Optimization, Automation and Validation of NOP Receptor PET radiopharmaceutical, [11C]NOP-1A for Human Use

637 Objectives: Nociceptin/orphanin FQ peptide (NOP) receptors have been implicated in the pathophysiology of emotional disorders and substance use disorder but in vivo human studies have been sparse due to the lack of relevant PET ligands. [11C]NOP-1A is a novel high-affinity PET ligand for imaging NOP receptors (Lohith et al. 2014). We report herein a fully automated loop radiomethylation of [11C]NOP-1A using GE TRACERlab™ FXC Pro synthesis module (Figure 1) and the validation for human PET studies according to FDA and USP regulations. Methods: [11C]-NOP-1A was produced on the GE TRACERlab™ FXC Pro radiosynthesis unit (Figure 1) by methylation of (S)-N-desmethyl precursor, 1 [(S)-FPSPPA] with [11C]CH3I. (S)-N-Desmethyl precursor, 1 (1.0 ± 0.1 mg) was dissolved in 120 µL of DMF, treated with sodium hydride (NaH) (5-8 mg) and vortexed for 2 minutes under nitrogen atmosphere. The precursor solution was then preloaded into the HPLC loop for radiomethylation. [11C]CH3I is flowed through the loop for 3 minutes (Scheme 1) followed by a 4 minute reaction. The reaction was immediately followed by semi-preparative isocratic RP-HPLC purification (Waters XSelect HSS T3 column, 5µ, 10 x 250 mm, flow at 7 mL/min with 30:70 ACN: 50mM Sodium Phosphate pH 4.5). The major radiochemical product (tR ∼15 min, i.e. [11C]NOP-1A) was collected into a HPLC fraction dilution vessel containing 24 mL water, then transferred to SPE cartridge and eluted with 1 mL of EtOH and 10 mL 0.9% sodium chloride. The [11C]NOP-1A was then passed through a 0.22µ PES sterilizing filter into a sterile vial. Results: The synthesis conditions for radiomethylation of (S)-N-desmethyl precursor, 1 were screened using 11CH3I/11CH3OTf with strong bases such as KOH and NaH. Both bases have an ability to deprotonate of 1 and results observed that both bases gave N-11CH3 product, 2. But in the presence of NaH and DMF yielded higher radiochemical conversion (RCC) (60% RCC by HPLC) than in the presence of KOH and DMSO (48% RCC by HPLC). Handling of KOH is easier than NaH because of sensitivity to air. However, in our hands using KOH as a base and DMSO as a solvent in the reactor method, [11C]NOP-1A was obtained in a lower RCY (3-5%) and did not give us reproducible production as previously described (Pike et al. 2011). Therefore, based on the screening reaction condition results with [11C]CH3I in the presence of NaH as a base and DMF as a solvent allowed for reliable and reproducible productions of [11C]NOP-1A with radiochemical yields in the range of what was reported in the literature. Three consecutive productions of [11C]NOP-1A were carried out to validate this radiopharmaceutical for human use (Table 1). HPLC analysis of the formulated product revealed high radiochemical (>97%) and high chemical purity. Formulated [11C]NOP-1A maintained stability, as measured by radio-HPLC as well as clarity and a pH of 5.5 over a period of 1 hour. The formulated product was free of pyrogens (Charles River Endosafe® PTS) and sterile. Volatile organic compound analysis was carried out by GC-FID showing residual acetone, CH3CN and DMF below the lower limit of detection, thereby within ICH requirements. Conclusions: Based on the results of our experiments, we identified a novel automated synthesis method of producing [11C]NOP-1A using NaH as a base. [11C]NOP-1A was validated for human use with a GE TRACERlab™ FXC Pro radiosynthesis module. The automated radiosynthesis of [11C]NOP-1A provides the radiotracer in a form that is ready for injection and in corrected radiochemical yield of 13 ± 2.5% (relative to starting [11CO2]), with a specific activity of 2147-4402 mCi/μmol at EOS (53 min; n = 3). Acknowledgements: We acknowledge NIH grant: P41EB022544 (PI: El Fakhri) and NARSAD Distinguished Investigator Award, Brain & Behavior Research Foundation (PI: Pizzagalli, grant #: 26950) for generously providing funding for this research. Keywords: Automation, NOP-1A, Carbon-11