Introduction
Prostate cancer (PCa) is one of the most common cancers globally, with approximately 1.41 million new cases and 370,000 deaths in 2020 [1]. The five-year survival rate for localized prostate cancer is nearly 100%, but 10–15% of patients have metastases at diagnosis, and the five-year survival rate for metastatic PCa is only 30% [2, 3]. After androgen deprivation therapy, metastatic PCa progresses to metastatic castration-resistant prostate cancer (mCRPC). Despite various treatments, such as androgen receptor-axis-targeted agents (abiraterone and enzalutamide) [4, 5], poly ADP-ribose polymerase inhibitors (olaparib) [6], chemotherapies (docetaxel and cabazitaxel) [7, 8], and radiotherapy (radium-223) [9], that prolong prognosis, mCRPC remains incurable. Therefore, novel additional treatment options are needed for mCRPC. Prostate-specific membrane antigen (PSMA) radioligand therapy (RLT) uses radionuclide-labelled ligands that bind to PSMA, the protein highly expressed on PCa cell membranes. These radionuclides deliver cytotoxic radiation (alpha- or beta-emitters) at short range, offering effective treatment with minimal side effects [10]. In 2021, [177Lu]Lu-PSMA, PSMA-RLT using the beta-emitter Lutetium-177 (177Lu) developed in Germany, was approved by the US Food and Drug Administration because it improved the overall survival of mCRPC patients [11‐13], though 54% of patients failed to achieve a sufficient prostatic-specific antigen response [14]. This insufficient therapeutic effect was attributed to incomplete DNA damage caused by beta-emitters, focusing attention on alpha-emitter therapy, which has higher energy and induces efficient DNA double-strand breaks [15]. [225Ac]Ac-PSMA-617, PSMA-RLT using the alpha-emitter actinium-225 (225Ac), has superior anti-tumour effects in patients with [177Lu]Lu-PSMA treatment failure [16‐18]. Unfortunately, 225Ac is only produced at 75 GBq per year from scarce thorium-229, and it is thus difficult to satisfy the global demand at this time [19]. Furthermore, the half-life of 225Ac is 10 days, and the four alpha-emitting daughter radionuclides released from the ligand during initial decay potentially accumulate in non-target tissue (off-target effects) [20]. Astatine-211 (211At) is another alpha-emitter that potentially resolves these issues with 225Ac. 211At belongs to the halogen groups, has a short half-life of 7.2 h, and releases only one daughter nuclide during decay, thus avoiding off-target effects because of daughter nuclides [21]. 211At is producible using a cyclotron with readily available and inexpensive bismuth-209. 211At also emits X-rays during the decay of polonium-211 (211Po), enabling in vivo imaging. Because of these characteristics, 211At is considered a promising nuclide for targeted alpha therapy (TAT) [22, 23]. However, the binding of 211At to conventional PSMA ligands is unstable in vivo, with easy deastatination, and 211At accumulates in non-target organs such as stomach, thyroid and salivary glands due to its molecular characteristics of belonging to the halogen groups [24, 25]. This accumulation of 211At in non-target organs can lead to side effects and potentially attenuates the therapeutic effect, because distribution to target tissue is decreased. Therefore, a PSMA ligand that binds stably to 211At in vivo is needed [25, 26].
To reduce the off-target effects of 211At, Suzuki et al. developed a neopentyl-glycol (NpG) structure that stabilizes 211At labelling [27]. This structure enhanced in vivo stability against deastatination due to its steric arrangement. In addition, they created neopentyl-glycol-PSMA (NpG-PSMA), a structural analogue to [18F]F-PSMA-1007 (Fig. 1a), using the NpG structure at the 211At labelling site [28, 29] (Fig. 1b, c). [211At]At-NpG-PSMA shows stable 211At binding with simple in vivo biodistribution, suggesting its potential for a high anti-tumour effect against PCa and minimal side effects. However, the biodistribution, stability, anti-tumour effect, and safety of [211At]At-NpG-PSMA have not been evaluated in detail in PCa animal models. In this study, [211At]At-NpG-PSMA was administered to PSMA-positive xenograft model mice to assess the detailed biodistribution and anti-tumour effect. In addition, relatively detailed histopathological analyses and blood tests were performed to evaluate the safety of [211At]At-NpG-PSMA, and discuss its potential clinical application.
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Fig. 1
Molecular structures: a [18F]F-PSMA-1007, b NpG-PSMA, c [211At]At-NpG-PSMA
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Materials and methods
Production of 211At and radiosynthesis of [211At]At-NpG-PSMA
211At was produced through the 209Bi (α, 2n) 211At reaction using the Sumitomo Multipurpose Cyclotron (MP-30, Sumitomo Heavy Industries, Ltd., Tokyo, Japan) in the Advanced Clinical Research Center at Fukushima Medical University School of Medicine, Japan [25]. 211At was isolated from the irradiated target using the dry distillation procedure and recovered in acetonitrile. NpG-PSMA was manufactured at Chiba University (Japan). [211At]At-NpG-PSMA was synthesized using a nucleophilic substitution reaction between the neopentyl-glycol structure and 211At, followed by hydrolysis with acid [29]. The radiochemical yield of [211At]At-NpG-PSMA was 41% (n = 3, decay-corrected). The radiochemical purity of the product was over 99.9% (Supplementary Fig. S1). The molar activity of the radioligand was 161 MBq/nmol. The structures of NpG-PSMA and [211At]At-NpG-PSMA are shown in Fig. 1. The radiochemical purity of [211At]At-NpG-PSMA was determined using reverse-phase radio-high-performance liquid chromatography (LC-20AD, Shimadzu, Kyoto, Japan).
Cell culture
PSMA-positive (PSMA+) PC-3 PIP cells and PSMA-negative (PSMA−) PC-3 flu cells were provided by Dr. Xinning Wang and Dr. Warren Heston (Case Western Reserve University, Cleveland, OH, USA) and were maintained as described [30‐32]. The androgen-independent PC-3 cell line was originally derived from a human mCRPC bone metastasis. The expression levels of PSMA in these cells were confirmed (Supplementary Fig. S2).
Cellular uptake analysis
PSMA + PC-3 PIP and PSMA-PC-3 flu cells were seeded at 2 × 105 cells/well in a 12-well plate and cultured for 24 h. The medium was then replaced with medium containing [211At]At-NpG-PSMA (20 kBq/200 µL) and incubated for 4 h at 37 °C. Non-specific binding was assessed with 100 µM 2-(phosphonomethyl) pentanedioic acid (2-PMPA), which inhibits the carboxypeptidase activity of PSMA. The medium was removed, and the cells were washed twice with phosphate-buffered saline (2 ml×2). Subsequently, the cells were lysed in 1 M NaOH (2 mL×2) for 10 min, and radioactivity was measured using a gamma-counter (2480 Wizard2, Perkin Elmer, Shelton, CT, USA). The protein concentration of the cell lysates was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA).
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Preparation of a PSMA + PC-3 PIP xenograft model
The experimental procedures and animal care were approved by the Fukushima Medical University School of Medicine Institute of Animal Care and Use Committee. Eight-week-old male athymic mice (BALB/c nu/nu; Japan-SLC, Shizuoka, Japan) were anesthetized with isoflurane and subcutaneously injected with 2.5 × 106 PSMA + PC-3 PIP cells into the flank.
Biodistribution study
Biodistribution study by gamma-counter
PSMA + PC-3 PIP xenograft mice (23.62 ± 0.90 g, 5 mice per time point) were injected intravenously with 100 µL of saline containing [211At]At-NpG-PSMA (0.076 ± 0.005 MBq). The mice were sacrificed at 5 min, and at 1, 3 and 24 h post-injection. Tissues were excised, weighed, and counted for 211At using a gamma-counter.
Biodistribution study by X-ray camera imaging
XCam-CdTe (iMAGINE-X, Japan) [33] was used to capture X-rays in the 75–95 keV range generated by 211Po during 211At decay. Imaging was performed 1, 3 and 24 h after administration of [211At]At-NpG-PSMA (1.06 ± 0.09 MBq, n = 3). Tumour radioactivity was measured using the free software Horos (Ver. 3.3.6, USA). The radioactivity measured at the three time points was integrated to calculate the tumour absorbed dose (Gy/MBq). Detailed calculation methods are described in Supplementary Fig. S3.
Monitoring tumour volume and body weight after [211At]At-NpG-PSMA treatment
Seven days post-implantation of PSMA + PC-3 PIP cells, when tumours reached about 50 mm3, mice (23.59 ± 0.96 g) received intravenous [211At]At-NpG-PSMA (0.32 ± 0.02, 1.00 ± 0.10, 1.93 ± 0.02 MBq; 5 mice per dose) or saline (5 mice). Tumour size and body weight were measured for 35 days. Tumour volume was calculated with the formula: tumour volume (mm3) = (length × width2)/2. Mice were euthanized under deep isoflurane anaesthesia when their body weight decreased over 20% from the day of administration, they exhibited signs of intolerable suffering, or their tumour volume exceeded 800 mm3. Statistical endpoints were defined as a body weight reduction of more than 20%, a tumour volume increase of more than 10-fold from the day of drug administration (Day 0), or a tumour volume exceeding 500 mm³.
Evaluation of side effects on day 3 post-administration
To assess temporary toxicity, xenograft mice were treated with [211At]At-NpG-PSMA (0.49 ± 0.02, 1.09 ± 0.03 and 1.79 ± 0.06 MBq; 3 mice per group) or saline (3 mice). After 3 days, mice were euthanized, blood samples were collected, and the thyroid, salivary glands, stomach, small intestine, spleen, kidney, femoral bone and PSMA + PC-3 PIP tumours were excised.
Evaluation of side effects on day 35 post-administration
Proteinuria was qualitatively analysed using urinalysis test strips (Multistix Ames 2820, Siemens Healthcare, Japan). After 35 days, blood samples were collected, and tissues were excised.
Pathological analysis
Tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 3 μm for haematoxylin and eosin (H&E) staining. Kidney Sect. (1.5 μm thick) were stained with periodic acid-Schiff (PAS) stain and assessed for glomerular size and nuclear atypia of tubular epithelium, a marker of regenerative tubules [34, 35]. The proportion of glomeruli that had decreased in size by 50% or more was compared to controls. Nuclear atypia was counted across 10 high-power fields (×200 magnification), and the average per field was calculated. PSMA + PC-3 PIP tumour Sect. (3 μm thick) were immunostained for Ki-67 with anti-Ki67 antibody [SP6] ab16667 (Abcam, Cambridge, UK). Histological evaluations were performed by a pathologist. The Ki-67 positivity rate was evaluated using the open-source software Qupath (Version 0.5.1-x64, University of Edinburgh, UK) [36].
Haematological analysis
Haemoglobin (Hb), white blood cell (WBC) counts and platelet (PLT) counts of collected blood samples were measured using a MEK-6550 Celltac α (NIHON KOHDEN, Tokyo, Japan). Plasma obtained by centrifuging the blood was analysed for kidney function markers such as blood urea nitrogen (BUN), creatinine and potassium using a DRI-CHEM NX500VIC (Fujifilm, Tokyo, Japan).
Statistical analysis
Data are presented as mean ± standard deviation (SD) values. In the anti-tumour effect study, tumour volumes were normalized to the volume on the day of drug administration (set as Day 0) to calculate relative tumour volume. Changes in relative tumour volume among groups were analysed by one-way ANOVA with repeated measures. Comparisons between groups in both the cellular uptake assay and anti-tumour effect study were made using Dunnett’s multiple comparison test. A p value of < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS (Version 27.0, IBM Japan, Japan).
Results
Cellular uptake analysis
In the cellular uptake analysis, [211At]At-NpG-PSMA showed significantly higher uptake in PSMA + PC-3 PIP cells than in PSMA-PC-3 flu cells and 2-PMPA-inhibited PSMA + PC-3 PIP cells (p < 0.01; Fig. 2). These results indicated that the cellular uptake of [211At]At-NpG-PSMA was dependent on the expression of PSMA on PCa cells and was suppressed when PSMA carboxypeptidase was blocked.
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Fig. 2
Cellular uptake of [211At]At-NpG-PSMA in PSMA + PC-3 PIP (high expression of PSMA) and PSMA-PC-3 flu (high expression of PSMA) cells
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Biodistribution study
Biodistribution study by gamma-counter
Figure 3 shows the biodistribution of [211At]At-NpG-PSMA in PSMA + PC-3 PIP xenograft mice. Details are provided in Supplementary Table S1. Tumour uptake peaked at 1 h (48.14 ± 11.69%ID/g) and remained until 24 h (41.08 ± 7.18%ID/g). Blood and bone uptake were rapidly cleared within 1 h (0.82 ± 0.27 and 0.05 ± 0.01%ID/g, respectively). Uptake was low in the stomach, salivary glands, and thyroid gland at 1 h (1.31 ± 0.33, 1.62 ± 0.29%ID/g and 0.44 ± 0.10%ID; respectively) and further decreased at 3 h (0.71 ± 0.12, 0.88 ± 0.10%ID/g and 0.28 ± 0.20%ID; respectively). In contrast, high uptake was observed in the spleen at 1 h (13.64 ± 2.16%ID/g). Renal uptake peaked at 1 h (251.23 ± 34.51%ID/g), decreased by 3 h (200.92 ± 8.11%ID/g), and was almost completely excreted by 24 h (0.66 ± 0.16%ID/g). At 24 h, uptake was almost completely cleared from normal organs except the tumour.
Fig. 3
Biodistribution of [211At]At-NpG-PSMA in PSMA + PC-3 PIP xenograft model (BALB/c nu/nu mice) The values are presented as percentage injected radioactivity dose per gram (%ID/g), except the thyroid values, which are percentage injected radioactivity dose (%ID)
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Biodistribution study by X-ray camera imaging
Representative X-ray camera images of the non-invasive biodistribution study are shown in Fig. 4a. Tumour uptake reached 70.9 kBq at 1 h and was maintained at 6.9 kBq at 24 h. Renal uptake reached 300 kBq at 1 h, followed by urinary excretion into the bladder, and was almost not observed in the kidneys at 24 h (Fig. 4b). The absorbed dose to the tumour was calculated to be 7.4 Gy/MBq.
Fig. 4
a X-ray camera images of PSMA + PC-3 PIP xenograft mice at 1, 3, and 24 h after [211At]At-NpG-PSMA (1.06 MBq) administration. b Region of interest analysis of radioactivity in the kidney and tumour
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Anti-tumour effects of [211At]At-NpG-PSMA
Tumour volume reduction after treatment with [211At]At-NpG-PSMA
On day 0, there was no significant difference in absolute tumour volume between the control and treated groups (p = 0.790). In the control group, rapid tumour growth was observed, with the relative tumour volume increasing 10.6 ± 4.9-fold by Day 18 (p < 0.01). In contrast, the treated groups showed a dose-dependent anti-tumour effect. Tumour suppression was maintained up to day 18 in the 0.32 MBq group (3.6 ± 2.6-fold increase, p = 0.091), up to day 21 in the 1.00 MBq group (2.8 ± 1.8-fold increase, p = 0.077) and up to day 35 in the 1.93 MBq group (2.6 ± 2.7-fold increase, p = 0.190). After day 5, tumour growth was significantly suppressed in all treatment groups compared to controls (p < 0.01; Fig. 5a). By day 15, the tumour volume in the control group had increased by 796.0 ± 437.6%, whereas the 0.32, 1.00 and 1.93 MBq groups had increased by 161.0 ± 213.4%, -76.4 ± 19.2% and − 59.5 ± 41.6%, respectively (p < 0.01 vs. control; Fig. 5b, c). In one of the five mice injected with 1.93 MBq, the tumour disappeared until around day 35. The absolute tumour volumes for all mice are shown in Supplementary Fig. S4.
Fig. 5
Anti-tumour effects of [211At]At-NpG-PSMA on PSMA + PC-3 PIP xenograft mice. a Relative tumour volume change after treatment with [211At]At-NpG-PSMA (0.32, 1.00, and 1.93 MBq; 5 mice per group) and control (0 MBq; 5 mice). b Representative images of mice on day 15 after treatment with [211At]At-NpG-PSMA (0.32, 1.00, and 1.93 MBq) and control (0 MBq). Dashed circles indicate tumours. c Tumour volume percentage change on day 15 after treatment with [211At]At-NpG-PSMA (0.32, 1.00, and 1.93 MBq; 5 mice per group) and control (0 MBq)
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PSMA + PC-3 PIP tumour cell changes
After 3 days of [211At]At-NpG-PSMA treatment, dose-dependent necrosis and a decrease in Ki-67-positive cells were observed in PSMA + PC-3 PIP tumour (Fig. 6, Supplementary Table S2).
Fig. 6
Temporal histological changes in PSMA + PC-3 PIP tumours after administration of [211At]At-NpG-PSMA (0.49, 1.09, and 1.79 MBq; 3 mice per group) and saline (3 mice). Representative images of H&E-stained sections (left) and Ki-67-stained sections (right) of PSMA + PC-3 PIP tumours on days 3 after administration
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Evaluation of side effects
Body weight change
By day 3 post-administration, the control and 0.32 and 1.00 MBq groups experienced less than 5% weight loss, whereas the 1.93 MBq group showed less than 10% loss; however, all groups later recovered. On day 15, no significant weight differences were observed between the control and treated groups (p = 0.071; Fig. 7). In the control group, weight loss due to cachexia was observed with tumour growth.
Fig. 7
Body weight changes after treatment with [211At]At-NpG-PSMA (0.32, 1.00, and 1.93 MBq; 5 mice per group) and control (0 MBq; 5 mice)
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Side effects on day 3 post-administration
Non-target organs showed no histological abnormalities (Fig. 8). Although WBC counts tended to decrease with higher doses, the changes were not significant and within the normal range (Supplementary Fig. S5).
Fig. 8
Histological analysis on day 3 post-administration for the control group receiving saline and the treatment groups receiving 0.49, 1.09, and 1.79 MBq of [211At]At-NpG-PSMA. Representative slices stained with H&E (3 μm thick) excluding the kidneys, and representative kidney slices stained with PAS (1.5 μm thick) are shown
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Side effects on day 35 post-administration
Histological abnormalities were only observed in the kidneys. The 0.32 MBq group showed no kidney abnormalities, whereas the 1.00 MBq group exhibited mild proteinaceous casts and nuclear atypia at approximately 1.3/HPF. The 1.93 MBq group had moderate proteinaceous casts and nuclear atypia at approximately 5.3/HPF, with 2% of glomeruli showing shrinkage (Fig. 9, Supplementary Table S3). Creatinine levels were 0.05 mg/dl in the control group and increased significantly to 0.11 mg/dl in the 1.93 MBq group, which was still within the normal range (< 0.12 mg/dl). BUN did not increase in any groups (Supplementary Fig. S6). Although proteinuria did not increase in the control and 0.32 MBq groups, it increased temporarily in the 1.00 MBq group and then improved. In contrast, severe proteinuria persisted in the 1.93 MBq group (Supplementary Table S4). The severity of proteinuria and histological changes correlated with dose.
Fig. 9
Histological analysis on day 35 post-administration for the control group receiving saline and the treatment groups receiving 0.32, 1.00, and 1.93 MBq of [211At]At-NpG-PSMA. Representative slices stained with H&E (3 μm thick) excluding the kidneys, and representative kidney slices stained with PAS (1.5 μm thick) are shown. The asterisks in the kidney sections indicate protein casts in tubules, the arrows indicate nuclear atypia in regenerative tubules, and the arrowheads represent shrunken glomeruli
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Discussion
[211At]At-NpG-PSMA showed high and prolonged tumour uptake in the biodistribution study and demonstrated a high anti-tumour effect. In contrast, uptake was low in organs where it accumulated due to the halogen characteristics of astatine, suggesting low deastatination. In the kidney, high early accumulation was observed, but moderate clearance was observed. It showed early clearance from the blood and bone marrow. [211At]At-NpG-PSMA was demonstrated to have high in vivo stability, anti-tumour effects, and few side effects.
It has been reported that 211At tends to accumulate in the thyroid, salivary glands, and stomach due to its halogen properties, and that the uptake in these organs reflects the accumulation of deastatinated 211At from 211At-labelled RLT drugs [24, 25]. Two previous astatine-labelled RLT drugs, [211At]PSMA-5 and 211At-Lu-3, have been reported [21, 37]. With [211At]PSMA-5, a 211At-labelled PSMA RLT drug with benzene ring derivatives, persistent uptake in thyroid, salivary glands, and stomach from 3 to 24 h (11, 11, and 5%ID/g at 3 h; respectively), which indicated deastatination, was observed in an in vivo study [37]. Deastatination potentially induces off-target effects and adverse events. In contrast, [211At]At-NpG-PSMA showed lower uptake in the thyroid, salivary glands, and stomach at 1 h (0.44%ID, 1.62%ID, and 1.3%ID/g, respectively), with most uptake diminished by 3 h (0.28%ID, 0.88%ID, and 0.71%ID/g, respectively). Furthermore, no histological abnormalities were observed in these organs, indicating stability of [211At]At-NpG-PSMA against deastatination in vivo. These results suggested that [211At]At-NpG-PSMA is more stable than [211At]PSMA-5 and potentially reduces the off-target effects by deastatination. In contrast, 211At-Lu-3 showed fast drug clearance and high stability, similar to [211At]At-NpG-PSMA. 211At-Lu-3 incorporates non-radioactive lutetium and a chelate structure in its design, which enhances its in vivo stability and improves renal clearance (90 and 2%ID/g at 1 and 4 h, respectively). However, as well as rapid clearance in the kidney, 211At-Lu-3 showed rapid clearance in the tumour (30, 17, and 10%ID/g at 1, 4, and 24 h respectively). Rapid clearance in the tumour results in a decrease in absorbed dose, which potentially leads to a lower anti-tumour effect [38]. In contrast, [211At]At-NpG-PSMA maintained high and prolonged tumour uptake (48, 42, and 41%ID/g at 1, 3, and 24 h, respectively). Although it is difficult to directly compare the anti-tumour effect of [211At]At-NpG-PSMA and 211At-Lu-3, the high and prolonged tumour uptake of [211At]At-NpG-PSMA indicated a higher absorbed dose in tumour, leading to a higher anti-tumour effect. In fact, administration of [211At]At-NpG-PSMA to xenograft model mice showed a dose-dependent anti-tumour effect. Even at doses as low as 0.32 MBq, [211At]At-NpG-PSMA exhibited anti-tumour effects. Pathological analysis showed tumour cell necrosis and a decrease in Ki67-positive cells.
PSMA-RLT drugs generally show renal uptake [39], which is attributed to PSMA expression in the renal proximal tubules [40]. This uptake potentially leads to renal impairment and dysfunction and results in dose-limiting toxicity. [211At]At-NpG-PSMA also showed high initial renal uptake, consistent with the properties of PSMA-RLT drugs such as [211At]PSMA-5. Renal uptake of [211At]At-NpG-PSMA was cleared faster than [211At]PSMA-5 and slower than 211At-Lu-3. This led to histopathological observations of mild renal toxicity. Although mild regenerative tubules (nuclear atypia 1.3/HPF) were observed in mice receiving 1.00 MBq, this was considered reversible, because creatinine was not elevated, and proteinuria was temporary. However, in mice receiving 1.93 MBq, irreversible proteinuria, elevated creatinine, and delayed radiation-induced tissue changes (nuclear atypia 5.3/HPF, glomerular shrinkage 2%) [34, 35] were observed. These results indicated that the 1.93-MBq dose potentially causes irreversible renal damage. Therefore, renal dysfunction is considered the dose-limiting toxicity of [211At]At-NpG-PSMA.
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211At-NpG-PSMA shows similar tumour uptake to 225Ac-PSMA-617, a PSMA-TAT, suggesting comparable antitumour effects. On the other hand 211At-NpG-PSMA exhibits higher renal uptake compared to 225Ac-PSMA-617, indicating potential nephrotoxicity, which warrants caution [41]. However it is difficult to directly compare anti-tumour effect and organtoxicity between 211At-NpG-PSMA and 225Ac-PSMA-617.
In the present study, [211At]At-NpG-PSMA was rapidly cleared from the blood and bone, and its toxicity to blood and bone marrow was minimal and recoverable. That suggests a potential to reduce haematological toxicity in clinical practice. However, since the present study did not evaluate a metastatic model, careful monitoring is necessary when administering it to mCRPC patients with diffuse bone metastases in clinical settings.
Species differences exist in the biodistribution of PSMA-RLT drugs [42]. In humans, uptake of PSMA ligands to salivary glands is significantly higher, and salivary gland damage has been identified as a dose-limiting toxicity in treatments using [225Ac]Ac-PSMA-617 [43, 44]. Although the mechanisms of PSMA RLT drug uptake in human salivary glands are unknown, nonspecific uptake pathways have been suggested [43, 45]. In the present study, [211At]At-NpG-PSMA showed very low mouse salivary gland uptake. However, due to differences in biodistribution between species, careful monitoring is required for clinical application. In addition, [211At]At-NpG-PSMA showed high uptake in the spleen (13.64% ID/g at 1 h); it is considered to be non-specific uptake commonly observed with PSMA-RLT drugs [46, 47] and not deastatination. Nonspecific uptake in the spleen may require caution in clinical applications.
Currently, beta-emitting 177Lu is widely used in PSMA-RLT and has shown high therapeutic efficacy [12]. In addition, alpha-emitting 225Ac used in PSMA-RLT is promising because it has higher energy and a shorter range compared to beta-emitters, allowing for more effective tumour cell destruction [18]. However, the availability of 225Ac is limited, which poses a significant constraint [19]. In this regard, 211At is producible by cyclotrons, facilitating its easy availability [23]. The [211At]At-NpG-PSMA with a neopentyl-glycol structure has addressed the instability of astatine in vivo, with high stability and reduced uptake by non-target organs. Furthermore, [211At]At-NpG-PSMA showed rapid clearance from normal tissues while maintaining high accumulation in tumours, indicating optimised pharmacokinetics. This resulted in a high anti-tumour effect and minimal toxicity to normal tissues. [211At]At-NpG-PSMA showed a balance between efficacy and safety, and is considered to be a promising therapeutic option for the treatment of mCRPC in the future.
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Limitations
This study had several limitations. In prostate cancer, the heterogeneity of PSMA expression has been noted as a potential factor that could compromise the treatment effect [48]. The PC-3 PIP cells used in this study artificially express PSMA strongly and uniformly, and their tumour uptake may differ between the biodistribution in this study and real-world clinical practice. For clinical application, it would be essential to assess PSMA expression individually using PSMA-PET to identify patients suitable for treatment. Furthermore, this study was designed to evaluate the anti-tumour effects and side effects of a single administration of [211At]At-NpG-PSMA. The toxicity associated with repeated dosing and the long-term side effects require further investigation in future studies.
Conclusion
[211At]At-NpG-PSMA is stable in vivo, has high tumour and low off-target accumulation, and shows fast clearance in a PCa xenograft model. Therefore, a high anti-tumour effect and lower side effects are expected. These findings suggest that [211At]At-NpG-PSMA has the potential to become a new therapeutic agent for PSMA-TAT in mCRPC.
Acknowledgements
The authors would like to thank Xinning Wang, Warren D.W. Heston, and Gopi (Case Western Reserve University) for providing the PSMA + PC-3 PIP and PSMA-PC-3 flu cells. They also thank Dr. Shinichiro Takeda (iMAGINE-X, Japan) for developing the XCam-CdTe and assisting with its installation and use in this study. Finally, the authors would like to thank H. Hiraki, E. Watanabe, and Y. Kuroki for their technical assistance. The authors would like to thank Xinning Wang, Warren D.W. Heston, and Gopi (Case Western Reserve University) for providing the PSMA + PC-3 PIP and PSMA-PC-3 flu cells. They also thank Dr. Shinichiro Takeda (iMAGINE-X, Japan) for developing the XCam-CdTe and assisting with its installation and use in this study. Finally, the authors would like to thank H. Hiraki, E. Watanabe and Y. Kuroki for their technical assistance.
Declarations
Ethical approval
Animal studies were conducted in accordance with the institutional guidelines approved by the Fukushima Medical University School of Medicine Institute of Animal Care and Use Committee (approval number: 2024-046).
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
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