Radiochemistry and Comparative In Vitro Assessment of PSMA-617 Labeled with Lead-212 (212Pb), Actinium-225 (225Ac), and Lutetium-177 (177Lu).

Radiochemistry and Comparative In Vitro Assessment of PSMA-617 Labeled with Lead-212 (212Pb), Actinium-225 (225Ac), and Lutetium-177 (177Lu). | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Radiochemistry and Comparative In Vitro Assessment of PSMA-617 Labeled with Lead-212 (212Pb), Actinium-225 (225Ac), and Lutetium-177 (177Lu). Abhijit Bera, Graham Ragland, Yuhan Zhang, Patricia G. Madel, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9419820/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background PSMA-targeted radioligand therapy is a promising approach for the treatment of advanced prostate cancer; however, the clinical efficacy of [ 177 Lu]Lu-PSMA-617 (Pluvicto®) is limited by the relatively low cytotoxic potency of the β-emitting radionuclide 177 Lu (t 1/2 : 6.65 d). This has driven high interest in α-emitting radionuclides, such as 212 Pb (t 1/2 : 10.64 h) and 225 Ac (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 212 Pb- and 225 Ac-labeled PSMA-617 compared with [ 177 Lu]LuPSMA-617, using two different PSMA-positive prostate cancer cell lines, LNCaP-AR and DU145-PSMA, along with paired negative controls. Results The radioligands were synthesized with an isolated radiochemical yield of > 95% for [ 177 Lu]Lu-PSMA-617 and about 45% for [ 212 Pb]Pb-PSMA-617 and [ 225 Ac]Ac-PSMA-617. The molar activity after Sep-Pak purification was about 37 MBq/nmol (1 mCi/nmol) for [ 177 Lu]Lu-PSMA-617, 4.4–8.9 MBq/nmol (120–240 µCi/nmol) for [ 212 Pb]Pb-PSMA-617, and 0.24–0.57 MBq/mol (6.5–15.5 µCi/nmol) for [ 225 Ac]Ac-PSMA-617. In vitro stability studies in PBS, human serum, and whole blood revealed ≥ 90% stability for [ 177 Lu]Lu-PSMA-617 (up to 5 days) and [ 212 Pb]Pb-PSMA-617 (24 hours), whereas [ 225 Ac]Ac-PSMA-617 exhibited ~ 72% stability in PBS and ~ 90% in serum and whole blood for 5 days. The uptake of [ 177 Lu]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 ± 0.5%, 65% internalized). Compared to [ 177 Lu]Lu-PSMA-617, [ 212 Pb]Pb-PSMA-617 showed about 2-fold higher uptake and internalization in LNCaP-AR cells at 4 h (14.2 ± 0.0%, 58% internalized), but a similar uptake in DU145-PSMA cells (29.3 ± 0.7%, 62% internalized). In contrast, [ 225 Ac]Ac-PSMA-617 exhibited lower uptake in both cell lines, with 1.1 ± 0.0% 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 ± 0.5 nM for [ 177 Lu]Lu-PSMA-617, 5.7 ± 0.5 nM and 5.8 ± 0.9 nM for [ 225 Ac]Ac-PSMA-617, and 2.7 ± 0.5 nM and 8.0 ± 1.1 nM for [ 212 Pb]Pb-PSMA-617, respectively. Conclusion [ 212 Pb]Pb-PSMA-617 showed similar or better uptake in PSMA-positive cell lines compared to [ 177 Lu]Lu-PSMA-617. While [ 225 Ac]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 [ 225 Ac]Ac-PSMA-617 compared to [ 212 Pb]Pb-PSMA-617 or [ 177 Lu]Lu-PSMA-617. PSMA-617 Targeted Alpha Therapy 177Lu 225Ac 212Pb Prostate Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Prostate cancer remains a leading cause of cancer-related mortality in men in the United States and worldwide (Kratzer et al., 2025 ; Sung et al., 2021 ). Early translational studies demonstrated that PSMA-617, which consists of a DOTA chelator conjugated to the Glu-urea-Lys pharmacophore, exhibits high binding affinity to prostate-specific membrane antigen (PSMA) and efficient internalization into PSMA-positive cells, laying the foundation for PSMA-targeted radioligand therapy (RLT) (Benesova et al., 2015 ). Subsequent medicinal chemistry efforts and preclinical studies further established PSMA-617 as a robust ligand for labeling with a variety of radionuclides for targeting PSMA expressing prostate cancers (Benesova et al., 2016 ). Clinical translation of lutetium-177 ( 177 Lu, t 1/2 = 6.65 d) labeled PSMA-617 began shortly thereafter, with early clinical studies demonstrating significant tumor regression, improved overall survival, and favorable safety profiles in men with metastatic castration-resistant prostate cancer (Hofman et al., 2021 ). The radiopharmaceutical [ 177 Lu]Lu-PSMA-617 (Pluvicto ®ฏ ) has been approved for the treatment of metastatic castration-resistant prostate cancer following the landmark VISION trial, which demonstrated improved overall survival and radiographic progression-free survival in patients with metastatic castration-resistant prostate cancer (Sartor et al., 2021 ). Despite its promising therapeutic efficacy, approximately 30–50% of patients exhibit limited or no therapeutic response to [ 177 Lu]Lu-PSMA-617 (Kafka et al., 2024 ; Sartor et al., 2021 ), highlighting the need for improved therapeutic strategies. To address these limitations, α-particle-emitting radionuclides such as actinium-225 ( 225 Ac, t 1/2 = 9.92 d) and lead-212 ( 212 Pb, t 1/2 = 10.64 h) have been investigated for PSMA-targeted RLT. Compared with β-emitting radionuclides, α-emitters such as 225 Ac deliver high linear energy transfer (LET, ~ 100 keV/µm) radiation with a short path length (50–100 µm), enabling highly localized tumor cell killing through DNA double-strand breaks while minimizing radiation exposure to surrounding healthy tissues (Kratochwil et al., 2016 ). In line with this, multiple clinical studies have confirmed that 225 Ac-labeled PSMA-617 can provide therapeutic benefits in men with metastatic castration-resistant prostate cancer, including those refractory to [ 177 Lu]Lu-PSMA-617 (Kratochwil et al., 2016 ; Yadav et al., 2020 ). Additionally, preclinical studies in mouse models of metastatic prostate cancer demonstrated that early treatment with [ 225 Ac]Ac-PSMA-617 prevented the development of liver metastases and significantly improved survival, whereas delayed treatment prolonged survival without significantly reducing tumor burden (Stuparu et al., 2020 ). Likewise, α-emitters with shorter half-lives, such as 212 Pb (10.64 h), have also attracted increasing interest for PSMA-targeted RLT. The relatively short half-life of 212 Pb enables rapid decay and localized dose delivery to tumor cells, potentially reducing α-recoil-driven redistribution of radioactive daughter isotopes (Yong & Brechbiel, 2015 ). In addition, 212 Pb exhibits favorable radiochemical properties, allowing reproducible synthesis and stable complexation with DOTA-based ligands, while its generator-based production supports scalable clinical availability (Zimmermann, 2024 ). The PSMA-targeting properties of [ 212 Pb]Pb-PSMA-617 have been investigated in PSMA-positive C4-2 prostate cancer cells, and its in vivo performance supports further exploration for therapeutic applications (Stenberg et al., 2020 ). While [ 177 Lu]Lu-PSMA-617 has established the clinical role of PSMA-targeted RLT, 225 Ac- and 212 Pb-labeled radioligands represent the next generation of RLTs, with the potential to improve therapeutic efficacy and clinical outcomes (Stenberg et al., 2020 ; Yadav et al., 2020 ). In this study, we compared the radiolabeling and in vitro properties of 225 Ac- and 212 Pb-labeled PSMA-617 with the clinically established [ 177 Lu]Lu-PSMA-617 (Fig. 1 ) using two pairs of PSMA-positive and -negative cell lines. Results Nonradioactive Chemistry To evaluate the inhibitory potency of the PSMA-617 conjugates, we synthesized nonradioactive reference analogues of [ 177 Lu]Lu-PSMA-617 and [ 212 Pb]Pb-PSMA-617 by complexing PSMA-617 with lutetium (Lu 3+ ) and lead (Pb 2+ ) using LuCl 3 and PbCl 2 , respectively ( Figures S1 and S2 ). The resulting nonradioactive complexes Lu-PSMA-617 and Pb-PSMA-617 were obtained in 37% and 48% yields, respectively, and with > 95% purity after purification using a Sep-Pak ®ฏ C18 cartridge. The identity of the nonradioactive complexes was verified by LC/MS analysis, which showed the expected molecular ion peaks corresponding to the respective nonradioactive PSMA-617 conjugates ( Figures S1 and S2 ). Nonradioactive Ac-PSMA-617 could not be synthesized due to the lack of a stable isotope for actinium. Radiochemistry Radiolabeling of PSMA-617 with 177 Lu, 225 Ac, and 212 Pb was performed following previously published procedures with some modifications (Hooijman et al., 2021 ; Wurzer et al., 2022 ). For [ 177 Lu]Lu-PSMA-617, the labeling yield increased from 63% with 0.25 nmol precursor (PSMA-617) to 95% with 1 nmol precursor at 37 MBq of 177 LuCl 3 ( Figure S3 ), providing a molar activity of approximately 37 MBq/nmol. Further increase in the amount of precursor (up to 10 nmol) led to ≥ 99% labeling efficiency for the labeled conjugate. In the case of [ 225 Ac]Ac-PSMA-617, instant thin-layer chromatography (iTLC) analysis of the crude reaction mixture indicated a labeling efficiency of 80–95% when the reactions were conducted at a 1–2 nmol scale (PSMA-617) using 0.93–1.85 MBq of [ 225 Ac]AcCl 3 ( Figure S4 ). However, subsequent purification of the crude reaction mixture via a Sep-Pak revealed only about 45% of the loaded activity retained on the column, which was eluted with ethanol and used for in vitro studies. The effective molar activity of [ 225 Ac]Ac-PSMA-617 after Sep-Pak purification was 0.35 ± 0.10 MBq/nmol (n = 4). Similarly, 212 Pb-labeling of PSMA-617 was accomplished by reacting 1 nmol PSMA-617 with 18.5 MBq of [ 212 Pb]PbCl 2 , eluted from a 224 Ra/ 212 Pb generator in 0.2 M sodium acetate buffer. Sep-Pak purification of the crude mixture after radiolabeling yielded [ 212 Pb]Pb-PSMA-617 in 46.3 ± 1.7% (n = 4) yield, which was consistent with that determined by the radio-HPLC analysis of the crude reaction mixture (46.5 ± 7.2%) ( Figure S5 ). The molar activity was 7.5 ± 2.3 MBq/nmol (n = 4). Radio-HPLC analysis of the purified [ 177 Lu]Lu-PSMA-617 and [ 212 Pb]Pb-PSMA-617 showed > 98% radiochemical purity, whereas iTLC was used for the [ 225 Ac]Ac-PSMA-617 conjugate, which showed a purity of approximately 95%. Distribution Coefficient ( D ) Values The lipophilicity of the radioconjugates was determined by measuring their distribution coefficient ( D ) values through partitioning between n-octanol and PBS (pH 7.4). The distribution coefficient was calculated as the ratio of radioactivity (CPM) in the octanol phase to that in the aqueous PBS phase and expressed as log D 7.4 values. The measured log D 7.4 values were − 3.29 ± 0.02 (n = 4) for [ 177 Lu]Lu-PSMA-617, -2.95 ± 0.03 (n = 4) for [ 225 Ac]Ac-PSMA-617, and − 3.01 ± 0.04 (n = 3) for [ 212 Pb]Pb-PSMA-617 (Table 1 ). These results indicate that the three radioligands exhibit comparable lipophilicity, with only minor differences observed among the three PSMA-617-based radioligands. In Vitro Stability Studies The in vitro stability of the three PSMA-617 radioligands was evaluated in PBS, human serum, and human whole blood by incubating the radioligands at 37°C for up to 120 h. [ 177 Lu]Lu-PSMA-617 demonstrated ≥ 90% stability in PBS, human serum, and human whole blood for up to 120 h, as confirmed by iTLC analysis ( Figure S6A ). [ 225 Ac]Ac-PSMA-617 exhibited > 94% stability in human serum over the 120 h incubation period. However, in human whole blood, stability decreased slightly at later time points, reaching 86.3% and 89.1% at 96 h and 120 h, respectively. In comparison, stability in PBS decreased more noticeably, reaching 72.3% at 120 h post-incubation ( Figure S6B ). Because of the short half-life of 212 Pb, the stability of [ 212 Pb]Pb-PSMA-617 was assessed only until 24 h, which revealed ≥ 90% stability in PBS, human serum, and human whole blood ( Figure S6C ). Cell Uptake and Internalization The uptake, specificity, selectivity, and internalization rate of [ 177 Lu]Lu-PSMA-617, [ 225 Ac]Ac-PSMA-617, and [ 212 Pb]Pb-PSMA-617 were compared in two pairs of PSMA-positive and negative prostate cancer cell lines: LNCaP-AR (PSMA-positive) and CWRR1-EnzR (PSMA-negative), and DU145-PSMA (PSMA-positive) and DU145 (PSMA-negative). Immunocytochemical staining followed by fluorescence imaging confirmed high PSMA expression in the two PSMA-positive cell lines LNCaP-AR and DU145-PSMA, with minimal expression in the respective control cell lines CWRR1-EnzR and DU145 (Fig. 2 ). Cell uptake studies showed that the uptake of [ 177 Lu]Lu-PSMA-617 in the LNCaP-AR cell line increased over time, from 2.86 ± 1.02% of added activity at 0.5 h post-incubation to 7.48 ± 0.65% at 4 h (Fig. 3 A). Uptake in the CWRR1-EnzR1 cell line was minimal at all time points, with 0.34 ± 0.03% uptake at 4 h. Co-incubation with excess unlabeled PSMA-617 (300 nM) blocked uptake by > 95%, limiting it to 0.12 ± 0.01% at 4 h in LNCaP-AR cells, confirming the specificity and selectivity of [ 177 Lu]Lu-PSMA-617 binding in the LNCaP-AR cell line. In contrast to [ 177 Lu]Lu-PSMA-617, uptake of [ 225 Ac]Ac-PSMA-617 in LNCaP-AR cells was low, with 0.64 ± 0.05% of added activity at 0.5 h and increasing to 1.58 ± 0.24% at 4 h (Fig. 3 B). Similar to [ 177 Lu]Lu-PSMA-617, co-incubation with excess unlabeled PSMA-617 (300 nM) reduced uptake of [ 225 Ac]Ac-PSMA-617 by > 95%, with 0.26 ± 0.06% at 4 h. Uptake in the PSMA-negative CWRR1-EnzR cells remained minimal at all time points, with 0.20 ± 0.05% uptake at 4 h, which reduced to 0.09 ± 0.04% with blocking. Similar to the above two radioligands, uptake of [ 212 Pb]Pb-PSMA-617 in LNCaP-AR cells increased over time, reaching 14.19 ± 0.03% at 4 h compared to 5.01 ± 0.45% at 0.5 h (Fig. 3 C). Co-incubation with excess unlabeled PSMA-617 (300 nM) reduced uptake to 0.58 ± 0.05% at 4 h, and uptake of [ 212 Pb]Pb-PSMA-617 in the PSMA-negative CWRR1-EnzR cells remained minimal at all time points, with 0.65 ± 0.03% at 4 h. Additionally, for [ 177 Lu]Lu-PSMA-617 and [ 212 Pb]Pb-PSMA-617, the fraction of internalized activity, calculated as the percentage of total uptake in cells, increased with time, from 14.2 ± 8.6% at 0.5 h to 33.3 ± 10.0% at 4 h for [ 177 Lu]Lu-PSMA-617 (Fig. 3 A) and from 31.6 ± 1.5% at 0.5 h to 47.1 ± 0.9% at 4 h for [ 212 Pb]Pb-PSMA-617 (Fig. 3 C). Compared to these, [ 225 Ac]Ac-PSMA-617 exhibited less increase in internalization over time from 40.3 ± 8.4% at 0.5 h to 51.2 ± 2.4% by 4 h (Fig. 3 B). Cell uptake and internalization of the three radioligands were also evaluated in a second pair of isogenic cell lines, a PSMA-overexpressing DU145-PSMA cell line (courtesy of Novartis Institutes for BioMedical Research) and the parental DU145 cell line (PSMA-negative). The results from these studies are consistent with the uptake trends observed for the three radioligands in the LNCaP-AR cell line, but with significantly higher uptake (by 2-3-fold) than in the LNCaP-AR cell line. The uptake of [ 177 Lu]Lu-PSMA-617 in DU145-PSMA cells at 4 h was 31.10 ± 0.64% compared to 0.29 ± 0.02% with blocking (300 nM PSMA-617) and 0.22 ± 0.03% in the parental DU145 cells (Fig. 4 A ). Consistent with results from the LNCaP-AR cell line, [ 225 Ac]Ac-PSMA-617 exhibited lower uptake in the DU145-PSMA cells; nonetheless, the uptake increased over time, from 2.26 ± 0.08% at 0.5 h to 4.63 ± 0.18% at 4 h (Fig. 4 B). Co-incubation of cells with an excess of PSMA-617 (300 nM) reduced the uptake to 0.31 ± 0.01% at 4 h, and the uptake in control DU145 cells remained minimal (0.12 ± 0.05%). In contrast, [ 212 Pb]Pb-PSMA-617 exhibited a high uptake that is comparable to [ 177 Lu]Lu-PSMA-617 in DU145-PSMA cells. Uptake at 0.5 h was 13.74 ± 1.07%, increasing with time to 29.26 ± 0.69% at 4 h. For comparison, uptake in the parental DU145 cell line was 0.32 ± 0.02%, and 0.70 ± 0.04% with blocking (300 nM PSMA-617) at 4 h. (Fig. 4 C). For all three radioligands, > 60% of uptake was found to be internalized in DU145-PSMA cells at 4 h, with the remaining activity (< 40%) being cell-surface bound (Fig. 4 ). Taken together, these results demonstrate high PSMA-specific uptake of the three PSMA-617 radioconjugates in PSMA-expressing cells, with minimal uptake observed in PSMA-negative controls. However, the magnitude of uptake was lower for [ 225 Ac]Ac-PSMA-617 relative to [ 212 Pb]Pb-PSMA-617 and the reference [ 177 Lu]Lu-PSMA-617 in both the cell lines ( Figure S7 ). Inhibitory Potency (IC) Assays The inhibitory potencies (IC 50 ) of the nonradioactive analogues Lu-PSMA-617 and Pb-PSMA-617 were evaluated by a competitive inhibition assay using [ 177 Lu]Lu-PSMA-617 in DU145-PSMA cells. Cells were incubated with [ 177 Lu]Lu-PSMA-617 in the presence of increasing concentrations of Lu-PSMA-617 or Pb-PSMA-617 (0.14–300 nM). Cell uptake, presented as the percentage of added activity, was assessed at each concentration to determine the half-maximal inhibitory concentration value (IC 50 ) for the two nonradioactive PSMA-617 inhibitors. The results demonstrated concentration-dependent inhibition of the radiotracer uptake in the DU145-PSMA cell line. The inhibitory potency (IC 50 ) values derived from these assays were 8.6 nM for Lu-PSMA-617 and 10.5 nM for Pb-PSMA-617 (Fig. 5 ), indicating similar inhibitory potency for the two PSMA-617 conjugates toward PSMA. Binding Affinity Studies The binding affinities ( K D ) of the three radioligands for PSMA were evaluated using the LNCaP-AR and DU145-PSMA cell lines by conducting saturation binding assays as described previously (Chitneni, Yan, & Zalutsky, 2018 ). Cells were incubated with increasing concentrations of 177 Lu-, 225 Ac-, or 212 Pb-labeled PSMA-617 (0.4–50.0 nM) for 2 h, and the cell-bound activity was assessed by gamma counting. For each concentration, nonspecific binding was measured in parallel by co-incubating cells with an excess of unlabeled PSMA-617 (300 nM). Specific binding data were generated by subtracting nonspecific binding from total binding for each concentration, and nonlinear regression analysis was conducted in GraphPad ®ฏ Prism (Fig. 6 ). Equilibrium dissociation constant values ( K D ), determined as the concentration needed to achieve half-maximum binding at equilibrium, were derived from these assays for the three radioligands in the two PSMA-positive cell lines. The K D values determined from these assays in LNCaP-AR and DU145-PSMA cells were 5.9 ± 0.7 nM and 2.3 ± 0.3 nM for [ 177 Lu]Lu-PSMA-617 (Fig. 6 A), compared to 5.7 ± 0.5 nM and 5.8 ± 0.9 nM for [ 225 Ac]Ac-PSMA-617 (Fig. 6 B) and 2.7 ± 0.5 nM and 8.0 ± 1.0 nM for [ 212 Pb]Pb-PSMA-617 (Fig. 6 C), respectively. Table 1 Overview of in vitro properties of the three PSMA-617 radioligands synthesized in this work, [ 177 Lu]Lu-PSMA-617, [ 225 Ac]Ac-PSMA-617, and [ 212 Pb]Pb-PSMA-617. Radioligand Lipophilicity (log D ) Molar Activity (per nmol) Cell Uptake (PSMA+, 4 h) Binding Affinity ( K D ) LNCaP-AR DU145-PSMA LNCaP-AR DU145-PSMA [ 177 Lu]Lu-PSMA-617 -3.29 37.0–74.0 MBq 7.5 ± 0.7% 31.1 ± 0.5% 5.9 nM 2.3 nM [ 225 Ac]Ac-PSMA-617 -2.95 0.22–0.55 MBq 1.6 ± 0.2% 4.6 ± 0.2% 5.7 nM 5.8 nM [ 212 Pb]Pb-PSMA-617 -3.01 4.4–11.1 MBq 14.2 ± 0.0% 29.3 ± 0.7% 2.7 nM 8.0 nM Discussion Our radiolabeling experiments of PSMA-617 with the β-emitter 177 Lu and the α-emitters 225 Ac and 212 Pb revealed important differences in radiolabeling characteristics and radiochemical yields for the corresponding radionuclides. Unlike 177 Lu, radio-HPLC-based QC analysis is less informative for 225 Ac due to its complex decay scheme and low levels of radioactivity employed, necessitating the use of iTLC-based methods to determine radiolabeling yields and/or purity of the labeled compounds. Although our iTLC analysis of the crude reaction mixture consistently showed > 95% labeling with less free 225 Ac ( Figure S4 ), subsequent Sep-Pak purification typically retained only ~ 45% of the loaded activity, with the remainder of the activity either unretained or washed off with the water rinse. Careful assessment of the iTLC chromatograms of the crude reaction mixture and the unretained activity fractions indicated the presence of a labeled species migrating at the front of the [ 225 Ac]Ac-PSMA-617 peak on the iTLC (at 40–50 mm, Figure S4 ). Thus, for all subsequent studies and in vitro evaluation of [ 225 Ac]Ac-PSMA-617, the reaction mixture was purified by Sep-Pak, and the radioligand was freshly synthesized on the day of each in vitro experiment. Additionally, all cell uptake studies were conducted in the presence of DTPA (0.1 mg/mL) in the incubation mixture to chelate any free 225 Ac or daughter isotopes, although comparable results were obtained without DTPA as well in select experiments ( Figure S8 ). In contrast, radiolabeling of PSMA-617 with 212 Pb proved more straightforward. However, despite optimization efforts and increasing the precursor amount (PSMA-617), the RCY could not be improved beyond ~ 50%. Nonetheless, the molar activities we obtained for [ 212 Pb]Pb-PSMA-617 in the present study proved sufficient (4.4–11.1 MBq/nmol) for efficient cell uptake and internalization and yielded similar or higher uptake than [ 177 Lu]Lu-PSMA-617 in the two PSMA-positive cell lines we evaluated (Figs. 3 and 4 ). Across the three radioligands, lipophilicity values were comparable (Table 1 ) and reflect the overall hydrophilicity of the molecule (PSMA-617), suggesting that substitution of 177 Lu in [ 177 Lu]Lu-PSMA-617 with 225 Ac or 212 Pb may not significantly alter the physicochemical properties of the resulting radioconjugates. In vitro stability studies demonstrated excellent radiochemical stability for [ 177 Lu]Lu-PSMA-617 and [ 225 Ac]Ac-PSMA-617, with ≥ 90% stability in PBS, human serum, and human whole blood up to 72 h. In view of the short half-life of 212 Pb (10.6 h), the stability of [ 212 Pb]Pb-PSMA-617 was evaluated only for 24 h, which showed ≥ 90% stability of the labeled conjugate. Comparative cell uptake studies in the two PSMA-expressing prostate cancer cell lines revealed distinct trends for the three isotopes or the radioligands. In LNCaP-AR cells, [ 212 Pb]Pb-PSMA-617 exhibited about 2-fold higher uptake than [ 177 Lu]Lu-PSMA-617, but similar levels of uptake were noted for both radioligands in the DU145-PSMA cell line. Despite the differences in the total uptake levels, all three radioligands exhibited a higher uptake and internalized fraction in the PSMA overexpressing cell line DU145-PSMA than in LNCaP-AR at 4 h post-incubation. Few studies have reported the radiosynthesis and in vivo evaluation of [ 225 Ac]Ac-PSMA-617 (Busslinger et al., 2022 ; Stuparu et al., 2020 ); however, systematic evaluation of the labeled conjugate in vitro models and head-to-head comparison with the established PSMA-targeted radioligand [ 177 Lu]Lu-PSMA-617 and [ 212 Pb]Pb-PSMA-617 is largely lacking. We believe that the lower uptake of [ 225 Ac]Ac-PSMA-617 compared to [ 177 Lu]Lu-PSMA-617 and [ 212 Pb]Pb-PSMA-617 in the two PSMA-positive cell lines was primarily due to the low molar activity of the radioligand, resulting in competitive inhibition of the radioligand by the unlabeled PSMA-617 in the final product. In this study, our initial attempts to achieve a molar activity of at least 3.7 MBq/nmol for [ 225 Ac]Ac-PSMA-617, to be on par with [ 212 Pb]Pb-PSMA-617, were unsuccessful. In contrast, an acceptable molar activity was achieved for [ 212 Pb]Pb-PSMA-617. The similar or higher uptake of [ 212 Pb]Pb-PSMA-617 compared to [ 177 Lu]Lu-PSMA-617 in our cell uptake studies indicates a desired molar activity of ≥ 3.7 MBq/nmol for efficient uptake of PSMA-617-based radioligands in PSMA expressing prostate cancer cells. Minimal uptake of the three radioligands in the PSMA-negative cell lines (CWRR1-EnzR and DU145) and efficient blocking with excess PSMA-617 in the two PSMA-positive cell lines confirms the specificity of the three radioligands to PSMA and the PSMA-mediated internalization of the radioligands in our cell models. Although molar activities, uptake, and internalization rates varied between cell lines, saturation binding assays demonstrated comparable binding affinity values ( K D ) in the nM range for 177 Lu-, 225 Ac-, and 212 Pb-labeled PSMA-617 in the two PSMA-positive cell lines LNCaP-AR and DU145-PSMA (Fig. 6 ). Our results strongly suggest that the change of radioisotope did not affect the binding affinity of labeled PSMA-617 in PSMA-expressing cells. Additionally, the observed binding affinity values are consistent with those reported in the literature, e.g., a K D of about 4.4 nM for [ 177 Lu]Lu-PSMA-617 to LNCaP cells (Peng, Chen, & Tang, 2025 ), 11 nM for [ 225 Ac]Ac-PSMA-617 to PC-3 PIP cells (Busslinger et al., 2022 ), and about 11.1 nM for [ 212 Pb]Pb-PSMA-617 to C4-2 cells (Stenberg et al., 2020 ). Collectively, our results indicate the suitability of [ 225 Ac]Ac-PSMA-617 and [ 212 Pb]Pb-PSMA-617 for further evaluation using in vivo models of PSMA-expressing prostate cancer in comparison with [ 177 Lu]Lu-PSMA-617. Although it may not be possible to achieve high molar activity for [ 225 Ac]Ac-PSMA-617 similar to that for 177 Lu- or 212 Pb-labeled PSMA-617, it remains to be tested if the reduced cell uptake from in vitro models translates to lower tumor uptake (e.g., percentage injected dose per gram (% ID/g)) in vivo compared to [ 177 Lu]Lu-PSMA-617 or [ 212 Pb]Pb-PSMA-617. To that end, our future studies will focus on extending these in vitro studies to in vivo by directly comparing tumor uptake of the three radioligands and the therapeutic efficacy of the ⍺-emitting PSMA-617 radioligands with [ 177 Lu]Lu-PSMA-617 in the same tumor models. Conclusion This study was designed to directly compare radiolabeling and in vitro characteristics of PSMA-617 labeled with α- and β-emitting radionuclides as a prelude to in vivo comparisons in the same tumor models. In this study, we successfully optimized radiolabeling protocols for [ 225 Ac]Ac-PSMA-617 and [ 212 Pb]Pb-PSMA-617, achieving high radiochemical purity and in vitro stability suitable for in vivo evaluation. Both the α-emitting radioligands demonstrated selective uptake, specificity, and efficient internalization in two different PSMA-expressing prostate cancer cell lines. Notably, [ 212 Pb]Pb-PSMA-617 exhibited an in vitro uptake comparable to or exceeding that of [ 177 Lu]Lu-PSMA-617 in PSMA-positive cells, highlighting its potential as a targeted α-therapy agent for PSMA-expressing prostate cancers. Although [ 225 Ac]Ac-PSMA-617 exhibited PSMA-specific uptake in the two cell lines we evaluated, the uptake levels were substantially lower, most likely due to the low molar activities achievable for 225 Ac-labeling versus 212 Pb- and 177 Lu-labeling. Collectively, the results from this study support further evaluation of [ 212 Pb]Pb-PSMA-617 and [ 225 Ac]Ac-PSMA-617 to directly compare their tumor uptake and therapeutic efficacy in prostate cancer models for targeted α-therapy in comparison with [ 177 Lu]Lu-PSMA-617. Experimental Section General methods All reagents and solvents were purchased from Fisher Scientific, MilliporeSigma, or Ambeed and used as received unless otherwise noted. PSMA-617 was supplied by Novartis Institutes for BioMedical Research (NIBR, Basel, Switzerland). TraceSELECT™ water was purchased from Honeywell, and Chelex® 100 resin was obtained from Bio-Rad. All buffers and reagents used in this work were prepared using Chelex-treated water unless specified. Nonradioactive complexes were analyzed using an expression-L compact mass spectrometer coupled to a high-performance liquid chromatography (HPLC) system equipped with a UV detector and operated using Mass Express software (Advion Interchim Scientific, Ithaca, NY). The following method was used for the analysis: Agilent Poroshell 120 C18 column (3 × 50 mm); mobile phases: A = water with 0.1% trifluoroacetic acid (TFA), B = acetonitrile (MeCN) with 0.1% TFA; flow rate: 0.3 mL/min; gradient conditions: 0-0.3 min, 5% B; 0.3-9 min, 5→95% B; 9-9.5 min, 95→5% B. All three isotopes, 177 Lu, 225 Ac, and 212 Pb, were purchased through the U.S. Department of Energy’s National Isotope Development Center (NIDC). 177 Lu was supplied as 177 LuCl 3 in 0.04 M HCl. 225 Ac was supplied as dry 225 AcNO 3 . 212 Pb was obtained in the form of a 224 Ra/ 212 Pb generator and eluted following the instructions provided with the generator. Briefly, a Pb resin QML cartridge (20–50 µm, Eichrom Technologies) was preconditioned with 2 M HCl (1 mL) and connected to the outlet of the generator. Then 1 mL of 2 M HCl was passed through the inlet line of the generator to elute and capture 212 Pb on the Pb cartridge. The tubing was purged with air, after which the Pb cartridge was disconnected from the generator. The generator was then rinsed with 2 mL of water and was left with water for storage until the next elution (the next day). The Pb resin cartridge, containing 212 Pb, was then rinsed with water (1 mL) to remove residual acid. The cartridge was reversed, and 212 Pb was eluted by passing 0.2-1.0 M sodium acetate (NaOAc) solution (0.5-1 mL), which was used directly for radiolabeling reactions without further modification. HPLC analysis of radioactive samples ( 177 Lu, 212 Pb) was conducted using an Agilent 1260 Infinity II quaternary pump system coupled to an Agilent 1260 Infinity II variable-wavelength UV detector and a Dual Scan-RAM radio-TLC and radio-HPLC detector (LabLogic, Chantilly, VA). The following method was used for the HPLC analysis: Kinetix® 5 µm EVO C18 column (4.6 × 150 mm, 100 Å); mobile phases: A = water with 0.1% TFA, B = MeCN with 0.1% TFA; flow rate: 1.0 mL/min; gradient conditions: 0 min, 5% B; 0–10 min, 5→70% B; 10–12 min, 70% B; 12–15 min, 70→5% B. Unless specified, all 225 Ac samples were counted (CPM) at approximately 16 h after collection to allow reaching secular equilibrium with gamma-emitting progeny isotopes (e.g., 213 Bi), in an automated gamma counter (Cobra II, Packard). Synthesis of Nonradioactive Lu-PSMA-617 and Pb-PSMA-617 Complexes For the synthesis of nonradioactive reference standards, PSMA-617 (1 mg, 1 µmol) was reacted with either LuCl 3 (0.6 mg, 1.4 µmol) in 0.25 M NaOAc buffer (pH 5.5, 150 µL) or PbCl 2 (0.42 mg, 1.5 µmol, 1.5 equiv.) in 0.25 M NaOAc buffer (pH 5.5, 150 µL). The reaction mixture was heated at 90°C for 30 min, and the resulting complexes, Lu-PSMA-617 and Pb-PSMA-617, were purified using an Oasis HLB Plus Light cartridge (30 mg sorbent, Waters), eluted with ethanol. The purified complexes were characterized using LC-MS. Lu-PSMA-617: white solid, isolated yield: 0.45 mg (37%). ESI-MS: calc. for C 49 H 69 LuN 9 O 16 [M + H]⁺: 1214.4; found, [M + H]⁺: 1214.1. Pb-PSMA-617: white solid, isolated yield: 0.6 mg (48%). ESI-MS, calc. for C 49 H 69 N 9 O 16 Pb [M + H]⁺: 1247.4629, [M + 2H] 2 ⁺: 624.8, found, [M + 2H] 2 ⁺: 624.8. Radiochemistry In a 1.5 mL protein Eppendorf tube (LoBind ®ฏ ), approximately 37 MBq of 177 LuCl 3 in 0.25 M NaOAc buffer (pH 5.5, 100 µL) or 18.5 MBq of 212 PbCl 2 in 0.2 M NaOAc buffer (pH 5.5, 100 µL) was mixed with PSMA-617 (~ 1 nmol, 1 µL). The reaction mixture was heated at 90°C for 30 min in a thermomixer (500 rpm) to obtain the corresponding radioligand. Radiolabeling efficiency was evaluated by radio-HPLC as described above. The reaction mixture was cooled to room temperature and purified using an Oasis HLB Plus Light cartridge (30 mg, Waters). The cartridge was washed with water (2 × 2 mL), and the radiolabeled product was eluted with ethanol (200 proof, 0.5 mL) into a vial for use in in vitro studies. For 225 Ac labeling of PSMA-617, 225 AcCl 3 (0.92–1.85 MBq) was added to 0.2 M NH 4 OAc buffer (pH 5.2, 100 µL) in a 0.5 mL Eppendorf tube (LoBind ®ฏ ), followed by the addition of PSMA-617 (1 nmol, 1 µL). The reaction mixture was heated at 95°C for 45 min in a thermomixer (500 rpm), after which time the reaction mixture was cooled to room temperature and quenched with 50 nM DTPA solution (4 µL). The crude mixture was purified using an Oasis HLB Plus Light cartridge (30 mg), washed with water (2 × 2 mL), and the labeled compound was eluted with ethanol (200 proof, 0.5 mL). Distribution Coefficient ( D ) The distribution coefficient ( D ) of the PSMA-617 radioligands was evaluated by the shake-flask method. Briefly, an aliquot of the radioligands was added to a mixture of n -octanol and PBS pH 7.4 (2 mL each, n = 3–4) in polypropylene tubes and vortexed for 1 min. The octanol and aqueous layers were separated by centrifugation at 3000 rpm for 5 min. From each tube, 0.5 mL aliquots of octanol and water phases were carefully withdrawn into pre-weighed Eppendorf tubes and measured for radioactivity in an automated gamma counter (Cobra II, Packard). Samples were weighed and normalized for density to get radioactivity counts as CPM/mL. Distribution coefficient ( D ) values were calculated as the ratio of CPM/mL in the octanol phase to that in the aqueous phase and presented as log D (mean ± SD) for each radioligand. In Vitro Stability Studies In a 1.5 mL Eppendorf tube (LoBind ®ฏ ), approximately 3.7 MBq of [ 177 Lu]Lu-PSMA-617, 74 kBq of [ 225 Ac]Ac-PSMA-617, or 0.37 MBq of [ 212 Pb]Pb-PSMA-617 were incubated with 0.2 mL of PBS (pH 7.4), human serum, or human whole blood at 37°C for up to 5 days in a thermomixer (500 rpm). For [ 212 Pb]Pb-PSMA-617, the incubation period was limited to 24 h due to its short physical half-life (10.6 h). At 24 h intervals, a 20 µL aliquot was withdrawn and analyzed by iTLC. The strips were developed in 0.1 M citrate buffer (pH 7.4), dried, cut into 1 cm sections, and measured for radioactivity using an automated gamma counter (Cobra II, Packard). For [ 225 Ac]Ac-PSMA-617, the iTLC strips were recounted after at least 16 h to allow secular equilibrium of ɣ-emitting progeny isotopes (e.g., 213 Bi). Immunocytochemistry Studies For immunocytochemistry, 1 × 10 6 cells were seeded in 6-well plates containing poly-L-lysine-coated coverslips (Fisher Scientific) and allowed to attach before staining. Cells were fixed with 2% methanol-free formaldehyde prepared in 1x PBS for 15 min at room temperature and washed three times with PBS. Cells were then permeabilized by incubation with methanol at -20°C for 15 min and blocked for 1 h at room temperature in PBS supplemented with 5% normal serum (Cell Signaling Technology) and 0.1% Triton X-100. The cells were incubated overnight at 4°C with primary antibody against PSMA (Cell Signaling Technology, catalog #12702) diluted 1:400 in PBS containing 0.1% Triton X-100. Following primary antibody incubation, samples were washed three times with PBS and incubated for 1 h at room temperature protected from light with Alexa Fluor 550-conjugated anti-rabbit IgG (H + L) F(ab') 2 fragment secondary antibody (Cell Signaling Technology) diluted 1:1000 in PBS containing 0.1% Triton X-100. Cells were washed three times with PBS and mounted using ProLong Diamond Antifade Mountant with DAPI (Invitrogen, #P36971). Fluorescence and bright-field images were acquired using an EVOS fluorescence microscope at 40× magnification. Cell Uptake and Internalization Studies LNCaP-AR, CWRR1-EnzR, DU145-PSMA, and DU145 cells were cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS (Corning) and 1% penicillin/streptomycin (ThermoFisher), with the additional supplements for the following cell lines: 1% HEPES (ThermoFisher), 1% sodium pyruvate (ThermoFisher), 1% Glutamax (ThermoFisher) for LNCaP-AR; 10 µM enzalutamide (MedChemExpress) for CWRR1-EnzR. For cell uptake and internalization studies, cells were seeded in 24-well plates at 150,000 cells per well in triplicate and allowed to adhere overnight in the growth medium. On the following day, the culture medium was removed, and cells were washed once with ice-cold PBS. Cells were then incubated at 37°C with 74 kBq of [ 177 Lu]Lu-PSMA-617, 7.4 kBq of [ 225 Ac]Ac-PSMA-617, or 18.5 kBq of [ 212 Pb]Pb-PSMA-617 in 0.5 mL serum-free medium, either in the presence or absence of an excess of unlabeled PSMA-617 (300 nM) to assess blocking. Unless specified, the cell uptake studies of [ 225 Ac]Ac-PSMA-617 were conducted in the presence of DTPA (0.1 mg/mL) in the incubation medium. At predetermined time points (0.5, 1, 2, and 4 h), the supernatant was collected, and cells were washed twice with 0.5 mL ice-cold PBS, collecting washes. Next, cells were incubated with 0.5 mL of ice-cold glycine-HCl buffer (50 mM, pH 2.5-3.0) for 5 min at room temperature to isolate cell surface-bound radioactivity, followed by an additional PBS wash. The glycine wash and PBS wash were pooled to represent the cell surface-bound fraction. Subsequently, cells were lysed using 0.25 mL Cell Lysis Reagent (Promega), and the wells were rinsed with 0.25 mL PBS, which was combined with the lysis fraction to obtain the internalized fraction. Radioactivity counts (CPM) in all fractions were measured using an automated gamma counter (Cobra II). Cell uptake was calculated as the percentage of the input dose (CPM) present in the cell fraction for each well and expressed as mean ± standard deviation (SD) for triplicate wells at each time point. Cell surface-bound (glycine wash) and internalized (lysis fraction) fractions were also determined as percentages of the initially bound activity to assess internalization rate for each radioligand at different time points. Inhibitory Potency (IC 50 ) Assays 50 The inhibitory potencies of the nonradioactive Lu-PSMA-617 and Pb-PSMA-617 analogues were determined by competitive inhibition assays using [ 177 Lu]Lu-PSMA-617 in the DU145-PSMA cell line. Cells seeded in 24-well plates (150,000 cells/well) were incubated with [ 177 Lu]Lu-PSMA-617 (2 µCi/well) in serum-free medium for 2 h under standard cell culture conditions in the presence of increasing concentrations of Lu-PSMA-617 or Pb-PSMA-617 (0.14–300 nM, 3-fold serial dilutions, n = 8). After incubation, the medium was collected, and cells were washed with cold PBS (2 × 0.5 mL). Cells were then lysed with 1× Cell Lysis Reagent (0.25 mL, Promega), followed by an additional PBS wash (0.25 mL). Radioactivity in each fraction was measured using an automated gamma counter. Cell-bound activity was determined by calculating the percentage of the input dose (CPM). Half-maximal inhibitory concentration values (IC 50 ) for the two nonradioactive conjugates were determined by nonlinear regression analysis of the inhibitory data using a log(inhibitor) versus response model in GraphPad Prism (v10.6). Saturation Binding Assays The binding affinities ( K D ) of the PSMA radioligands were determined by saturation binding assays using the two PSMA-positive cell lines LNCaP-AR and DU145-PSMA. Cells seeded in 24-well plates (150,000 cells/well) were incubated with [ 177 Lu]Lu-PSMA-617, [ 225 Ac]Ac-PSMA-617, or [ 212 Pb]Pb-PSMA-617 for 2 h at 37°C in serum-free medium containing a series of increasing concentrations of the corresponding radioligand (0.40–50 nM, n = 8), prepared by two-fold serial dilutions starting from 50 nM. For each concentration, nonspecific binding was assessed in parallel by co-incubation with an excess of unlabeled PSMA-617 (300 nM). After incubation, the supernatant was collected, and the cells were washed with ice-cold PBS and lysed as described for cell uptake studies. Radioactivity (CPM) in the cell fractions was measured using an automated gamma counter (Cobra II). The equilibrium dissociation constant ( K D ) was calculated by nonlinear regression analysis of the data in GraphPad Prism (v10.6). Abbreviations PSMA prostate-specific membrane antigen RLT radioligand therapy LET linear energy transfer Lu lutetium Ac actinium Pb lead Bi bismuth mCi millicurie µCi microcurie kBq kilobecquerel MBq megabecquerel nmol nanomoles M molar nM nanomolar µM micromolar µmol micromole PBS phosphate-buffered saline K D equilibrium dissociation constant mCRPC metastatic castration-resistant prostate cancer keV kiloelectronvolt µm micrometer DNA deoxyribonucleic acid HPLC high-performance liquid chromatography LC-MS liquid chromatography-mass spectrometry iTLC instant thin-layer chromatography D distribution coefficient CPM counts per minute h hours min minutes DTPA diethylenetriaminepentaacetic acid mg milligram mL milliliter µL microliter α alpha β beta γ gamma QC quality control RCY radiochemical yield HCl hydrochloric acid NaOAc sodium acetate NH 4 OAc ammonium acetate ESI-MS electrospray ionization mass spectrometry MeCN acetonitrile TFA trifluoroacetic acid Å angstrom. Declarations Supplementary Information The online version contains supplementary material Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors have no financial or proprietary interests in any material discussed in this article. Funding The authors acknowledge funding from the Novartis Institutes for BioMedical Research (NIBR) that supported this research. Authors’ Contributions AB, GR, YZ, PM, JB’L, and AS contributed to the experimental design, data collection, and analysis. RZS, CTC, and SKC contributed to the study design and data interpretation and provided resources. All authors have read and approved the manuscript. Acknowledgements The authors would like to thank the Novartis Institutes for BioMedical Research for providing the DU145-PSMA cell line. Data availability The data is available upon request References Benesova M, Bauder-Wust U, Schafer M, Klika KD, Mier W, Haberkorn U, et al. Linker modification strategies to control the prostate-specific membrane antigen (PSMA)-targeting and pharmacokinetic properties of DOTA-conjugated PSMA inhibitors. J Med Chem. 2016;59(5):1761–75. Benesova M, Schafer M, Bauder-Wust U, Afshar-Oromieh A, Kratochwil C, Mier W, et al. Preclinical evaluation of a tailor-made DOTA-conjugated PSMA inhibitor with optimized linker moiety for imaging and endoradiotherapy of prostate cancer. J Nucl Med. 2015;56(6):914–20. Busslinger SD, Tschan VJ, Richard OK, Talip Z, Schibli R, Müller C. [ 225 Ac]Ac-SibuDAB for targeted alpha therapy of prostate cancer: preclinical evaluation and comparison with [ 225 Ac]Ac-PSMA-617. Cancers. 2022;14(22):5651. Chitneni SK, Yan H, Zalutsky MR. Synthesis and evaluation of a 18 F-labeled triazinediamine analogue for imaging mutant IDH1 expression in gliomas by PET. ACS Med Chem Lett. 2018;9(7):606–11. Hofman MS, Emmett L, Sandhu S, Iravani A, Joshua AM, Goh JC et al. Prostate Cancer Trials, G. (2021). [ 177 Lu]Lu-PSMA-617 versus cabazitaxel in patients with metastatic castration-resistant prostate cancer (TheraP): a randomised, open-label, phase 2 trial. Lancet, 397 (10276), 797–804. Hooijman EL, Chalashkan Y, Ling SW, Kahyargil FF, Segbers M, Bruchertseifer F, et al. Development of [ 225 Ac]Ac-PSMA-I&T for targeted alpha therapy according to GMP guidelines for treatment of mCRPC. Pharmaceutics. 2021;13(5):715. Kafka M, Horninger A, di Santo G, Virgolini I, Neuwirt H, Unterrainer LM, et al. Real-world outcomes and predictive biomarkers for 177 Lu-lutetium prostate-specific membrane antigen ligand treatment in metastatic castration-resistant prostate cancer: a european association of urology young academic urologists prostate cancer working group multi-institutional observational study. Eur Urol Oncol. 2024;7(3):421–9. Kratochwil C, Bruchertseifer F, Giesel FL, Weis M, Verburg FA, Mottaghy F, et al. ). 225 Ac-PSMA-617 for PSMA-targeted alpha-radiation therapy of metastatic castration-resistant prostate cancer. J Nucl Med. 2016;57(12):1941–4. Kratzer TB, Mazzitelli N, Star J, Dahut WL, Jemal A, Siegel RL. Prostate cancer statistics, 2025. CA Cancer J Clin. 2025;75(6):485–97. Peng CL, Chen CT, Tang IC. Exploring the therapeutic potential of Lu-PSMA-617 in a mouse model of prostate cancer bone metastases. Int J Mol Sci. 2025;26(13):5970. Sartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177-PSMA-617 for metastatic castration-resistant prostate cancer. N Engl J Med. 2021;385(12):1091–103. Stenberg VY, Juzeniene A, Chen Q, Yang X, Bruland ØS, Larsen RH. Preparation of the alpha-emitting prostate-specific membrane antigen targeted radioligand [ 212 Pb]Pb-NG001 for prostate cancer. J Label Comp Radiopharm. 2020;63(3):129–43. Stuparu AD, Meyer CAL, Evans-Axelsson SL, Luckerath K, Wei LH, Kim W, et al. Targeted alpha therapy in a systemic mouse model of prostate cancer - a feasibility study. Theranostics. 2020;10(6):2612–20. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49. Wurzer A, Kunert JP, Fischer S, Felber V, Beck R, de Rose F, et al. Synthesis and preclinical evaluation of 177 Lu-labeled radiohybrid PSMA ligands for endoradiotherapy of prostate cancer. J Nucl Med. 2022;63(10):1489–95. Yadav MP, Ballal S, Sahoo RK, Tripathi M, Seth A, Bal C. Efficacy and safety of 225 Ac-PSMA-617 targeted alpha therapy in metastatic castration-resistant Prostate Cancer patients. Theranostics. 2020;10(20):9364–77. Yong K, Brechbiel M. Application of 212 Pb for targeted alpha-particle therapy (TAT): pre-clinical and mechanistic understanding through to clinical translation. AIMS Med Sci. 2015;2(3):228–45. Zimmermann R. Is 212 Pb really happening? The post- 177 Lu/ 225 Ac blockbuster? J Nucl Med. 2024;65(2):176–7. Supplementary Files PSMA617RadiochemManuscriptSupplementary041426.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revision 04 May, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers invited by journal 22 Apr, 2026 Editor assigned by journal 21 Apr, 2026 First submitted to journal 17 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9419820","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627585855,"identity":"54941da6-beb6-466e-81a6-79e8da48b916","order_by":0,"name":"Abhijit Bera","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Abhijit","middleName":"","lastName":"Bera","suffix":""},{"id":627585857,"identity":"31a39f41-0fa3-4297-8815-31825450ed02","order_by":1,"name":"Graham Ragland","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Graham","middleName":"","lastName":"Ragland","suffix":""},{"id":627585859,"identity":"a4d1bb54-5b6b-4f6e-9da6-a605fa8bcf4b","order_by":2,"name":"Yuhan Zhang","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Yuhan","middleName":"","lastName":"Zhang","suffix":""},{"id":627585860,"identity":"3e99a5b4-7871-46dc-8d51-ac8d033c2ba7","order_by":3,"name":"Patricia G. Madel","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"G.","lastName":"Madel","suffix":""},{"id":627585864,"identity":"df719978-290b-405e-bf7e-6611f100f4ab","order_by":4,"name":"Jasmine B'Lanton","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Jasmine","middleName":"","lastName":"B'Lanton","suffix":""},{"id":627585865,"identity":"d46a1706-400a-4dcd-a297-510f1f662948","order_by":5,"name":"Atchimnaidu Siriki","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Atchimnaidu","middleName":"","lastName":"Siriki","suffix":""},{"id":627585866,"identity":"15cf308a-7f30-41d2-9ed8-fd0730480b5d","order_by":6,"name":"Chin-Tu Chen","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Chin-Tu","middleName":"","lastName":"Chen","suffix":""},{"id":627585869,"identity":"d6b4fde9-14c8-47fd-98f3-3632814e5962","order_by":7,"name":"Russell Z. Szmulewitz","email":"","orcid":"","institution":"The University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Russell","middleName":"Z.","lastName":"Szmulewitz","suffix":""},{"id":627585870,"identity":"8a1d3735-6858-49c7-a4c4-61ccbda0f118","order_by":8,"name":"Satish K. Chitneni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYFACHoYDIIqNvYGBmcEALkhAC0gPG88BErRArJFIAGpBEsQJ5NvPHjz8cYdNHp/kG8PPBQV28vLuDYwP3rbh1mJwJi/hwMEzacVs0jnG0jMMkg03njnAbDgXnxYJHoMDB9sOJ7ZJ55gxA9mMG2cksEnz4tEiPwOs5X9im+QZsBZ7oBb23/i0MNwAazmQ2CbBA9aSOF8igY0ZnxaDMzkGB862JSe28aQVS/MYJCdv4DnYLDnnHB6HtZ8x/lDZZpc4v/3wxs88f+xs57c3H/zwpgyPwzDtPcDYQIp6kL2kahgFo2AUjIJhDwAxlFG6XP6jIAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1183-2286","institution":"The University of Chicago","correspondingAuthor":true,"prefix":"","firstName":"Satish","middleName":"K.","lastName":"Chitneni","suffix":""}],"badges":[],"createdAt":"2026-04-14 22:21:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9419820/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9419820/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108198881,"identity":"a95d2dbf-107d-4c38-88e6-8fe08b835540","added_by":"auto","created_at":"2026-04-30 11:30:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2355694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical structures of the three PSMA-617 radioligands synthesized and evaluated in this work.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/11fef71f6452b4505b107055.png"},{"id":108198882,"identity":"80b6ec97-728c-483d-9173-2254c915bea3","added_by":"auto","created_at":"2026-04-30 11:30:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3967156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunocytochemical imaging of PSMA expression in cell line pairs.\u003c/strong\u003e Images are paired as (\u003cstrong\u003eA\u003c/strong\u003e) the LNCaP-AR (PSMA-positive) and CWRR1-EnzR (PSMA-negative) pair, and (\u003cstrong\u003eB\u003c/strong\u003e) the isogenic DU145-PSMA (PSMA-positive) and DU145 (PSMA-negative) pair.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/6b7fa8688fe5f07d2a914498.png"},{"id":108491433,"identity":"2aace907-f60f-436f-b1c4-215d9c3b09c6","added_by":"auto","created_at":"2026-05-05 09:53:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17732532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of cell uptake and internalization in the LNCaP-AR and CWRR1-EnzR cell line pair.\u003c/strong\u003e Uptake and internalization of (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in the PSMA-positive prostate adenocarcinoma cell line LNCaP-AR. For comparison, the uptake of the three radioligands in the PSMA-negative CWRR1-EnzR cell line is also shown (center column). Uptake is presented on different scales (y-axis) for each radioligand.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/5c39058c450be3c710d592f1.png"},{"id":108198884,"identity":"5231d9d5-29cc-4b81-9b3e-91bcdd9290c9","added_by":"auto","created_at":"2026-04-30 11:30:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17859960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of cell uptake and internalization in the DU145-PSMA and DU145 cell line pair.\u003c/strong\u003e Uptake and internalization of (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in the PSMA overexpressing DU145-PSMA cell line. For comparison, the uptake of the three radioligands in the PSMA-negative parental DU145 cell line is also shown (center column). Uptake is presented on a different scale (y-axis) for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/c6199d533d377305cc7e20dc.png"},{"id":108198885,"identity":"4f6425d5-3d93-4f5d-9c71-47913ac131d9","added_by":"auto","created_at":"2026-04-30 11:30:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4966808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermination of inhibitory potency of nonradioactive analogues against PSMA using [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e177\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eLu]Lu-PSMA-617.\u003c/strong\u003e Competitive inhibition of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 uptake in DU145-PSMA cells by the nonradioactive Lu-PSMA-617 or Pb-PSMA-617, with the corresponding half-maximal inhibitory value (IC\u003csub\u003e50\u003c/sub\u003e) shown in parentheses.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/f2c40d1a19b805d9378ac911.png"},{"id":108491483,"identity":"4c5e3622-8fab-4954-a63a-de5cdc5bad18","added_by":"auto","created_at":"2026-05-05 09:54:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2056199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermination of binding affinity of the three radioligands in the LNCaP-AR and DU-145-PSMA cell lines.\u003c/strong\u003e Saturation binding of (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e[\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617\u003csup\u003e \u003c/sup\u003ein the PSMA-positive LNCaP-AR and DU145-PSMA cell lines. Equilibrium dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) values determined from these assays are shown for the corresponding radioligand.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/774dace9f28830260f29e481.png"},{"id":108491036,"identity":"00938bef-4fc3-46d5-8f8c-0283ed65f8ba","added_by":"auto","created_at":"2026-05-05 09:51:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29513731,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/4f89dab2-1998-4395-9bf0-deffc9976373.pdf"},{"id":108198887,"identity":"5eed4afb-a85b-45e1-a5ce-630a4d329c54","added_by":"auto","created_at":"2026-04-30 11:30:04","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3451352,"visible":true,"origin":"","legend":"","description":"","filename":"PSMA617RadiochemManuscriptSupplementary041426.docx","url":"https://assets-eu.researchsquare.com/files/rs-9419820/v1/c1ba15261d6dabb10f5a1498.docx"}],"financialInterests":"","formattedTitle":"Radiochemistry and Comparative In Vitro Assessment of PSMA-617 Labeled with Lead-212 (212Pb), Actinium-225 (225Ac), and Lutetium-177 (177Lu).","fulltext":[{"header":"Background","content":"\u003cp\u003eProstate cancer remains a leading cause of cancer-related mortality in men in the United States and worldwide (Kratzer et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sung et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Early translational studies demonstrated that PSMA-617, which consists of a DOTA chelator conjugated to the Glu-urea-Lys pharmacophore, exhibits high binding affinity to prostate-specific membrane antigen (PSMA) and efficient internalization into PSMA-positive cells, laying the foundation for PSMA-targeted radioligand therapy (RLT) (Benesova et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Subsequent medicinal chemistry efforts and preclinical studies further established PSMA-617 as a robust ligand for labeling with a variety of radionuclides for targeting PSMA expressing prostate cancers (Benesova et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Clinical translation of lutetium-177 (\u003csup\u003e177\u003c/sup\u003eLu, t\u003csub\u003e1/2\u003c/sub\u003e = 6.65 d) labeled PSMA-617 began shortly thereafter, with early clinical studies demonstrating significant tumor regression, improved overall survival, and favorable safety profiles in men with metastatic castration-resistant prostate cancer (Hofman et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The radiopharmaceutical [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Pluvicto\u003csup\u003e\u0026reg;ฏ\u003c/sup\u003e) has been approved for the treatment of metastatic castration-resistant prostate cancer following the landmark VISION trial, which demonstrated improved overall survival and radiographic progression-free survival in patients with metastatic castration-resistant prostate cancer (Sartor et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite its promising therapeutic efficacy, approximately 30\u0026ndash;50% of patients exhibit limited or no therapeutic response to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Kafka et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sartor et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), highlighting the need for improved therapeutic strategies.\u003c/p\u003e \u003cp\u003eTo address these limitations, α-particle-emitting radionuclides such as actinium-225 (\u003csup\u003e225\u003c/sup\u003eAc, t\u003csub\u003e1/2\u003c/sub\u003e = 9.92 d) and lead-212 (\u003csup\u003e212\u003c/sup\u003ePb, t\u003csub\u003e1/2\u003c/sub\u003e = 10.64 h) have been investigated for PSMA-targeted RLT. Compared with β-emitting radionuclides, α-emitters such as \u003csup\u003e225\u003c/sup\u003eAc deliver high linear energy transfer (LET, ~\u0026thinsp;100 keV/\u0026micro;m) radiation with a short path length (50\u0026ndash;100 \u0026micro;m), enabling highly localized tumor cell killing through DNA double-strand breaks while minimizing radiation exposure to surrounding healthy tissues (Kratochwil et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In line with this, multiple clinical studies have confirmed that \u003csup\u003e225\u003c/sup\u003eAc-labeled PSMA-617 can provide therapeutic benefits in men with metastatic castration-resistant prostate cancer, including those refractory to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Kratochwil et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, preclinical studies in mouse models of metastatic prostate cancer demonstrated that early treatment with [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 prevented the development of liver metastases and significantly improved survival, whereas delayed treatment prolonged survival without significantly reducing tumor burden (Stuparu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Likewise, α-emitters with shorter half-lives, such as \u003csup\u003e212\u003c/sup\u003ePb (10.64 h), have also attracted increasing interest for PSMA-targeted RLT. The relatively short half-life of \u003csup\u003e212\u003c/sup\u003ePb enables rapid decay and localized dose delivery to tumor cells, potentially reducing α-recoil-driven redistribution of radioactive daughter isotopes (Yong \u0026amp; Brechbiel, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, \u003csup\u003e212\u003c/sup\u003ePb exhibits favorable radiochemical properties, allowing reproducible synthesis and stable complexation with DOTA-based ligands, while its generator-based production supports scalable clinical availability (Zimmermann, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The PSMA-targeting properties of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 have been investigated in PSMA-positive C4-2 prostate cancer cells, and its in vivo performance supports further exploration for therapeutic applications (Stenberg et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 has established the clinical role of PSMA-targeted RLT, \u003csup\u003e225\u003c/sup\u003eAc- and \u003csup\u003e212\u003c/sup\u003ePb-labeled radioligands represent the next generation of RLTs, with the potential to improve therapeutic efficacy and clinical outcomes (Stenberg et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, we compared the radiolabeling and in vitro properties of \u003csup\u003e225\u003c/sup\u003eAc- and \u003csup\u003e212\u003c/sup\u003ePb-labeled PSMA-617 with the clinically established [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using two pairs of PSMA-positive and -negative cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNonradioactive Chemistry\u003c/h2\u003e \u003cp\u003eTo evaluate the inhibitory potency of the PSMA-617 conjugates, we synthesized nonradioactive reference analogues of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 by complexing PSMA-617 with lutetium (Lu\u003csup\u003e3+\u003c/sup\u003e) and lead (Pb\u003csup\u003e2+\u003c/sup\u003e) using LuCl\u003csub\u003e3\u003c/sub\u003e and PbCl\u003csub\u003e2\u003c/sub\u003e, respectively (\u003cb\u003eFigures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2\u003c/b\u003e). The resulting nonradioactive complexes Lu-PSMA-617 and Pb-PSMA-617 were obtained in 37% and 48% yields, respectively, and with \u0026gt;\u0026thinsp;95% purity after purification using a Sep-Pak\u003csup\u003e\u0026reg;ฏ\u003c/sup\u003e C18 cartridge. The identity of the nonradioactive complexes was verified by LC/MS analysis, which showed the expected molecular ion peaks corresponding to the respective nonradioactive PSMA-617 conjugates (\u003cb\u003eFigures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2\u003c/b\u003e). Nonradioactive Ac-PSMA-617 could not be synthesized due to the lack of a stable isotope for actinium.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRadiochemistry\u003c/h3\u003e\n\u003cp\u003eRadiolabeling of PSMA-617 with \u003csup\u003e177\u003c/sup\u003eLu, \u003csup\u003e225\u003c/sup\u003eAc, and \u003csup\u003e212\u003c/sup\u003ePb was performed following previously published procedures with some modifications (Hooijman et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wurzer et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, the labeling yield increased from 63% with 0.25 nmol precursor (PSMA-617) to 95% with 1 nmol precursor at 37 MBq of \u003csup\u003e177\u003c/sup\u003eLuCl\u003csub\u003e3\u003c/sub\u003e (\u003cb\u003eFigure S3\u003c/b\u003e), providing a molar activity of approximately 37 MBq/nmol. Further increase in the amount of precursor (up to 10 nmol) led to \u0026ge;\u0026thinsp;99% labeling efficiency for the labeled conjugate. In the case of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, instant thin-layer chromatography (iTLC) analysis of the crude reaction mixture indicated a labeling efficiency of 80\u0026ndash;95% when the reactions were conducted at a 1\u0026ndash;2 nmol scale (PSMA-617) using 0.93\u0026ndash;1.85 MBq of [\u003csup\u003e225\u003c/sup\u003eAc]AcCl\u003csub\u003e3\u003c/sub\u003e (\u003cb\u003eFigure S4\u003c/b\u003e). However, subsequent purification of the crude reaction mixture via a Sep-Pak revealed only about 45% of the loaded activity retained on the column, which was eluted with ethanol and used for in vitro studies. The effective molar activity of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 after Sep-Pak purification was 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 MBq/nmol (n\u0026thinsp;=\u0026thinsp;4). Similarly, \u003csup\u003e212\u003c/sup\u003ePb-labeling of PSMA-617 was accomplished by reacting 1 nmol PSMA-617 with 18.5 MBq of [\u003csup\u003e212\u003c/sup\u003ePb]PbCl\u003csub\u003e2\u003c/sub\u003e, eluted from a \u003csup\u003e224\u003c/sup\u003eRa/\u003csup\u003e212\u003c/sup\u003ePb generator in 0.2 M sodium acetate buffer. Sep-Pak purification of the crude mixture after radiolabeling yielded [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in 46.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% (n\u0026thinsp;=\u0026thinsp;4) yield, which was consistent with that determined by the radio-HPLC analysis of the crude reaction mixture (46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2%) (\u003cb\u003eFigure S5\u003c/b\u003e). The molar activity was 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 MBq/nmol (n\u0026thinsp;=\u0026thinsp;4). Radio-HPLC analysis of the purified [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 showed\u0026thinsp;\u0026gt;\u0026thinsp;98% radiochemical purity, whereas iTLC was used for the [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 conjugate, which showed a purity of approximately 95%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution Coefficient (\u003c/b\u003e \u003cb\u003eD\u003c/b\u003e \u003cb\u003e) Values\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe lipophilicity of the radioconjugates was determined by measuring their distribution coefficient (\u003cem\u003eD\u003c/em\u003e) values through partitioning between n-octanol and PBS (pH 7.4). The distribution coefficient was calculated as the ratio of radioactivity (CPM) in the octanol phase to that in the aqueous PBS phase and expressed as log\u003cem\u003eD\u003c/em\u003e\u003csub\u003e7.4\u003c/sub\u003e values. The measured log\u003cem\u003eD\u003c/em\u003e\u003csub\u003e7.4\u003c/sub\u003e values were \u0026minus;\u0026thinsp;3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (n\u0026thinsp;=\u0026thinsp;4) for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, -2.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (n\u0026thinsp;=\u0026thinsp;4) for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and \u0026minus;\u0026thinsp;3.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (n\u0026thinsp;=\u0026thinsp;3) for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results indicate that the three radioligands exhibit comparable lipophilicity, with only minor differences observed among the three PSMA-617-based radioligands.\u003c/p\u003e\n\u003ch3\u003eIn Vitro Stability Studies\u003c/h3\u003e\n\u003cp\u003eThe in vitro stability of the three PSMA-617 radioligands was evaluated in PBS, human serum, and human whole blood by incubating the radioligands at 37\u0026deg;C for up to 120 h. [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 demonstrated\u0026thinsp;\u0026ge;\u0026thinsp;90% stability in PBS, human serum, and human whole blood for up to 120 h, as confirmed by iTLC analysis (\u003cb\u003eFigure S6A\u003c/b\u003e). [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 exhibited\u0026thinsp;\u0026gt;\u0026thinsp;94% stability in human serum over the 120 h incubation period. However, in human whole blood, stability decreased slightly at later time points, reaching 86.3% and 89.1% at 96 h and 120 h, respectively. In comparison, stability in PBS decreased more noticeably, reaching 72.3% at 120 h post-incubation (\u003cb\u003eFigure S6B\u003c/b\u003e). Because of the short half-life of \u003csup\u003e212\u003c/sup\u003ePb, the stability of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 was assessed only until 24 h, which revealed\u0026thinsp;\u0026ge;\u0026thinsp;90% stability in PBS, human serum, and human whole blood (\u003cb\u003eFigure S6C\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003eCell Uptake and Internalization\u003c/h3\u003e\n\u003cp\u003eThe uptake, specificity, selectivity, and internalization rate of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 were compared in two pairs of PSMA-positive and negative prostate cancer cell lines: LNCaP-AR (PSMA-positive) and CWRR1-EnzR (PSMA-negative), and DU145-PSMA (PSMA-positive) and DU145 (PSMA-negative). Immunocytochemical staining followed by fluorescence imaging confirmed high PSMA expression in the two PSMA-positive cell lines LNCaP-AR and DU145-PSMA, with minimal expression in the respective control cell lines CWRR1-EnzR and DU145 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cell uptake studies showed that the uptake of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in the LNCaP-AR cell line increased over time, from 2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02% of added activity at 0.5 h post-incubation to 7.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65% at 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Uptake in the CWRR1-EnzR1 cell line was minimal at all time points, with 0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% uptake at 4 h. Co-incubation with excess unlabeled PSMA-617 (300 nM) blocked uptake by \u0026gt;\u0026thinsp;95%, limiting it to 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% at 4 h in LNCaP-AR cells, confirming the specificity and selectivity of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 binding in the LNCaP-AR cell line. In contrast to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, uptake of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 in LNCaP-AR cells was low, with 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% of added activity at 0.5 h and increasing to 1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24% at 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, co-incubation with excess unlabeled PSMA-617 (300 nM) reduced uptake of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 by \u0026gt;\u0026thinsp;95%, with 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06% at 4 h. Uptake in the PSMA-negative CWRR1-EnzR cells remained minimal at all time points, with 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% uptake at 4 h, which reduced to 0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% with blocking. Similar to the above two radioligands, uptake of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in LNCaP-AR cells increased over time, reaching 14.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% at 4 h compared to 5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45% at 0.5 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Co-incubation with excess unlabeled PSMA-617 (300 nM) reduced uptake to 0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% at 4 h, and uptake of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in the PSMA-negative CWRR1-EnzR cells remained minimal at all time points, with 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% at 4 h. Additionally, for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617, the fraction of internalized activity, calculated as the percentage of total uptake in cells, increased with time, from 14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6% at 0.5 h to 33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0% at 4 h for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and from 31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% at 0.5 h to 47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% at 4 h for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Compared to these, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 exhibited less increase in internalization over time from 40.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4% at 0.5 h to 51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% by 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell uptake and internalization of the three radioligands were also evaluated in a second pair of isogenic cell lines, a PSMA-overexpressing DU145-PSMA cell line (courtesy of Novartis Institutes for BioMedical Research) and the parental DU145 cell line (PSMA-negative). The results from these studies are consistent with the uptake trends observed for the three radioligands in the LNCaP-AR cell line, but with significantly higher uptake (by 2-3-fold) than in the LNCaP-AR cell line. The uptake of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in DU145-PSMA cells at 4 h was 31.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64% compared to 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% with blocking (300 nM PSMA-617) and 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% in the parental DU145 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e Consistent with results from the LNCaP-AR cell line, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 exhibited lower uptake in the DU145-PSMA cells; nonetheless, the uptake increased over time, from 2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08% at 0.5 h to 4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18% at 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Co-incubation of cells with an excess of PSMA-617 (300 nM) reduced the uptake to 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% at 4 h, and the uptake in control DU145 cells remained minimal (0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%). In contrast, [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 exhibited a high uptake that is comparable to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in DU145-PSMA cells. Uptake at 0.5 h was 13.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07%, increasing with time to 29.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69% at 4 h. For comparison, uptake in the parental DU145 cell line was 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02%, and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% with blocking (300 nM PSMA-617) at 4 h. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). For all three radioligands, \u0026gt;\u0026thinsp;60% of uptake was found to be internalized in DU145-PSMA cells at 4 h, with the remaining activity (\u0026lt;\u0026thinsp;40%) being cell-surface bound (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Taken together, these results demonstrate high PSMA-specific uptake of the three PSMA-617 radioconjugates in PSMA-expressing cells, with minimal uptake observed in PSMA-negative controls. However, the magnitude of uptake was lower for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 relative to [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 and the reference [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in both the cell lines (\u003cb\u003eFigure S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInhibitory Potency (IC) Assays\u003c/h3\u003e\n\u003cp\u003eThe inhibitory potencies (IC\u003csub\u003e50\u003c/sub\u003e) of the nonradioactive analogues Lu-PSMA-617 and Pb-PSMA-617 were evaluated by a competitive inhibition assay using [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in DU145-PSMA cells. Cells were incubated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in the presence of increasing concentrations of Lu-PSMA-617 or Pb-PSMA-617 (0.14\u0026ndash;300 nM). Cell uptake, presented as the percentage of added activity, was assessed at each concentration to determine the half-maximal inhibitory concentration value (IC\u003csub\u003e50\u003c/sub\u003e) for the two nonradioactive PSMA-617 inhibitors. The results demonstrated concentration-dependent inhibition of the radiotracer uptake in the DU145-PSMA cell line. The inhibitory potency (IC\u003csub\u003e50\u003c/sub\u003e) values derived from these assays were 8.6 nM for Lu-PSMA-617 and 10.5 nM for Pb-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating similar inhibitory potency for the two PSMA-617 conjugates toward PSMA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBinding Affinity Studies\u003c/h2\u003e \u003cp\u003eThe binding affinities (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) of the three radioligands for PSMA were evaluated using the LNCaP-AR and DU145-PSMA cell lines by conducting saturation binding assays as described previously (Chitneni, Yan, \u0026amp; Zalutsky, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cells were incubated with increasing concentrations of \u003csup\u003e177\u003c/sup\u003eLu-, \u003csup\u003e225\u003c/sup\u003eAc-, or \u003csup\u003e212\u003c/sup\u003ePb-labeled PSMA-617 (0.4\u0026ndash;50.0 nM) for 2 h, and the cell-bound activity was assessed by gamma counting. For each concentration, nonspecific binding was measured in parallel by co-incubating cells with an excess of unlabeled PSMA-617 (300 nM). Specific binding data were generated by subtracting nonspecific binding from total binding for each concentration, and nonlinear regression analysis was conducted in GraphPad\u003csup\u003e\u0026reg;ฏ\u003c/sup\u003e Prism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Equilibrium dissociation constant values (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e), determined as the concentration needed to achieve half-maximum binding at equilibrium, were derived from these assays for the three radioligands in the two PSMA-positive cell lines. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values determined from these assays in LNCaP-AR and DU145-PSMA cells were 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 nM and 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nM for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), compared to 5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM and 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 nM for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM and 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 nM for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverview of in vitro properties of the three PSMA-617 radioligands synthesized in this work, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRadioligand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLipophilicity\u003c/p\u003e \u003cp\u003e(log \u003cem\u003eD\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMolar Activity (per nmol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCell Uptake (PSMA+, 4 h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eBinding Affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLNCaP-AR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDU145-PSMA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLNCaP-AR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDU145-PSMA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.0\u0026ndash;74.0 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.9 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.3 nM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-2.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.22\u0026ndash;0.55 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.7 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.8 nM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.4\u0026ndash;11.1 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e29.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.7 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.0 nM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur radiolabeling experiments of PSMA-617 with the β-emitter \u003csup\u003e177\u003c/sup\u003eLu and the α-emitters \u003csup\u003e225\u003c/sup\u003eAc and \u003csup\u003e212\u003c/sup\u003ePb revealed important differences in radiolabeling characteristics and radiochemical yields for the corresponding radionuclides. Unlike \u003csup\u003e177\u003c/sup\u003eLu, radio-HPLC-based QC analysis is less informative for \u003csup\u003e225\u003c/sup\u003eAc due to its complex decay scheme and low levels of radioactivity employed, necessitating the use of iTLC-based methods to determine radiolabeling yields and/or purity of the labeled compounds. Although our iTLC analysis of the crude reaction mixture consistently showed\u0026thinsp;\u0026gt;\u0026thinsp;95% labeling with less free \u003csup\u003e225\u003c/sup\u003eAc (\u003cb\u003eFigure S4\u003c/b\u003e), subsequent Sep-Pak purification typically retained only\u0026thinsp;~\u0026thinsp;45% of the loaded activity, with the remainder of the activity either unretained or washed off with the water rinse. Careful assessment of the iTLC chromatograms of the crude reaction mixture and the unretained activity fractions indicated the presence of a labeled species migrating at the front of the [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 peak on the iTLC (at 40\u0026ndash;50 mm, \u003cb\u003eFigure S4\u003c/b\u003e). Thus, for all subsequent studies and in vitro evaluation of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, the reaction mixture was purified by Sep-Pak, and the radioligand was freshly synthesized on the day of each in vitro experiment. Additionally, all cell uptake studies were conducted in the presence of DTPA (0.1 mg/mL) in the incubation mixture to chelate any free \u003csup\u003e225\u003c/sup\u003eAc or daughter isotopes, although comparable results were obtained without DTPA as well in select experiments (\u003cb\u003eFigure S8\u003c/b\u003e). In contrast, radiolabeling of PSMA-617 with \u003csup\u003e212\u003c/sup\u003ePb proved more straightforward. However, despite optimization efforts and increasing the precursor amount (PSMA-617), the RCY could not be improved beyond ~\u0026thinsp;50%. Nonetheless, the molar activities we obtained for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in the present study proved sufficient (4.4\u0026ndash;11.1 MBq/nmol) for efficient cell uptake and internalization and yielded similar or higher uptake than [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in the two PSMA-positive cell lines we evaluated (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAcross the three radioligands, lipophilicity values were comparable (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and reflect the overall hydrophilicity of the molecule (PSMA-617), suggesting that substitution of \u003csup\u003e177\u003c/sup\u003eLu in [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 with \u003csup\u003e225\u003c/sup\u003eAc or \u003csup\u003e212\u003c/sup\u003ePb may not significantly alter the physicochemical properties of the resulting radioconjugates. In vitro stability studies demonstrated excellent radiochemical stability for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, with \u0026ge;\u0026thinsp;90% stability in PBS, human serum, and human whole blood up to 72 h. In view of the short half-life of \u003csup\u003e212\u003c/sup\u003ePb (10.6 h), the stability of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 was evaluated only for 24 h, which showed\u0026thinsp;\u0026ge;\u0026thinsp;90% stability of the labeled conjugate.\u003c/p\u003e \u003cp\u003eComparative cell uptake studies in the two PSMA-expressing prostate cancer cell lines revealed distinct trends for the three isotopes or the radioligands. In LNCaP-AR cells, [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 exhibited about 2-fold higher uptake than [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, but similar levels of uptake were noted for both radioligands in the DU145-PSMA cell line. Despite the differences in the total uptake levels, all three radioligands exhibited a higher uptake and internalized fraction in the PSMA overexpressing cell line DU145-PSMA than in LNCaP-AR at 4 h post-incubation. Few studies have reported the radiosynthesis and in vivo evaluation of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 (Busslinger et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Stuparu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); however, systematic evaluation of the labeled conjugate in vitro models and head-to-head comparison with the established PSMA-targeted radioligand [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 is largely lacking. We believe that the lower uptake of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in the two PSMA-positive cell lines was primarily due to the low molar activity of the radioligand, resulting in competitive inhibition of the radioligand by the unlabeled PSMA-617 in the final product. In this study, our initial attempts to achieve a molar activity of at least 3.7 MBq/nmol for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, to be on par with [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617, were unsuccessful. In contrast, an acceptable molar activity was achieved for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617. The similar or higher uptake of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in our cell uptake studies indicates a desired molar activity of \u0026ge;\u0026thinsp;3.7 MBq/nmol for efficient uptake of PSMA-617-based radioligands in PSMA expressing prostate cancer cells. Minimal uptake of the three radioligands in the PSMA-negative cell lines (CWRR1-EnzR and DU145) and efficient blocking with excess PSMA-617 in the two PSMA-positive cell lines confirms the specificity of the three radioligands to PSMA and the PSMA-mediated internalization of the radioligands in our cell models.\u003c/p\u003e \u003cp\u003eAlthough molar activities, uptake, and internalization rates varied between cell lines, saturation binding assays demonstrated comparable binding affinity values (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e) in the nM range for \u003csup\u003e177\u003c/sup\u003eLu-, \u003csup\u003e225\u003c/sup\u003eAc-, and \u003csup\u003e212\u003c/sup\u003ePb-labeled PSMA-617 in the two PSMA-positive cell lines LNCaP-AR and DU145-PSMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our results strongly suggest that the change of radioisotope did not affect the binding affinity of labeled PSMA-617 in PSMA-expressing cells. Additionally, the observed binding affinity values are consistent with those reported in the literature, e.g., a \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e of about 4.4 nM for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 to LNCaP cells (Peng, Chen, \u0026amp; Tang, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), 11 nM for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 to PC-3 PIP cells (Busslinger et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and about 11.1 nM for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 to C4-2 cells (Stenberg et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCollectively, our results indicate the suitability of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 for further evaluation using in vivo models of PSMA-expressing prostate cancer in comparison with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617. Although it may not be possible to achieve high molar activity for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 similar to that for \u003csup\u003e177\u003c/sup\u003eLu- or \u003csup\u003e212\u003c/sup\u003ePb-labeled PSMA-617, it remains to be tested if the reduced cell uptake from in vitro models translates to lower tumor uptake (e.g., percentage injected dose per gram (% ID/g)) in vivo compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 or [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617. To that end, our future studies will focus on extending these in vitro studies to in vivo by directly comparing tumor uptake of the three radioligands and the therapeutic efficacy of the ⍺-emitting PSMA-617 radioligands with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in the same tumor models.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study was designed to directly compare radiolabeling and in vitro characteristics of PSMA-617 labeled with α- and β-emitting radionuclides as a prelude to in vivo comparisons in the same tumor models. In this study, we successfully optimized radiolabeling protocols for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617, achieving high radiochemical purity and in vitro stability suitable for in vivo evaluation. Both the α-emitting radioligands demonstrated selective uptake, specificity, and efficient internalization in two different PSMA-expressing prostate cancer cell lines. Notably, [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 exhibited an in vitro uptake comparable to or exceeding that of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in PSMA-positive cells, highlighting its potential as a targeted α-therapy agent for PSMA-expressing prostate cancers. Although [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 exhibited PSMA-specific uptake in the two cell lines we evaluated, the uptake levels were substantially lower, most likely due to the low molar activities achievable for \u003csup\u003e225\u003c/sup\u003eAc-labeling versus \u003csup\u003e212\u003c/sup\u003ePb- and \u003csup\u003e177\u003c/sup\u003eLu-labeling. Collectively, the results from this study support further evaluation of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 to directly compare their tumor uptake and therapeutic efficacy in prostate cancer models for targeted α-therapy in comparison with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617.\u003c/p\u003e "},{"header":"Experimental Section","content":"\u003ch2\u003eGeneral methods\u003c/h2\u003e\u003cp\u003eAll reagents and solvents were purchased from Fisher Scientific, MilliporeSigma, or Ambeed and used as received unless otherwise noted. PSMA-617 was supplied by Novartis Institutes for BioMedical Research (NIBR, Basel, Switzerland). TraceSELECT™ water was purchased from Honeywell, and Chelex® 100 resin was obtained from Bio-Rad. All buffers and reagents used in this work were prepared using Chelex-treated water unless specified. Nonradioactive complexes were analyzed using an expression-L compact mass spectrometer coupled to a high-performance liquid chromatography (HPLC) system equipped with a UV detector and operated using Mass Express software (Advion Interchim Scientific, Ithaca, NY). The following method was used for the analysis: Agilent Poroshell 120 C18 column (3 × 50 mm); mobile phases: A = water with 0.1% trifluoroacetic acid (TFA), B = acetonitrile (MeCN) with 0.1% TFA; flow rate: 0.3 mL/min; gradient conditions: 0-0.3 min, 5% B; 0.3-9 min, 5→95% B; 9-9.5 min, 95→5% B.\u003c/p\u003e\u003cp\u003eAll three isotopes, \u003csup\u003e177\u003c/sup\u003eLu, \u003csup\u003e225\u003c/sup\u003eAc, and \u003csup\u003e212\u003c/sup\u003ePb, were purchased through the U.S. Department of Energy’s National Isotope Development Center (NIDC). \u003csup\u003e177\u003c/sup\u003eLu was supplied as \u003csup\u003e177\u003c/sup\u003eLuCl\u003csub\u003e3\u003c/sub\u003e in 0.04 M HCl. \u003csup\u003e225\u003c/sup\u003eAc was supplied as dry \u003csup\u003e225\u003c/sup\u003eAcNO\u003csub\u003e3\u003c/sub\u003e. \u003csup\u003e212\u003c/sup\u003ePb was obtained in the form of a \u003csup\u003e224\u003c/sup\u003eRa/\u003csup\u003e212\u003c/sup\u003ePb generator and eluted following the instructions provided with the generator. Briefly, a Pb resin QML cartridge (20–50 µm, Eichrom Technologies) was preconditioned with 2 M HCl (1 mL) and connected to the outlet of the generator. Then 1 mL of 2 M HCl was passed through the inlet line of the generator to elute and capture \u003csup\u003e212\u003c/sup\u003ePb on the Pb cartridge. The tubing was purged with air, after which the Pb cartridge was disconnected from the generator. The generator was then rinsed with 2 mL of water and was left with water for storage until the next elution (the next day). The Pb resin cartridge, containing \u003csup\u003e212\u003c/sup\u003ePb, was then rinsed with water (1 mL) to remove residual acid. The cartridge was reversed, and \u003csup\u003e212\u003c/sup\u003ePb was eluted by passing 0.2-1.0 M sodium acetate (NaOAc) solution (0.5-1 mL), which was used directly for radiolabeling reactions without further modification. HPLC analysis of radioactive samples (\u003csup\u003e177\u003c/sup\u003eLu, \u003csup\u003e212\u003c/sup\u003ePb) was conducted using an Agilent 1260 Infinity II quaternary pump system coupled to an Agilent 1260 Infinity II variable-wavelength UV detector and a Dual Scan-RAM radio-TLC and radio-HPLC detector (LabLogic, Chantilly, VA). The following method was used for the HPLC analysis: Kinetix® 5 µm EVO C18 column (4.6 × 150 mm, 100 Å); mobile phases: A = water with 0.1% TFA, B = MeCN with 0.1% TFA; flow rate: 1.0 mL/min; gradient conditions: 0 min, 5% B; 0–10 min, 5→70% B; 10–12 min, 70% B; 12–15 min, 70→5% B. Unless specified, all \u003csup\u003e225\u003c/sup\u003eAc samples were counted (CPM) at approximately 16 h after collection to allow reaching secular equilibrium with gamma-emitting progeny isotopes (e.g., \u003csup\u003e213\u003c/sup\u003eBi), in an automated gamma counter (Cobra II, Packard).\u003c/p\u003e\u003ch2\u003eSynthesis of Nonradioactive Lu-PSMA-617 and Pb-PSMA-617 Complexes\u003c/h2\u003e\u003cp\u003eFor the synthesis of nonradioactive reference standards, PSMA-617 (1 mg, 1 µmol) was reacted with either LuCl\u003csub\u003e3\u003c/sub\u003e (0.6 mg, 1.4 µmol) in 0.25 M NaOAc buffer (pH 5.5, 150 µL) or PbCl\u003csub\u003e2\u003c/sub\u003e (0.42 mg, 1.5 µmol, 1.5 equiv.) in 0.25 M NaOAc buffer (pH 5.5, 150 µL). The reaction mixture was heated at 90°C for 30 min, and the resulting complexes, Lu-PSMA-617 and Pb-PSMA-617, were purified using an Oasis HLB Plus Light cartridge (30 mg sorbent, Waters), eluted with ethanol. The purified complexes were characterized using LC-MS. Lu-PSMA-617: white solid, isolated yield: 0.45 mg (37%). ESI-MS: calc. for C\u003csub\u003e49\u003c/sub\u003eH\u003csub\u003e69\u003c/sub\u003eLuN\u003csub\u003e9\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e [M + H]⁺: 1214.4; found, [M + H]⁺: 1214.1. Pb-PSMA-617: white solid, isolated yield: 0.6 mg (48%). ESI-MS, calc. for C\u003csub\u003e49\u003c/sub\u003eH\u003csub\u003e69\u003c/sub\u003eN\u003csub\u003e9\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003ePb [M + H]⁺: 1247.4629, [M + 2H]\u003csup\u003e2\u003c/sup\u003e⁺: 624.8, found, [M + 2H]\u003csup\u003e2\u003c/sup\u003e⁺: 624.8.\u003c/p\u003e\u003ch2\u003eRadiochemistry\u003c/h2\u003e\u003cp\u003eIn a 1.5 mL protein Eppendorf tube (LoBind\u003csup\u003e®ฏ\u003c/sup\u003e), approximately 37 MBq of \u003csup\u003e177\u003c/sup\u003eLuCl\u003csub\u003e3\u003c/sub\u003e in 0.25 M NaOAc buffer (pH 5.5, 100 µL) or 18.5 MBq of \u003csup\u003e212\u003c/sup\u003ePbCl\u003csub\u003e2\u003c/sub\u003e in 0.2 M NaOAc buffer (pH 5.5, 100 µL) was mixed with PSMA-617 (~ 1 nmol, 1 µL). The reaction mixture was heated at 90°C for 30 min in a thermomixer (500 rpm) to obtain the corresponding radioligand. Radiolabeling efficiency was evaluated by radio-HPLC as described above. The reaction mixture was cooled to room temperature and purified using an Oasis HLB Plus Light cartridge (30 mg, Waters). The cartridge was washed with water (2 × 2 mL), and the radiolabeled product was eluted with ethanol (200 proof, 0.5 mL) into a vial for use in in vitro studies.\u003c/p\u003e\u003cp\u003eFor \u003csup\u003e225\u003c/sup\u003eAc labeling of PSMA-617, \u003csup\u003e225\u003c/sup\u003eAcCl\u003csub\u003e3\u003c/sub\u003e (0.92–1.85 MBq) was added to 0.2 M NH\u003csub\u003e4\u003c/sub\u003eOAc buffer (pH 5.2, 100 µL) in a 0.5 mL Eppendorf tube (LoBind\u003csup\u003e®ฏ\u003c/sup\u003e), followed by the addition of PSMA-617 (1 nmol, 1 µL). The reaction mixture was heated at 95°C for 45 min in a thermomixer (500 rpm), after which time the reaction mixture was cooled to room temperature and quenched with 50 nM DTPA solution (4 µL). The crude mixture was purified using an Oasis HLB Plus Light cartridge (30 mg), washed with water (2 × 2 mL), and the labeled compound was eluted with ethanol (200 proof, 0.5 mL).\u003c/p\u003e\u003cp\u003e \u003cb\u003eDistribution Coefficient (\u003c/b\u003e \u003cb\u003eD\u003c/b\u003e \u003cb\u003e)\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe distribution coefficient (\u003cem\u003eD\u003c/em\u003e) of the PSMA-617 radioligands was evaluated by the shake-flask method. Briefly, an aliquot of the radioligands was added to a mixture of \u003cem\u003en\u003c/em\u003e-octanol and PBS pH 7.4 (2 mL each, n = 3–4) in polypropylene tubes and vortexed for 1 min. The octanol and aqueous layers were separated by centrifugation at 3000 rpm for 5 min. From each tube, 0.5 mL aliquots of octanol and water phases were carefully withdrawn into pre-weighed Eppendorf tubes and measured for radioactivity in an automated gamma counter (Cobra II, Packard). Samples were weighed and normalized for density to get radioactivity counts as CPM/mL. Distribution coefficient (\u003cem\u003eD\u003c/em\u003e) values were calculated as the ratio of CPM/mL in the octanol phase to that in the aqueous phase and presented as log\u003cem\u003eD\u003c/em\u003e (mean ± SD) for each radioligand.\u003c/p\u003e\u003ch2\u003eIn Vitro Stability Studies\u003c/h2\u003e\u003cp\u003eIn a 1.5 mL Eppendorf tube (LoBind\u003csup\u003e®ฏ\u003c/sup\u003e), approximately 3.7 MBq of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, 74 kBq of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, or 0.37 MBq of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 were incubated with 0.2 mL of PBS (pH 7.4), human serum, or human whole blood at 37°C for up to 5 days in a thermomixer (500 rpm). For [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617, the incubation period was limited to 24 h due to its short physical half-life (10.6 h). At 24 h intervals, a 20 µL aliquot was withdrawn and analyzed by iTLC. The strips were developed in 0.1 M citrate buffer (pH 7.4), dried, cut into 1 cm sections, and measured for radioactivity using an automated gamma counter (Cobra II, Packard). For [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, the iTLC strips were recounted after at least 16 h to allow secular equilibrium of ɣ-emitting progeny isotopes (e.g., \u003csup\u003e213\u003c/sup\u003eBi).\u003c/p\u003e\u003ch2\u003eImmunocytochemistry Studies\u003c/h2\u003e\u003cp\u003eFor immunocytochemistry, 1 × 10\u003csup\u003e6\u003c/sup\u003e cells were seeded in 6-well plates containing poly-L-lysine-coated coverslips (Fisher Scientific) and allowed to attach before staining. Cells were fixed with 2% methanol-free formaldehyde prepared in 1x PBS for 15 min at room temperature and washed three times with PBS. Cells were then permeabilized by incubation with methanol at -20°C for 15 min and blocked for 1 h at room temperature in PBS supplemented with 5% normal serum (Cell Signaling Technology) and 0.1% Triton X-100. The cells were incubated overnight at 4°C with primary antibody against PSMA (Cell Signaling Technology, catalog #12702) diluted 1:400 in PBS containing 0.1% Triton X-100. Following primary antibody incubation, samples were washed three times with PBS and incubated for 1 h at room temperature protected from light with Alexa Fluor 550-conjugated anti-rabbit IgG (H + L) F(ab')\u003csub\u003e2\u003c/sub\u003e fragment secondary antibody (Cell Signaling Technology) diluted 1:1000 in PBS containing 0.1% Triton X-100. Cells were washed three times with PBS and mounted using ProLong Diamond Antifade Mountant with DAPI (Invitrogen, #P36971). Fluorescence and bright-field images were acquired using an EVOS fluorescence microscope at 40× magnification.\u003c/p\u003e\u003ch2\u003eCell Uptake and Internalization Studies\u003c/h2\u003e\u003cp\u003eLNCaP-AR, CWRR1-EnzR, DU145-PSMA, and DU145 cells were cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS (Corning) and 1% penicillin/streptomycin (ThermoFisher), with the additional supplements for the following cell lines: 1% HEPES (ThermoFisher), 1% sodium pyruvate (ThermoFisher), 1% Glutamax (ThermoFisher) for LNCaP-AR; 10 µM enzalutamide (MedChemExpress) for CWRR1-EnzR. For cell uptake and internalization studies, cells were seeded in 24-well plates at 150,000 cells per well in triplicate and allowed to adhere overnight in the growth medium. On the following day, the culture medium was removed, and cells were washed once with ice-cold PBS. Cells were then incubated at 37°C with 74 kBq of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, 7.4 kBq of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, or 18.5 kBq of [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 in 0.5 mL serum-free medium, either in the presence or absence of an excess of unlabeled PSMA-617 (300 nM) to assess blocking. Unless specified, the cell uptake studies of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 were conducted in the presence of DTPA (0.1 mg/mL) in the incubation medium. At predetermined time points (0.5, 1, 2, and 4 h), the supernatant was collected, and cells were washed twice with 0.5 mL ice-cold PBS, collecting washes. Next, cells were incubated with 0.5 mL of ice-cold glycine-HCl buffer (50 mM, pH 2.5-3.0) for 5 min at room temperature to isolate cell surface-bound radioactivity, followed by an additional PBS wash. The glycine wash and PBS wash were pooled to represent the cell surface-bound fraction. Subsequently, cells were lysed using 0.25 mL Cell Lysis Reagent (Promega), and the wells were rinsed with 0.25 mL PBS, which was combined with the lysis fraction to obtain the internalized fraction. Radioactivity counts (CPM) in all fractions were measured using an automated gamma counter (Cobra II). Cell uptake was calculated as the percentage of the input dose (CPM) present in the cell fraction for each well and expressed as mean ± standard deviation (SD) for triplicate wells at each time point. Cell surface-bound (glycine wash) and internalized (lysis fraction) fractions were also determined as percentages of the initially bound activity to assess internalization rate for each radioligand at different time points.\u003c/p\u003e\u003ch2\u003eInhibitory Potency (IC\u003csub\u003e50\u003c/sub\u003e) Assays\u003c/h2\u003e\u003csub\u003e50\u003c/sub\u003e\u003cp\u003eThe inhibitory potencies of the nonradioactive Lu-PSMA-617 and Pb-PSMA-617 analogues were determined by competitive inhibition assays using [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in the DU145-PSMA cell line. Cells seeded in 24-well plates (150,000 cells/well) were incubated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (2 µCi/well) in serum-free medium for 2 h under standard cell culture conditions in the presence of increasing concentrations of Lu-PSMA-617 or Pb-PSMA-617 (0.14–300 nM, 3-fold serial dilutions, n = 8). After incubation, the medium was collected, and cells were washed with cold PBS (2 × 0.5 mL). Cells were then lysed with 1× Cell Lysis Reagent (0.25 mL, Promega), followed by an additional PBS wash (0.25 mL). Radioactivity in each fraction was measured using an automated gamma counter. Cell-bound activity was determined by calculating the percentage of the input dose (CPM). Half-maximal inhibitory concentration values (IC\u003csub\u003e50\u003c/sub\u003e) for the two nonradioactive conjugates were determined by nonlinear regression analysis of the inhibitory data using a log(inhibitor) versus response model in GraphPad Prism (v10.6).\u003c/p\u003e\u003ch2\u003eSaturation Binding Assays\u003c/h2\u003e\u003cp\u003eThe binding affinities (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) of the PSMA radioligands were determined by saturation binding assays using the two PSMA-positive cell lines LNCaP-AR and DU145-PSMA. Cells seeded in 24-well plates (150,000 cells/well) were incubated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, or [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 for 2 h at 37°C in serum-free medium containing a series of increasing concentrations of the corresponding radioligand (0.40–50 nM, n = 8), prepared by two-fold serial dilutions starting from 50 nM. For each concentration, nonspecific binding was assessed in parallel by co-incubation with an excess of unlabeled PSMA-617 (300 nM). After incubation, the supernatant was collected, and the cells were washed with ice-cold PBS and lysed as described for cell uptake studies. Radioactivity (CPM) in the cell fractions was measured using an automated gamma counter (Cobra II). The equilibrium dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) was calculated by nonlinear regression analysis of the data in GraphPad Prism (v10.6).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprostate-specific membrane antigen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRLT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradioligand therapy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elinear energy transfer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLu\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elutetium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eactinium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePb\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elead\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebismuth\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emCi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emillicurie\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;Ci\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrocurie\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ekBq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekilobecquerel\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMBq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emegabecquerel\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003enmol\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enanomoles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emolar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003enM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enanomolar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;M\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicromolar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;mol\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicromole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eequilibrium dissociation constant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emCRPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emetastatic castration-resistant prostate cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ekeV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekiloelectronvolt\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;m\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrometer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eliquid chromatography-mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiTLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einstant thin-layer chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eD\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edistribution coefficient\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecounts per minute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eh\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehours\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emin\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eminutes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ediethylenetriaminepentaacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emilligram\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emilliliter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;L\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicroliter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eα\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eβ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebeta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eγ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egamma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equality control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRCY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradiochemical yield\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehydrochloric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNaOAc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium acetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNH\u003csub\u003e4\u003c/sub\u003eOAc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eammonium acetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESI-MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelectrospray ionization mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMeCN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacetonitrile\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrifluoroacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026Aring;\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eangstrom.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupplementary Information\u003c/h2\u003e \u003cp\u003eThe online version contains supplementary material\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors acknowledge funding from the Novartis Institutes for BioMedical Research (NIBR) that supported this research.\u003c/p\u003e\u003ch2\u003eAuthors\u0026rsquo; Contributions\u003c/h2\u003e \u003cp\u003eAB, GR, YZ, PM, JB\u0026rsquo;L, and AS contributed to the experimental design, data collection, and analysis. RZS, CTC, and SKC contributed to the study design and data interpretation and provided resources. All authors have read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors would like to thank the Novartis Institutes for BioMedical Research for providing the DU145-PSMA cell line.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data is available upon request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBenesova M, Bauder-Wust U, Schafer M, Klika KD, Mier W, Haberkorn U, et al. Linker modification strategies to control the prostate-specific membrane antigen (PSMA)-targeting and pharmacokinetic properties of DOTA-conjugated PSMA inhibitors. J Med Chem. 2016;59(5):1761\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenesova M, Schafer M, Bauder-Wust U, Afshar-Oromieh A, Kratochwil C, Mier W, et al. Preclinical evaluation of a tailor-made DOTA-conjugated PSMA inhibitor with optimized linker moiety for imaging and endoradiotherapy of prostate cancer. J Nucl Med. 2015;56(6):914\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusslinger SD, Tschan VJ, Richard OK, Talip Z, Schibli R, M\u0026uuml;ller C. [\u003csup\u003e225\u003c/sup\u003eAc]Ac-SibuDAB for targeted alpha therapy of prostate cancer: preclinical evaluation and comparison with [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617. Cancers. 2022;14(22):5651.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChitneni SK, Yan H, Zalutsky MR. Synthesis and evaluation of a \u003csup\u003e18\u003c/sup\u003eF-labeled triazinediamine analogue for imaging mutant IDH1 expression in gliomas by PET. ACS Med Chem Lett. 2018;9(7):606\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofman MS, Emmett L, Sandhu S, Iravani A, Joshua AM, Goh JC et al. Prostate Cancer Trials, G. (2021). [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 versus cabazitaxel in patients with metastatic castration-resistant prostate cancer (TheraP): a randomised, open-label, phase 2 trial. \u003cem\u003eLancet, 397\u003c/em\u003e(10276), 797\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHooijman EL, Chalashkan Y, Ling SW, Kahyargil FF, Segbers M, Bruchertseifer F, et al. Development of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-I\u0026amp;T for targeted alpha therapy according to GMP guidelines for treatment of mCRPC. Pharmaceutics. 2021;13(5):715.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKafka M, Horninger A, di Santo G, Virgolini I, Neuwirt H, Unterrainer LM, et al. Real-world outcomes and predictive biomarkers for \u003csup\u003e177\u003c/sup\u003eLu-lutetium prostate-specific membrane antigen ligand treatment in metastatic castration-resistant prostate cancer: a european association of urology young academic urologists prostate cancer working group multi-institutional observational study. Eur Urol Oncol. 2024;7(3):421\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKratochwil C, Bruchertseifer F, Giesel FL, Weis M, Verburg FA, Mottaghy F, et al. ). \u003csup\u003e225\u003c/sup\u003eAc-PSMA-617 for PSMA-targeted alpha-radiation therapy of metastatic castration-resistant prostate cancer. J Nucl Med. 2016;57(12):1941\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKratzer TB, Mazzitelli N, Star J, Dahut WL, Jemal A, Siegel RL. Prostate cancer statistics, 2025. CA Cancer J Clin. 2025;75(6):485\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng CL, Chen CT, Tang IC. Exploring the therapeutic potential of Lu-PSMA-617 in a mouse model of prostate cancer bone metastases. Int J Mol Sci. 2025;26(13):5970.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177-PSMA-617 for metastatic castration-resistant prostate cancer. N Engl J Med. 2021;385(12):1091\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStenberg VY, Juzeniene A, Chen Q, Yang X, Bruland \u0026Oslash;S, Larsen RH. Preparation of the alpha-emitting prostate-specific membrane antigen targeted radioligand [\u003csup\u003e212\u003c/sup\u003ePb]Pb-NG001 for prostate cancer. J Label Comp Radiopharm. 2020;63(3):129\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStuparu AD, Meyer CAL, Evans-Axelsson SL, Luckerath K, Wei LH, Kim W, et al. Targeted alpha therapy in a systemic mouse model of prostate cancer - a feasibility study. Theranostics. 2020;10(6):2612\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWurzer A, Kunert JP, Fischer S, Felber V, Beck R, de Rose F, et al. Synthesis and preclinical evaluation of \u003csup\u003e177\u003c/sup\u003eLu-labeled radiohybrid PSMA ligands for endoradiotherapy of prostate cancer. J Nucl Med. 2022;63(10):1489\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav MP, Ballal S, Sahoo RK, Tripathi M, Seth A, Bal C. Efficacy and safety of \u003csup\u003e225\u003c/sup\u003eAc-PSMA-617 targeted alpha therapy in metastatic castration-resistant Prostate Cancer patients. Theranostics. 2020;10(20):9364\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYong K, Brechbiel M. Application of \u003csup\u003e212\u003c/sup\u003ePb for targeted alpha-particle therapy (TAT): pre-clinical and mechanistic understanding through to clinical translation. AIMS Med Sci. 2015;2(3):228\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmermann R. Is \u003csup\u003e212\u003c/sup\u003ePb really happening? The post-\u003csup\u003e177\u003c/sup\u003eLu/\u003csup\u003e225\u003c/sup\u003eAc blockbuster? J Nucl Med. 2024;65(2):176\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PSMA-617, Targeted Alpha Therapy, 177Lu, 225Ac, 212Pb, Prostate Cancer","lastPublishedDoi":"10.21203/rs.3.rs-9419820/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9419820/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePSMA-targeted radioligand therapy is a promising approach for the treatment of advanced prostate cancer; however, the clinical efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (Pluvicto\u0026reg;) is limited by the relatively low cytotoxic potency of the β-emitting radionuclide \u003csup\u003e177\u003c/sup\u003eLu (t\u003csub\u003e1/2\u003c/sub\u003e: 6.65 d). This has driven high interest in α-emitting radionuclides, such as \u003csup\u003e212\u003c/sup\u003ePb (t\u003csub\u003e1/2\u003c/sub\u003e: 10.64 h) and \u003csup\u003e225\u003c/sup\u003eAc (t\u003csub\u003e1/2\u003c/sub\u003e: 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 \u003csup\u003e212\u003c/sup\u003ePb- and \u003csup\u003e225\u003c/sup\u003eAc-labeled PSMA-617 compared with [\u003csup\u003e177\u003c/sup\u003eLu]LuPSMA-617, using two different PSMA-positive prostate cancer cell lines, LNCaP-AR and DU145-PSMA, along with paired negative controls.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe radioligands were synthesized with an isolated radiochemical yield of \u0026gt;\u0026thinsp;95% for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 and about 45% for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617. The molar activity after Sep-Pak purification was about 37 MBq/nmol (1 mCi/nmol) for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, 4.4\u0026ndash;8.9 MBq/nmol (120\u0026ndash;240 \u0026micro;Ci/nmol) for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617, and 0.24\u0026ndash;0.57 MBq/mol (6.5\u0026ndash;15.5 \u0026micro;Ci/nmol) for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617. In vitro stability studies in PBS, human serum, and whole blood revealed\u0026thinsp;\u0026ge;\u0026thinsp;90% stability for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 (up to 5 days) and [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 (24 hours), whereas [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 exhibited\u0026thinsp;~\u0026thinsp;72% stability in PBS and ~\u0026thinsp;90% in serum and whole blood for 5 days. The uptake of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in LNCaP-AR cells was 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;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\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%, 65% internalized). Compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 showed about 2-fold higher uptake and internalization in LNCaP-AR cells at 4 h (14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0%, 58% internalized), but a similar uptake in DU145-PSMA cells (29.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%, 62% internalized). In contrast, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 exhibited lower uptake in both cell lines, with 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0% in LNCaP-AR cells and 4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;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 (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) in LNCaP-AR and DU145-PSMA cell lines, with 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 nM and 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617, 5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM and 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 nM for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617, and 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nM and 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 nM for [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 showed similar or better uptake in PSMA-positive cell lines compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617. While [\u003csup\u003e225\u003c/sup\u003eAc]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 [\u003csup\u003e225\u003c/sup\u003eAc]Ac-PSMA-617 compared to [\u003csup\u003e212\u003c/sup\u003ePb]Pb-PSMA-617 or [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617.\u003c/p\u003e","manuscriptTitle":"Radiochemistry and Comparative In Vitro Assessment of PSMA-617 Labeled with Lead-212 (212Pb), Actinium-225 (225Ac), and Lutetium-177 (177Lu).","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 11:29:58","doi":"10.21203/rs.3.rs-9419820/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2026-05-04T17:28:46+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-04-22T10:04:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T08:00:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-21T13:35:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2026-04-17T18:57:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f5c6524a-ecf0-4a7e-8dea-18a76b1b05fd","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Minor revision","date":"2026-05-04T17:28:46+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T14:57:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 11:29:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9419820","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9419820","identity":"rs-9419820","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}