Targeting of Metastatic Prostate Cancer with Alpha Particle Generating 212Pb-Labeled PSMA Seeking Ligands

65 Introduction: Patients with metastatic castration-resistant prostate cancer (mCRPC) often experience bone and extraskeletal metastases, resulting in the poor prognosis. Late-stage mCRPC often has a high expression of prostate-specific membrane antigen (PSMA) which can be targeted by radioligands. Here, we present a novel liquid 224Ra/212Pb-generator solution, useful for producing 212Pb-labeled PSMA seeking ligands in situ. Purified 212Pb-labeled ligands can be prepared from the 224Ra/212Pb solution by standard gel filtration. The 212Pb is a source of highly cytotoxic alpha particles via its decay to alpha-emitting 212Bi. Methods: Labeling efficiency of two novel PSMA targeting ligands p-SCN-Bn-TCMC-PSMA ligand 1 (NG001) and p-SCN-Bn-DOTA-PSMA ligand 2 (NG002) with 212Pb using the 224Ra/212Pb-generator was determined and the subsequent removal of 224Ra from the solution using Sephadex G-10 column was evaluated. The PSMA targeting properties of 212Pb-labeled NG001 and NG002 were compared to that of commonly used PSMA-617 labeled with 212Pb. Cell binding and internalization were evaluated in PSMA-positive C4-2 human prostate cancer cells, while tumor-targeting ability was investigated in athymic nude mice bearing C4-2 human prostate cancer xenografts. Results: All PSMA ligands were efficiently labeled with 212Pb using the liquid 224Ra/212Pb generator (radiochemical purity >94% at concentrations of ≥15 µg/ml). The radioligands were purified from the 224Ra solutions with less than 1% breakthrough of 224Ra. Similar binding and internalization of 212Pb-labeled NG001 and PSMA-617 were observed in C4-2 cells (binding ratios of around 32 %IA/106 cells and internalization ratios of around 22 %IA/106 cells at 3 nM of radioligand). All three PSMA ligands displayed comparable tumor uptake after 2 h (18-28 %ID/g), but NG001 showed a 2.5-3.3-fold lower kidney uptake than PSMA-617 and NG002 (kidney uptakes of 21.1±10.3, 52.8±26.6 and 70.1±5.7 %ID/g, respectively). Biodistribution studies of purified [212Pb]Pb-NG001 showed that clearance of the radioligand from non-targeted tissues was rapid (below 1.2 %ID/g in most organs and 10 %ID/g in kidneys after 4 h; 5 %ID/g in kidneys after 24 h), while the clearance from tumor was slow (over 10 %ID/g after 24 h), resulting in improved tumor-to-kidney ratios over time. Conclusions: The 224Ra/212Pb liquid generator is suitable for preparing 212Pb-labeled radioligands, including [212Pb]Pb-NG001 which targets PSMA-expressing cells. In mice, the radioligand accumulated rapidly in tumor and showed a high retention over 24 h, while it rapidly cleared from non-targeted tissues. The obtained results warrant further preclinical studies to evaluate the therapeutic efficacy of [212Pb]Pb-NG001. Acknowledgment: The study was financially supported by Nucligen AS (Oslo, Norway) and the Industrial PhD project number 290639 of The Norwegian Research Council (Oslo, Norway).