo, with (R)-¹⁸F-OF-NB1 showing slow clearance from the brain. The (S)-enantiomer in particular exhibited appropriate tissue kinetics and high GluN2B-specific binding that was unaffected by additional blockade of the sigma-1 receptor. As such, (S)-¹⁸F-OF-NB1 is a promising radioligand for clinical translation. Research support: NIH Grant U01-MH107803. Reference: 1. Ahmed H. et al. jnumed.120.246785