Comparison of Nuclear Medicine Therapeutics Targeting PSMA among Alpha-Emitting Nuclides

Currently, targeted alpha therapy (TAT) is a new therapy involving the administration of a therapeutic drug that combines a substance of α-emitting nuclides that kill cancer cells and a drug that selectively accumulates in cancer cells. It is known to be effective against cancers that are difficult to treat with existing methods, such as cancer cells that are widely spread throughout the whole body, and there are high expectations for its early clinical implementation. The nuclides for TAT, including 149Tb, 211At, 212/213Bi, 212Pb (for 212Bi), 223Ra, 225Ac, 226/227Th, and 230U, are known. However, some nuclides encounter problems with labeling methods and lack sufficient preclinical and clinical data. We labeled the compounds targeting prostate specific membrane antigen (PSMA) with 211At and 225Ac. PSMA is a molecule that has attracted attention as a theranostic target for prostate cancer, and several targeted radioligands have already shown therapeutic effects in patients. The results showed that 211At, which has a much shorter half-life, is no less cytotoxic than 225Ac. In 211At labeling, our group has also developed an original method (Shirakami Reaction). We have succeeded in obtaining a highly purified labeled product in a short timeframe using this method.


Introduction
There are many nuclides that could be used in targeted α therapy (TAT) (Table 1) [1].Among them, astatine-211 ( 211 At) and actinium-225 ( 225 Ac) are thought to be useful αemitting nuclides (Table 2).This is because these nuclides can be produced in relatively large quantities [2].Prostate specific membrane antigen (PSMA) is highly expressed in metastatic and castration-resistant prostate cancers and is a well-known therapeutic target for prostate cancer in radioligand therapy [3][4][5].Although the functions of PSMA in cancer cells are still unclear, its expression increases in correlation with the degree of cancer progression, making it an extremely useful theranostic target for prostate cancer.Prostate cancer is mainly seen in people over 60 years old, and it is known that more than half of men over 80 years old have latent prostate cancer [6].Remarkable results in TAT have been reported using 225 Ac-labeled therapeutics, and their usefulness is clear [7].However, the supply of 225 Ac is insufficient for widespread clinical application.The current supply of 225 Ac is mostly due to the decay of uranium-223 ( 233 U), with a worldwide supply of approximately 63 GBq (2 Ci)/year [8].Therefore, lutetium-177 ( 177 Lu), a β-ray emitting nuclide, is used and [ 177 Lu]PSMA-617 has already been approved and is commercially available in the US and Europe [9].Initial treatment for prostate cancer generally includes surgery or radiation therapy, followed by hormone therapy.Recently, various treatments have been implemented for hormone-sensitive prostate cancer (HSPC).Castration-resistant prostate cancer (CRPC) has a poor prognosis, and taxane chemotherapy, such as docetaxel (DTX) and cabazitaxel (CBZ), is administered, but the treatment effect may not be sufficient.In the TheraP trial, it was reported that 177 Lu-PSMA-617 treatment had fewer side effects and lowered PSA levels compared to CBZ [10].We are currently conducting research with 211 At, which can be produced using an accelerator, and have successfully labeled astatine as a highly selective PSMA compound [11].We previously showed the strong therapeutic effect of this compound and its promising potential for clinical applications.Th: thorium, Ra: radium, Tb: terbium, Nd: neodymium, Ac: actinium, Bi: bismuth, At: astatine, Pb: lead, U: uran.
This study aimed to clarify the performance of the PSMA-targeting compound labeled with 211 At ( 211 At-PSMA-5) in comparison to 225 Ac-PSMA-617.These nuclides differ in their physical half-lives ( 225 Ac; 10 days, 211 At; 7.2 h), the number of α-particle emissions ( 225 Ac; 4, 211 At; 1), and in the properties of the elements themselves ( 225 Ac; actinoid, 211 At; halogen), resulting in the need for different binding domains for labeling.Even when using the same α-particle emitting nuclide, it is thought that there are other problems of practical application other than the amount of supply.Examples include the ease of labeling, the amount of compound used, and efficacy.If a small amount of the compound is used, the side effects caused by the compound might be reduced to a minimum.However, no comparison has been made to date between α-emitting nuclear medicine therapeutics that have the same molecular targets, e.g., PSMA.We have conducted this experiment in the hope that it will be useful for selecting the optimal nuclide for each situation.
Nuclear medicine involves minimally invasive procedures.However, by fully understanding the principles and properties of nuclear medicine therapeutics, we believe that they are effective.Furthermore, through an investigation of the dynamics and pathological analysis of nuclear medicine, it has been shown that the side effects are not significantly different from those of other drugs [12][13][14][15].Nuclear medicine therapeutics are expected to become new treatment options for patients for whom existing therapeutic drugs are not suitable.To expand patient options, we hope to demonstrate scientific evidence of its effectiveness, especially in TAT.

Evaluation of Effects on Cell Viability
We seeded the cell line LNCaP, which is known for its high PSMA expression, and the cell line PC3, characterized by low PSMA expression, at a density of 1 × 10 4 cells/well in a 96-well plate.The cells were treated with 225 Ac-PSMA-617 or 211 At-PSMA-5 for 3 days, and cell viability was evaluated.The toxicity of the labeled PSMA was more pronounced in LNCaP cells than in PC3 cells (Figure 1).The 225 Ac-PSMA-617 nuclide had a stronger effect on cell viability than 211 At-PSMA-5; however, above a certain concentration, cell viability did not decrease in a dose-dependent manner.

Evaluation of Effects on Cell Viability
We seeded the cell line LNCaP, which is known for its high PSMA expression, and the cell line PC3, characterized by low PSMA expression, at a density of 1 × 10 4 cells/well in a 96-well plate.The cells were treated with 225 Ac-PSMA-617 or 211 At-PSMA-5 for 3 days, and cell viability was evaluated.The toxicity of the labeled PSMA was more pronounced in LNCaP cells than in PC3 cells (Figure 1).The 225 Ac-PSMA-617 nuclide had a stronger effect on cell viability than 211 At-PSMA-5; however, above a certain concentration, cell viability did not decrease in a dose-dependent manner.

Evaluation of Effect on Replication
We performed a colony formation assay to assess the extent to which 225 Ac-PSMA-617 and 211 At-PSMA-5 affected the replication ability of the cells.Figure 2 shows a numerical graph based on the photographs shown in Figure 3. Cytotoxicity occurred in both PC3 (PSMA low ) and LNCaP (PSMA high ) cells in a concentration-dependent manner (Figures 2A  and 3A).This effect appeared to be stronger in cells with high PSMA expression.When the cells were treated with 225 Ac-PSMA-617, dose-dependent inhibition of replication was observed in both PC3 and LNCaP cells.After treatment with 211 At-PSMA-5, no effect was observed in PC3 cells (Figures 2B and 3B).

Evaluation of Effect on Replication
We performed a colony formation assay to assess the extent to which 225 Ac-PSMA-617 and 211 At-PSMA-5 affected the replication ability of the cells.Figure 2 shows a numerical graph based on the photographs shown in Figure 3. Cytotoxicity occurred in both PC3 (PSMA low ) and LNCaP (PSMA high ) cells in a concentration-dependent manner (Figures 2A and 3A).This effect appeared to be stronger in cells with high PSMA expression.When the cells were treated with 225 Ac-PSMA-617, dose-dependent inhibition of replication was observed in both PC3 and LNCaP cells.After treatment with 211 At-PSMA-5, no effect was observed in PC3 cells (Figures 2B and 3B

Evaluation of Cytotoxicity
DNA double strand breaks (DSBs) were observed in both PC3 (PSMA low ) and LNCaP (PSMA high ) cells (Figure 4). Figure 5 depicts a numerical graph based on the photographs

Evaluation of Cytotoxicity
DNA double strand breaks (DSBs) were observed in both PC3 (PSMA low ) and LNCaP (PSMA high ) cells (Figure 4). Figure 5 depicts a numerical graph based on the photographs shown in Figure 4. Green fluorescence indicates the presence of γH2AX, a marker of DSB.Focies of γH2AX were strongly induced by 225 Ac-PSMA-617 (Figure 5A), and many focies were induced by 211 At-PSMA-5 (Figure 5B).When comparing their effects on (a) PC3 and (b) LNCaP cells, more foci were found in the LNCaP cells, indicating that many DSBs appeared in the LNCaP cells.2.4.Uptake of 225 Ac-PSMA-617 or 211 At-PSMA-5 The 225 Ac-PSMA-617 nuclide had a low background of intracellular uptake and was hardly taken up by PC3 (PSMA low ) cells (Figure 6a).In contrast, 211 At-PSMA-5 was taken  2.4.Uptake of 225 Ac-PSMA-617 or 211 At-PSMA-5 The 225 Ac-PSMA-617 nuclide had a low background of intracellular uptake and was hardly taken up by PC3 (PSMA low ) cells (Figure 6a).In contrast, 211 At-PSMA-5 was taken  The 225 Ac-PSMA-617 nuclide had a low background of intracellular uptake and was hardly taken up by PC3 (PSMA low ) cells (Figure 6a).In contrast, 211 At-PSMA-5 was taken up in a certain amount by PC3 cells and large amounts of uptake were observed in LNCaP (PSMA high ) cells (Figure 6b). up in a certain amount by PC3 cells and large amounts of uptake were observed in LNCaP (PSMA high ) cells (Figure 6b).

Inhibition of Unlabeled Chemicals
The inhibition of uptake by unlabeled chemicals using LNCaP cells (PSMA high ) is shown in Figure 7.When we confirmed the presence of competitive inhibition by unlabeled chemicals for uptake at the same dose, it was clear that 211 At-PSMA-5 (Figure 7b) was inhibited at a lower concentration than 225 Ac-PSMA-617 (Figure 7a).The IC50 of 225 Ac-PSMA-617 was 2.64 nM and that of 211 At-PSMA-5 was 0.32 nM.Non-specific binding was evaluated using PC3 cells (PSMA low ).The effects of non-labeled chemicals on both the uptake of 225 Ac-PSMA-617 (Figure 7c) and 211 At-PSMA-5 (Figure 7d) in PC3 cells were thought to be minimal.

Inhibition of Unlabeled Chemicals
The inhibition of uptake by unlabeled chemicals using LNCaP cells (PSMA high ) is shown in Figure 7.When we confirmed the presence of competitive inhibition by unlabeled chemicals for uptake at the same dose, it was clear that 211 At-PSMA-5 (Figure 7b) was inhibited at a lower concentration than 225 Ac-PSMA-617 (Figure 7a).The IC 50 of 225 Ac-PSMA-617 was 2.64 nM and that of 211 At-PSMA-5 was 0.32 nM.Non-specific binding was evaluated using PC3 cells (PSMA low ).The effects of non-labeled chemicals on both the uptake of 225 Ac-PSMA-617 (Figure 7c) and 211 At-PSMA-5 (Figure 7d) in PC3 cells were thought to be minimal.up in a certain amount by PC3 cells and large amounts of uptake were observed in LNCaP (PSMA high ) cells (Figure 6b).

Inhibition of Unlabeled Chemicals
The inhibition of uptake by unlabeled chemicals using LNCaP cells (PSMA high ) is shown in Figure 7.When we confirmed the presence of competitive inhibition by unlabeled chemicals for uptake at the same dose, it was clear that 211 At-PSMA-5 (Figure 7b) was inhibited at a lower concentration than 225 Ac-PSMA-617 (Figure 7a).The IC50 of 225 Ac-PSMA-617 was 2.64 nM and that of 211 At-PSMA-5 was 0.32 nM.Non-specific binding was evaluated using PC3 cells (PSMA low ).The effects of non-labeled chemicals on both the uptake of 225 Ac-PSMA-617 (Figure 7c) and 211 At-PSMA-5 (Figure 7d) in PC3 cells were thought to be minimal.The stability of 211 At-PSMA5 was evaluated using HPLC, TLC, and electrophoresis.Its stability in blood and urine, along with the absence of metabolites and degradation products, was detected in vitro experiments.In in vivo experiments, slight (<1%) deastatination was observed.The same evaluations were conducted on 225 Ac-PSMA-617, confirming that it was stable.

Discussion
In this study, 225 Ac-PSMA-617 and 211 At-PSMA-5 were compared in terms of their nuclide decay number (Bq) in order to focus on the performance of the labeled chemicals.This study revealed that 225 Ac, which contains many α-particles, is highly cytotoxic, as was expected (Figure 1).The strong cytotoxic effect of 225 Ac-PSMA-617 was reflected in the loss of replication ability (Figures 2 and 3).At first glance, 225 Ac-PSMA-617 seemed to have stronger cytotoxicity than 211 At-PSMA-5.However, considering the AUC and α-ray emission ratio, it was difficult to say that 225 Ac-PSMA-617 was more effective than 211 At-PSMA-5 because there was a 25 to 83-fold difference even with the same radioactivity (Bq).We also compared γH2AX as an indicator of radiation-induced DNA damage (DNA double strand breaks, DSBs), and it became clear that 211 At-PSMA-5 induces DNA damage at the same levels of 225 Ac-PSMA-617 (Figures 4 and 5).This was thought to be because 211 At-PSMA-5 was taken up by cells in larger amounts and acted closer to the nucleus (Figure 6).Labeled chemicals appear to work more effectively as nuclear medicine therapeutics for intracellular uptake.It is interesting to note that more 211 At-PSMA-5 was taken up by the cells even though both chemical structures are similar (Figures 8 and 9).However, considering their structures, there seems to be a slight difference in fat solubility.Since 211 At-PSMA-5 is slightly more lipophilic, it is presumed to have a higher affinity for the cell membrane (Figure 7).The correction for non-specific binding concerning IC 50 should be strictly calculated using the inhibition value in an experimental system using PC3 cells (PSMA low ), Xenopus oocytes, or HEK293 cells overexpressing PSMA.However, for both 225 Ac-PSMA-617 and 211 At-PSMA-5, the ratio of PC3 to LNCaP cell uptake at 120 min did not change (approximately 30%) (Figure 6).Even when the background was subtracted at the same rate, the relationship between the IC 50 of 225 Ac-PSMA-617 and 211 At-PSMA-5 was similar.Thus, the IC 50 was calculated from the LNCaP value (Figure 7).The IC 50 value of 225 Ac-PSMA-617 was approximately eight-fold higher than that of 211 At.The lipid solubility of chemicals is an important factor in nuclear medicine.This is because a certain degree of fat solubility improves tumors [18][19][20][21].However, if fat solubility is too high, adsorption onto plastic experimental tools will be high.For example, an increase in the amount of adsorption on the purification column results in a poor collection yield.Additionally, when administered to animals, it has been observed that excretion from the intestinal tract increases and the amount excreted in feces increases.Screening for suitable compounds should consider both accumulation and excretion. 225Ac is an excellent therapeutic-emitting nuclide, but its current supply is limited.PSMA is also highly specific and only has a few side effects (caused by damage to the salivary and lacrimal glands, such as dry eye [22] and xerostomia [23]).Non-specific accumulation over a long period would induce side effects.If the ratio of non-specific accumulation is the same, the nuclide with a shorter half-time is less likely to induce side effects due to non-specific accumulation.The 211 At-PSMA-5 nuclide requires significantly fewer chemicals for labeling than 225 Ac-PSMA-617, and its cost of manufacturing is also lower.In this study, we attempted to compare two α-emitting nuclides used in TAT.We believe that our results suggest that, even though its half-life is much shorter than 225 Ac, 211 At can be sufficiently tolerated in clinical use by effectively utilizing its properties.If the half-life is short, the hospitalization period for nuclide decay can be shortened.This might reduce the burden on patients.  22Ac is an excellent therapeutic-emitting nuclide, but its current supply is limited.PSMA is also highly specific and only has a few side effects (caused by damage to the salivary and lacrimal glands, such as dry eye [22] and xerostomia [23]).Non-specific accumulation over a long period would induce side effects.If the ratio of non-specific accumulation is the same, the nuclide with a shorter half-time is less likely to induce side effects due to non-specific accumulation.The 211 At-PSMA-5 nuclide requires significantly fewer chemicals for labeling than 225 Ac-PSMA-617, and its cost of manufacturing is also lower.In this study, we attempted to compare two α-emitting nuclides used in TAT.We believe that our results suggest that, even though its half-life is much shorter than 225 Ac, 211 At can be sufficiently tolerated in clinical use by effectively utilizing its properties.If the half-life is short, the hospitalization period for nuclide decay can be shortened.This might reduce the burden on patients.
Although there are many clinical reports of 225 Ac-PSMA-617 use in humans, there are only a few reports on animal experiments using 225 Ac.Additionally, these reports were published later than the human reports.The 225 Ac-PSMA-617 nuclide demonstrated high levels of accumulation in the liver.However, it is well known that 225 Ac alone accumulates in the liver.There are doubts as to whether the results showing accumulation only in the  225 Ac is an excellent therapeutic-emitting nuclide, but its current supply is limited.PSMA is also highly specific and only has a few side effects (caused by damage to the salivary and lacrimal glands, such as dry eye [22] and xerostomia [23]).Non-specific accumulation over a long period would induce side effects.If the ratio of non-specific accumulation is the same, the nuclide with a shorter half-time is less likely to induce side effects due to non-specific accumulation.The 211 At-PSMA-5 nuclide requires significantly fewer chemicals for labeling than 225 Ac-PSMA-617, and its cost of manufacturing is also lower.In this study, we attempted to compare two α-emitting nuclides used in TAT.We believe that our results suggest that, even though its half-life is much shorter than 225 Ac, 211 At can be sufficiently tolerated in clinical use by effectively utilizing its properties.If the half-life is short, the hospitalization period for nuclide decay can be shortened.This might reduce the burden on patients.
Although there are many clinical reports of 225 Ac-PSMA-617 use in humans, there are only a few reports on animal experiments using 225 Ac.Additionally, these reports were published later than the human reports.The 225 Ac-PSMA-617 nuclide demonstrated high levels of accumulation in the liver.However, it is well known that 225 Ac alone accumulates in the liver.There are doubts as to whether the results showing accumulation only in the Although there are many clinical reports of 225 Ac-PSMA-617 use in humans, there are only a few reports on animal experiments using 225 Ac.Additionally, these reports were published later than the human reports.The 225 Ac-PSMA-617 nuclide demonstrated high levels of accumulation in the liver.However, it is well known that 225 Ac alone accumulates in the liver.There are doubts as to whether the results showing accumulation only in the liver can depict metastasis [24].A previous study used a combination of the PD-L1 antibody and 225 Ac-PSMA-617 in RM1-PGLS mice.Although it is used in combination with an immune checkpoint inhibitor, it has not been as effective as expected [25,26].Rodent metabolism is different from that of primates, meaning that research results on rodents are not guaranteed to be applicable to humans.In contrast, 211 At-PSMA-5 was investigated for imaging in cynomolgus monkeys by Watabe et al.Additionally, no acute inflammation was observed, and the side effects were expected to be minimal.Although clinical trials using 211 At-PSMA- 5 have not yet been conducted, animal studies have shown positive results [11].Thus, we expect that future clinical trials of 211 At-PSMA-5 will yield good results.
Irradiation to the target with high linear energy transfer (LET) radiation is highly lethal.Thus, resistance to this radiation is unlikely to occur.Therefore, selecting a specific and excellent molecular target, such as PSMA, is essential for nuclear medicine.To achieve a stable supply of nuclides, selecting optimal molecular targets, establishing chemicals that recognize and label these targets, and collaborating across departments, such as medicine, science, and nuclear physics, is necessary.
We also confirmed the utility of 225 Ac in another molecular target, fibroblast activate protein α (FAPα) [27,28].However, as previously mentioned, its supply is insufficient for clinical use [8].Various research groups are attempting to find methods to produce it in large quantities; however, no satisfactory method has yet been established.Therefore, there is an urgent need to establish a method for producing 225 Ac.Currently, we believe that the most promising method in Japan is the use of an electron linear accelerator.The production of 225 Ac was conducted using 226 Ra and a linear accelerator at Tohoku university [ 226 Ra(γ, n) 225 Ra→ 225 Ac].In Japan, the National Institute for Quantum Science and Technology (QST) and Nihon Medi-physics, Co. Ltd. are also trying to produce it with the transmutation of 226 Ra to obtain 225 Ac [ 226 Ra(p, 2n) 225 Ac] [29].In Canada's particle accelerator center (TRIUMF), a method of obtaining 225 Ac through the nuclear spallation of thorium-232 [ 232 Th(p, spall) 225 Ra→ 225 Ac] is being attempted [30].In contrast, 211 At has a short half-life; therefore, it must be manufactured in large quantities.This must be performed efficiently because it uses an accelerator.It is desirable to create multiple bases within a country where supplies are needed.However, to transport them to places where transportation is difficult, it is necessary to create large quantities.In 2023, Haba et al. aimed to develop targeted irradiation technology that minimizes the loss of 211 At due to radioactive decay and increases the production efficiency of 211 At through high-intensity beam irradiation.Their results may be useful for the mass production and supply of 211 At.
The half-life of 211 At is 7.2 h and α decays to bismuth-207 ( 207 Bi with a half-life of 32 years) with a probability of 41.8%.Therefore, with a probability of 58.2%, it can become polonium-211 ( 211 Po) through electron capture decay.Since 211 Po undergoes α decay immediately (with a half-life of 0.52 s) to the stable isotope lead-207 ( 207 Pb), 211 At emits α-particles with virtually 100% probability.On the other hand, there are many daughter nuclides of 225 Ac, such as francium-221 ( 221 Fr, with a half-life of 0.12 µ seconds), 217 At (with a half-life of 20 milliseconds), 213 Bi (with a half-life of 47 min), 213 Po (with a half-life of 3.65 µ seconds), thallium-209 ( 209 Tl, with a half-life of 1 h), 209 Pb (with a half-life of 3.3 h), and 209 Bi (stable), all of which have short lifetimes.Because none of these generate radon as daughter nuclide, which is a gas, post-administration management is easy.For practical use, it is very important that the nuclides are easily handled.In addition to the two nuclides discussed in this research, there are several other α-emitting nuclides that may be used for TAT, as shown in Table 1.However, various choices should be made with consideration of the balance between demand and supply.
We are currently developing nuclear medicine therapeutics using the 211 At and 225 Ac nuclides.During this period, we have observed the advantages and disadvantages of each nuclide.Initially, we thought that 225 Ac-PSMA-617 would provide better results than 211 At-PSMA-5 and we hoped that 211 At-PSMA-5 would be the next choice.However, the performance of 211 At-PSMA-5 was unexpectedly good.High-power accelerators are currently needed to obtain 211 At.However, since it can be obtained using accelerators, it is easier to obtain than 225 Ac, particularly in Japan.Although the availability of nuclides differs from country to country, it can be said that, at least in Japan, astatine has been shown to be extremely useful as a labeled nuclide for nuclear medicine therapeutics.We are currently conducting an investigator-initiated clinical trial of astatine-labeled chemicals (Na 211 At, targeting thyroid cancer) at Osaka University Hospital.By demonstrating the usefulness of nuclear medicine therapeutics, especially α-emitting nuclear medicine therapeutics, we hope that this will become a new choice for patients who are currently unable to undergo surgery or for whom existing drugs are not effective.
The most important step in the development of nuclear medicine is the selection of molecular targets.If expressed in normal tissues as well as in cancer tissues, nuclear medicine therapeutics can accumulate in normal tissues and damage them, causing side effects.Therefore, it is desirable to use molecular targets with a higher specificity.In the development of compounds, even for the same molecular target, selectivity can vary significantly depending on the structure of the compound.In addition, side effects can occur when the labeled nuclide is released from the compound.This is because 211 At behaves in a similar fashion to iodine and 225 Ac accumulates in the bones and liver [31].Consideration is also required in order to ensure safety.Key considerations in the clinical application of 225 Ac-PSMA-617 include liver uptake, which is assumed to result from the decay products of 225 Ac-PSMA-617.DTPA is an abbreviation for diethylenetriamine pentaacetic acid, a chelate compound with affinity for metals, and is a type of chemical protective agent against radiation damage.For example, Techne ® DTPA Kits (PDRadiopharma, Inc., Tokyo, Japan) or Indium ( 111 In) DTPA Injections (Nihon Medi-Physics Co., Ltd., Tokyo, Japan) exist.This protective agent has the function of removing radioactive materials from the body and is said to be most effective in excreting radioisotopes.DTPA may be added to the formulation of 225 Ac-PSMA-617 to prevent uptake in the liver (faster renal clearance).In the case of 211 At-PSMA-5, the properties of the element are different from 225 Ac; therefore, it may be necessary to use a different drug.
The functions of PSMA itself in cancer tissues are gradually being elucidated.For example, research conducted by Watanabe, et al. revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhance tumor angiogenesis in prostate cancer tissues [32].The elucidation of the role of PSMA in cancer tissues has supported its importance as a molecular target.On the other hand, reports on the structure and dynamics of chemicals are also being considered [33].We are also conducting studies, but because the in vivo environment and the stability of compounds are interrelated, the results are often not what we expected.We hope that similar studies conducted by various groups will clarify this relationship.

Materials and Methods
The 211 At nuclide was acquired from RIKEN through a supply platform for short-lived radioisotopes.The 211 At nuclide was produced according to the 209 Bi(α, 2n) 211 At reaction and was separated from the Bi target using the dry distillation method.The separated 211 At was then dissolved in pure water [11].The 225 Ac nuclide was produced mainly by members of Tohoku University [34,35].Experiments after isolation were conducted at Osaka University.The intensity of the nuclides were measured using a germanium semiconductor detector (BE2020, Canberra, Mirion Technologies, Inc., Atlanta, GA.USA) and a γ-counter (Wizard 2 2480, PerkinElmer, Inc., Shelton, CT, USA).The samples treated with 225 Ac were maintained until radiative equilibrium was reached before the measurements were taken, according to a previous study [31].The PSMA-selected chemicals were labeled with each nuclide using previously reported procedures.These structures are shown in Figures 8 and 9.The PSMA-5 precursor was synthesized at the Peptide Institute.Inc. (Osaka, Japan) for the Shirakami Reaction.The labeling method is explained in detail in the next section.To evaluate the quality of the labeled chemicals, we used a previously reported method.
4.2.Nuclide Production and Chemical Labeling 4.2.1.Production and PSMA-5 Labeling of 211 At PSMA-5 was used as a highly selective PSMA compound to label with 211 At.This compound has been previously reported by Watabe et al.We determined this compound to be optimal based on the labeling efficiency and in vitro and in vivo experimental results [11].The method we used for labeling PSMA-5 was the "borono group-astatine exchange reaction", also known as the "Shirakami reaction" [36].

Production and PSMA-617 Labeling of 225 Ac
The labeling method for 225 Ac was established according to a previous study [37].The 225 Ac used for labeling was separated at the Tohoku University Institute for Materials Research and was then transported to Osaka University for use.PSMA-617 was dissolved in DMSO (1 mg/mL) and a 10% DMSO aqueous solution was prepared.This solution was mixed with 0.2 M AcONH 4 and 10% Ascorbic acid and was incubated in 80 • C for 2 h.After measuring the dose using a Curie meter (ICG-8; ALOKA, Ltd., Tokyo, Japan), the quality was confirmed using electrophoresis and it was then used in experiments.

Cell Culture
The PC3 and LNCaP cells were obtained from RIKEN and ATCC cell banks, respectively.The cells were maintained in RPMI1640 (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical).Sodium pyruvate (Fujifilm Wako Pure Chemical) was added to the LNCaP culture medium at a concentration of 1%.The cells were maintained using trypsin-EDTA (Fujifilm Wako Pure Chemical), according to standard methods.Both cell lines were in the logarithmic growth phase at the time of experimental preparation.

Evaluation of Cell Viability
Two days before treatment, the cells were seeded in 1 × 10 4 cells/mL in 96-well culture plates.The cell numbers were measured using TC-20 TM (Bio-Rad Laboratories, Inc., Hercules, CA, USA).After 1 h of treatment, the cells were cultured for three days.Cell viability was evaluated using a cell counting kit-8 (Dojin, Kumamoto, Japan), according to the manufacturer's protocol.The absorbance was measured at 450 nm using a MultiSkan FC (Thermo Fisher Scientific).

Colony Formation Assay
Both LNCaP (PSMA high ) and PC3 (PSMA low ) cells were seeded in 24-well plates and treated with labeled PSMAs at various concentrations.After 1 h of treatment, the cells were peeled and seeded at 1000 cells per well.Colony formation was observed for two weeks.After observation, the cells were fixed with 1% crystal violet solution.Colony formation was calculated by analyzing the coverage ratio of cells based on photographs using ImageJ software (https://imagej.net/downloads,accessed on 1 April 2023) [38].

Evaluation of DNA Double Strand Breaks
The cells were seeded in a culture chamber (WATSON, Tokyo, Japan).After treatment with labeled chemicals, the cells were fixed and did the immunofluorescence staining, according to a previously reported protocol [37].The cells were observed using a BZ-X810 microscope (KEYENCE, Osaka, Japan).The fluorescence intensities were analyzed using ImageJ software [38].Both LNCaP (PSMA high ) and PC3 (PSMA low ) cells were collected after 5, 30, 60, and 120 min of treatment, washed with PBS (-) three times, lysed using 0.1 N NaOH solutions, and collected into microtubes.The sample counts were measured using a γ-counter and their counts were corrected for the amounts of cell protein, according to a previous published protocol [39].We also measured the protein amounts in the cell suspension using a protein assay BCA kit (Fujifilm Wako Pure Chemical), according to the manufacture's protocol.The absorbance was measured at 570 nm using a MultiSkan FC.

Inhibition Assay
Based on previous research [40], the LNCaP cells were simultaneously treated with unlabeled chemicals (PSMA-617 or PSMA-5).For the inhibition assay, the cell uptake experiments and procedures were the same, except for treatment with the compound as an inhibitor.The amount of unlabeled compound added was more than 100 times that of the labeled compound.

Conclusions
Let us consider the respective absorbed doses of 211 At-PSMA-5 and 225 Ac-PSMA617 in the cell experiments.Since the cell treatment conditions were the same, it can be assumed that the masses of cells were the same.In this case, the energy value and the absorbed dose was proportional.In other words, it is thought that comparisons can be made in becquerel, considering the half-life.For example, assuming that there is no washout from the cells, considering the area under curve (AUC) for 3 days, 225 Ac-PSMA-617 is about 6.3 times more powerful than 211 At-PSMA-5.Considering that 225 Ac emits four α rays, it is about 25 times more powerful.However, the amount of uptake itself is halved (Figure 6), which is approximately 12 times as much.There is no more than a 10-fold difference in their effects on the cells (Figure 4).Therefore, it can be said that PSMA-5 is superior as a compound.It might be better to consider the stability of PSMA-5 in more detail, but we consider that this stability might be increased by inducing three unnatural amino acids (Figure 9).PSMA-5 is expected to be sufficient for clinical use.
It might be said that 211 At-PSMA-5 is more easily taken up by LNCaP cells than 225 Ac-PSMA-617, suggesting that PSMA-5 might be superior as a compound (Figures 6 and 7).On the other hand, whether it is better in clinical practice may depend on the balance between effectiveness and potential side effects, aside from the availability of the nuclide.Although more studies may be needed, our findings indicate the possibility of completing treatment regimens faster because of the shorter half-life of 211 At-PSMA-5 for repeated administration compared to 225 Ac-PSMA-617.

Figure 1 .
Figure 1.Cell viability following PSMA-targeted radioligand administration in LNCaP (PSMA high ) and PC3 (PSMA low ) cell lines.Diamonds represent PC3 cell lines and circles represent LNCaP cell lines.The mean ± S.E of the triplicate results is shown.

Figure 1 .
Figure 1.Cell viability following PSMA-targeted radioligand administration in LNCaP (PSMA high ) and PC3 (PSMA low ) cell lines.Diamonds represent PC3 cell lines and circles represent LNCaP cell lines.The mean ± S.E of the triplicate results is shown. ).
Int. J. Mol.Sci.2024,25,  x FOR PEER REVIEW 5 of 14shown in Figure4.Green fluorescence indicates the presence of γH2AX, a marker of DSB.Focies of γH2AX were strongly induced by225 Ac-PSMA-617 (Figure5A), and many focies were induced by 211 At-PSMA-5 (Figure5B).When comparing their effects on (a) PC3 and (b) LNCaP cells, more foci were found in the LNCaP cells, indicating that many DSBs appeared in the LNCaP cells.

Figure 4 . 5 Figure 5 .
Figure 4. Percentage of cells with >5γH2AX foci/cells.(A) The white bar represents PC3 (PSMA low ) cells treated with 225 Ac-PSMA-617, the gray bar represents LNCaP (PSMA high ) cells treated with 225 Ac-PSMA-617.(B) the white bar represents PC3 cells treated with 211 At-PSMA-5, and the gray bar represents LNCaP cells treated with 211 At-PSMA-5.The mean ± S.E of the triplicate results is shown.

Figure 4 .
Figure 4. Percentage of cells with >5γH2AX foci/cells.(A) The white bar represents PC3 (PSMA low ) cells treated with 225 Ac-PSMA-617, the gray bar represents LNCaP (PSMA high ) cells treated with 225 Ac-PSMA-617.(B) the white bar represents PC3 cells treated with 211 At-PSMA-5, and the gray bar represents LNCaP cells treated with 211 At-PSMA-5.The mean ± S.E of the triplicate results is shown.

Figure 4 . 5 Figure 5 .
Figure 4. Percentage of cells with >5γH2AX foci/cells.(A) The white bar represents PC3 (PSMA low ) cells treated with 225 Ac-PSMA-617, the gray bar represents LNCaP (PSMA high ) cells treated with 225 Ac-PSMA-617.(B) the white bar represents PC3 cells treated with 211 At-PSMA-5, and the gray bar represents LNCaP cells treated with 211 At-PSMA-5.The mean ± S.E of the triplicate results is shown.

Figure 6 .
Figure 6.Uptake of the cell lines.(a) PC3 (PSMA low ) and LNCaP (PSMA high ) cells treated with 225 Ac-PSMA-617.(b) PC3 and LNCaP cells treated with 211 At-PSMA-5.The y axis indicates the uptake of radioactivity (% of dose/mg protein).The mean ± S.E of the triplicate results is shown.

Figure 6 .
Figure 6.Uptake of the cell lines.(a) PC3 (PSMA low ) and LNCaP (PSMA high ) cells treated with 225 Ac-PSMA-617.(b) PC3 and LNCaP cells treated with 211 At-PSMA-5.The y axis indicates the uptake of radioactivity (% of dose/mg protein).The mean ± S.E of the triplicate results is shown.

Figure 6 .
Figure 6.Uptake of the cell lines.(a) PC3 (PSMA low ) and LNCaP (PSMA high ) cells treated with 225 Ac-PSMA-617.(b) PC3 and LNCaP cells treated with 211 At-PSMA-5.The y axis indicates the uptake of radioactivity (% of dose/mg protein).The mean ± S.E of the triplicate results is shown.

Table 2 .
Comparison of physical properties between 211 At and 225 Ac.

Table 2 .
Comparison of physical properties between 211 At and 225 Ac. 2