Radiochemistry and comparative in vitro assessment of PSMA-617 labeled with lead-212 (²¹²Pb), actinium-225 (²²⁵Ac),...

PSMA-targeted radioligand therapy is a promising approach for the treatment of advanced prostate cancer; however, the clinical efficacy of [¹⁷⁷Lu]Lu-PSMA-617 (Pluvicto^®) is limited by the relatively low cytotoxic potency of the β-emitting radionuclide ¹⁷⁷Lu (t_1/2: 6.65 d). This has driven high interest in α-emitting radionuclides, such as ²¹²Pb (t_1/2: 10.64 h) and ²²⁵Ac (t_1/2: 9.92 d), which deliver high Linear Energy Transfer (LET) and cause more potent tumor cell killing. In this work, we assessed the radiolabeling and in vitro characteristics of ²¹²Pb- and ²²⁵Ac-labeled PSMA-617 compared with [¹⁷⁷Lu]LuPSMA-617, using two different PSMA-positive prostate cancer cell lines, LNCaP-AR and DU145-PSMA, along with paired negative controls.

The radioligands were synthesized with an isolated radiochemical yield of > 95% for [¹⁷⁷Lu]Lu-PSMA-617 and about 45% for [²¹²Pb]Pb-PSMA-617 and [²²⁵Ac]Ac-PSMA-617. The molar activity after Sep-Pak purification was about 37.0 MBq/nmol for [¹⁷⁷Lu]Lu-PSMA-617, 4.4-11.1 MBq/nmol for [²¹²Pb]Pb-PSMA-617, and 0.22-0.55 MBq/nmol for [²²⁵Ac]Ac-PSMA-617. In vitro stability studies in PBS, human serum, and whole blood revealed ≥ 90% stability for [¹⁷⁷Lu]Lu-PSMA-617 (up to 5 days) and [²¹²Pb]Pb-PSMA-617 (24 h), whereas [²²⁵Ac]Ac-PSMA-617 exhibited ~ 72% stability in PBS, > 94% in serum, and > 86% in whole blood for 5 days. The uptake of [¹⁷⁷Lu]Lu-PSMA-617 in LNCaP-AR cells was 7.5 ± 0.6%, with about 33% of the cell-bound activity internalized at 4 h. The uptake and internalization were significantly higher in the PSMA-overexpressing cell line DU145-PSMA (31.1 ± 0.6%, 65% internalized). Compared to [¹⁷⁷Lu]Lu-PSMA-617, [²¹²Pb]Pb-PSMA-617 showed about 2-fold higher uptake and increased internalization in LNCaP-AR cells at 4 h (14.2 ± 0.0%, 47.1% internalized), but a similar uptake in DU145-PSMA cells (29.3 ± 0.7%, 62% internalized). In contrast, [²²⁵Ac]Ac-PSMA-617 exhibited lower uptake in both cell lines, with 1.6 ± 0.2% in LNCaP-AR cells and 4.6 ± 0.2% in DU145-PSMA cells after 4 h incubation. For all 3 radioligands, the uptake could be fully blocked by co-incubation with unlabeled PSMA-617 (300 nM), confirming the specificity of binding to PSMA, and the uptake was minimal in the paired PSMA-negative cell lines CWRR1-EnzR and DU145. In saturation binding assays, the three radioligands exhibited comparable binding affinities (K_D) in LNCaP-AR and DU145-PSMA cell lines, with 5.9 ± 0.7 nM and 2.3 ± 0.3 nM for [¹⁷⁷Lu]Lu-PSMA-617, 5.7 ± 0.5 nM and 5.8 ± 0.9 nM for [²²⁵Ac]Ac-PSMA-617, and 2.7 ± 0.5 nM and 8.0 ± 1.1 nM for [²¹²Pb]Pb-PSMA-617, respectively.

[²¹²Pb]Pb-PSMA-617 showed similar or better uptake in PSMA-positive cell lines compared to [¹⁷⁷Lu]Lu-PSMA-617. While [²²⁵Ac]Ac-PSMA-617 also showed selective and specific uptake in the two PSMA-positive cell lines, the uptake levels were substantially lower, likely due to the low molar activities achievable for [²²⁵Ac]Ac-PSMA-617 compared to [²¹²Pb]Pb-PSMA-617 or [¹⁷⁷Lu]Lu-PSMA-617.