Targeted α-therapy using astatine (211At)-labeled PSMA1, 5, and 6: a preclinical evaluation as a novel compound

Purpose Targeted α-therapy (TAT) for prostate-specific membrane antigen (PSMA) is a promising treatment for metastatic castration-resistant prostate cancer (CRPC). Astatine is an α-emitter (half-life=7.2 h) that can be produced by a 30-MeV cyclotron. This study evaluated the treatment effect of 211At-labeled PSMA compounds in mouse xenograft models. Methods Tumor xenograft models were established by subcutaneous transplantation of human prostate cancer cells (LNCaP) in NOD/SCID mouse. [211At]PSMA1, [211At]PSMA5, or [211At]PSMA6 was administered to LNCaP xenograft mice to evaluate biodistribution at 3 and 24 h. The treatment effect was evaluated by administering [211At]PSMA1 (0.40 ± 0.07 MBq), [211At]PSMA5 (0.39 ± 0.03 MBq), or saline. Histopathological evaluation was performed for the at-risk organs at 3 and 6 weeks after administration. Results [211At]PSMA5 resulted in higher tumor retention compared to [211At]PSMA1 and [211At]PSMA6 (30.6 ± 17.8, 12.4 ± 4.8, and 19.1 ± 4.5 %ID/g at 3 h versus 40.7 ± 2.6, 8.7 ± 3.5, and 18.1 ± 2.2%ID/g at 24 h, respectively), whereas kidney excretion was superior in [211At]PSMA1 compared to [211At]PSMA5 and [211At]PSMA6. An excellent treatment effect on tumor growth was observed after [211At]PSMA5 administration. [211At]PSMA1 also showed a substantial treatment effect; however, the tumor size was relatively larger compared to that with [211At]PSMA5. In the histopathological evaluation, regenerated tubules were detected in the kidneys at 3 and 6 weeks after the administration of [211At]PSMA5. Conclusion TAT using [211At]PSMA5 resulted in excellent tumor growth suppression with minimal side effects in the normal organs. [211At]PSMA5 should be considered a new possible TAT for metastatic CRPC, and translational prospective trials are warranted. Supplementary Information The online version contains supplementary material available at 10.1007/s00259-022-06016-z.

However, prostate cancer finally becomes resistant as hormone-resistant cells can survive and have been selected during treatment, which is called castration-resistant prostate cancer (CRPC) [3]. The prognosis of metastatic CRPC is poor, and the median survival period is 9-13 months [4]. Although new androgen receptor inhibitors or chemotherapy using docetaxel or cabazitaxel can be provided to patients with non-metastatic or metastatic CRPC, some of them are progressive with a short doubling time of serum markers of prostate-specific antigen [5,6].
The prostate-specific membrane antigen (PSMA) is an excellent target for theranostics. PSMA-positron emission tomography (PET) is useful for the detection of recurrent lesions, especially in biochemical recurrence after surgery or radiation therapy [7,8]. PSMA uptake in recurrent lesions is usually remarkably high, and small metastases can be detected, which are difficult to detect using conventional computed tomography and bone scintigraphy [9]. For therapeutic applications, [ 177 Lu]PSMA therapy has been recently approved by the US Food and Drug Administration in 2022 [10]. It significantly prolongs the overall survival of patients with metastatic CRPC compared with standard treatment alone [11]. Targeted α-therapy for PSMA is a promising therapy for metastatic CRPC [12]. [ 225 Ac]PSMA is significantly effective even in refractory cases of [ 177 Lu]PSMA therapy, despite the need to balance its dose due to its adverse effect of xerostomia [13].
[ 225 Ac] has attracted attention for its labeling utility as a theranostic companion with [ 68 Ga] and [ 177 Lu]. However, its supply remains limited worldwide because its production requires nuclear fuel materials ([ 229 Th] or [ 232 Th]) or rare radioisotopes ([ 226 Ra]) [14]. Astatine is an α-emitter (half-life = 7.2 h) that can be produced by a 30 MeV cyclotron with a reasonable cost and labeled to small molecules and peptides [15]. Sodium astatine ([ 211 At]NaAt) and labeled amino acid analogs ([ 211 At]PA and [ 211 At]AAMT) are useful for the treatment of thyroid cancer, malignant glioma, pancreatic cancer, and malignant melanoma [16][17][18]. An investigator-initiated clinical trial using [ 211 At]NaAt in patients with refractory thyroid cancer (Clini calTr ials. gov Identifier: NCT05275946) is in progress [19]. We also developed a novel labeling method using the substitution reaction of 211 At with dihydroxyboryl groups [20]. Moreover, we developed a newly designed precursor based on the structure of [ 18 F]PSMA-1007, which we believe is suitable for 211 At-labeling [21]. In this study, we evaluated the characteristics of a novel 211

Synthesis of [ 211 At]PSMA1, [ 211 At]PSMA5, and [ 211 At] PSMA6
Precursor molecules of PSMA1, PSMA5, and PSMA6 were synthesized based on solid-phase peptide synthesis by Peptide Institute, Inc. (Osaka, Japan). 211 At was produced by a nuclear reaction of 209 Bi(α, 2n) 211 At using a cyclotron and purified by a dry distillation method, providing the aqueous solution of 211 At (0.1-1 MBq/μL) [20]. 211 At-labeled PSMA1, PSMA5, and PSMA6 were synthesized by the substitution reaction of 211 At with the dihydroxyboryl groups introduced to the corresponding precursor molecules, as described in a previous paper [20].

In vitro cellular uptake analysis
Human prostate cancer cell lines, prostatic carcinoma-3 (PC-3) (low expression of PSMA), and lymph node carcinoma of the prostate (LNCaP) (high expression of PSMA) were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were maintained in a culture medium, Roswell Park Memorial Institute 1640 medium (FUJI-FILM Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin-streptomycin (FUJIFILM Wako Pure Chemical). The medium for LNCaP was supplemented with 1% sodium pyruvate (FUJIFILM Wako Pure Chemical) in a culture medium. Cells were seeded in 24-well plates (5 × 10 4 /well) and cultured for 2 days. After washing twice with phosphate-buffered saline (PBS) (−), the culture medium was changed to Hanks' balanced salt solution (+

Preparation of xenograft models
Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (5 weeks old, male) were purchased from Charles River Japan, Inc. (Atsugi, Japan). LNCaP cells were suspended in a 1:1 mixture of medium and Matrigel (Corning, USA), subcutaneously implanted into the unilateral flank of the mice (approximately 6-10 × 10 6 cells), and used approximately 5 weeks later (range, 4-8 weeks). Institute of Cancer Research (ICR) mice (6 weeks old, male) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and used as a non-tumor-bearing cohort for the evaluation of biodistribution and histology.
Euthanasia was performed under deep anesthesia using isoflurane inhalation. The criteria for euthanasia were as follows: (1) animals showed signs of intolerable suffering, (2) a significant decrease in activity or a marked decrease in food and water intake was observed, (3) the tumor size reached 2 cm in diameter, and (4) the observation period ended.  High PSMA expression was already confirmed in LNCaP xenografts in our previous study [22]. The brain, thyroid, salivary gland, lung, heart, liver, spleen, pancreas, stomach, small intestine, colon, kidney, bone, testis, blood, urine, feces, and tumor were excised and weighed to evaluate biodistribution after euthanasia at 3 and 24 h after administration. Urine excretion was determined from absorption to filter paper or by urine collection in the cage, and feces were collected from the cage. Radioactivity was measured using a 2480 Wizard 2 γ counter. The detection efficiency for 211 At with the γ counter was calibrated by measurement of the 211 At source whose radioactivity was determined with a Ge semiconductor detector (BE2020, Mirion Technologies (Canberra), Connecticut, USA). Uptake was calculated as the percentage of injected dose (%ID).
Planar imaging was performed using a γ camera system (E-cam, Siemens) at 3 and 24 h after administration, targeting the X-rays emitted from the daughter nuclide 211 Po (energy window: 79 keV ± 20%) [16]. Image analysis was performed by setting the regions of interest in the tumor and kidneys using AMIDE software (version 1.0.4).
Plasma was obtained by centrifuging a portion of the blood sample collected at the time of euthanasia and was measured using a dry clinical chemistry analyzer (SPOT-CHEM D-00 QR D-02; ARKRAY, Inc., Kyoto, Japan). Blood urea nitrogen (BUN) and creatinine (Cre) levels were also measured. Cre values less than 0.2 were considered 0.2 in the statistical analysis. Urine analysis was also performed using urinalysis test strips (Multistix Ames 2820, Siemens Healthcare, Tokyo, Japan) during the observation period in normal ICR mice after the administration of [ 211 At]PSMA5.

Statistical analyses
Comparisons between two groups were performed using the unpaired t-test in SPSS (version 25.0, IBM Corp., Armonk, NY, USA). For multiple comparisons among the three groups, Bonferroni correction was performed. Differences were considered statistically significant at P < 0.05.

Results
In the cellular uptake analysis, [ 211 At]PSMA1 and [ 211 At] PSMA5 were more highly incorporated into LNCaP cells with high PSMA expression than in PC-3 cells with low PSMA expression (Fig. 2), suggesting PSMA-mediated uptake of both compounds. Moreover, [ 211 At]PSMA5 showed higher uptake than [ 211 At]PSMA1.
As shown in Fig. 3 Fig. 4. High uptake was observed in the tumor xenografts and kidneys at 3 and 24 h post-injection.
Regarding the treatment effect, excellent tumor growth suppression was observed in LNCaP xenograft after the administration of [ 211 At]PSMA5 (Fig. 5a, b). [ 211 At]PSMA1 also showed a good treatment effect, but it showed relatively larger tumor size than [ 211 At]PSMA5 did. No significant changes in body weight were observed among the three groups (Fig. 5c).   (Fig. 6a). In one out of four ICR mice administered [ 211 At]PSMA5 (1 MBq), regenerated tubules were observed in the cortical area (Fig. 6b). In NOD/SCID mice, regenerated tubules were observed in the kidneys 3 and 6 weeks after administration in LNCaP xenograft mice (Fig. 6c). No significant changes were observed in the salivary glands or stomach.

Discussion
In this study, we evaluated the novel 211 [7][8][9]. We introduced an aryl boronic acid for the 211 At labeling of our PSMA precursors instead of N,N,N-trimethyl-2-pyridinaminium moiety for 18 F labeling. The three PSMA analogs have different amino acid residues in their side chains, Gly-Lys, ©-G©(R)-Glu, and (S)-Glu-(S)-Glu in PSMA1, PSMA5, and PSMA6, respectively. We evaluated the effects of the differences in amino acid residues on the properties of tumor retention, biodistribution, and in vivo treatment effects.    Supplementary Fig. S1), the antitumor effect of [ 211 At]PSMA5 was attributed to the α-particle emission from 211 At.
In the cellular uptake analysis, [ 211 At]PSMA5 showed higher uptake than [ 211 At]PSMA1, corresponding to the in vivo uptake in tumor xenograft models. In addition, uptake was higher in LNCaP cells than in PC-3 cells, suggesting that PSMA mediates the uptake of [ 211 At]PSMA1 and [ 211 At]PSMA5. In the whole-body biodistribution of [ 211 At]PSMA compounds, the kidneys showed remarkably high uptake, similar to the other PSMA compounds, reflecting PSMA expression in the proximal tubule and urine excretion [24]. Mild uptake was observed in the thyroid, spleen, and stomach. These uptakes were the physiological uptake of sodium astatide (NaAt), suggesting dehalogenation of [ 211 At] from [ 211 At]PSMA5/6 [16]. [ 211 At]PSMA5 was subjected to slow deastatination in mice, resulting in not more than 1.0% of the injected doses of the metabolites, including astatide ions, to be present in urine at 3 h after injections of the agents. In the thyroid, variable uptake was observed, possibly due to the excision of the surrounding tissues, including the trachea, which influenced the variability in organ weight. Although we did not observe histological changes in the thyroid, it can be a risk organ for radioligand therapy using [ 211 At]-labeled compounds. We have an option to use iodine blocking in clinical applications to protect the thyroid by inhibiting its uptake [17,25]. Furthermore, if we increase the injected dose, the non-radiolabeled mass in the solution also increases proportionately to the radioactivity. This may affect the biodistribution of the [ 211 At]-labeled compound due to competitive binding.
In the histopathological evaluation of the kidneys after administration of [ 211 At]PSMA5, regenerated tubules were observed in the cortical area in all NOD/ SCID mice, although most of them showed mild changes. These changes were not observed in the same dose group Regenerated tubules are characterized by tubule basophilia, nuclear crowding, and increased mitoses. They were reported to occur as a reparative response to previous degeneration and/or necrosis of renal tubular epithelium [26]. It was also observed in the chronic phase after internal irradiation with α-emitting daughter nuclides of 225 Ac [27]. This may be due to the radiation-induced toxicity of [ 211 At]PSMA5, as PSMA expression was observed in the proximal tubule of the kidney [24]. In a previous study by Pomper et al., late nephrotoxicity was reported in PSMA-targeted 211 At-labeled α-particle radiotherapy [28]. They showed its uptake in the cortical area of the kidney by α-camera imaging, subcortical atrophy, and degenerative loss of proximal tubules after treatment with 211 At-6 (1.5 MBq). They also reported that all animals treated with 1.5 MBq developed proteinuria 1-2 months after treatment, and animals treated with 37 kBq developed mild proteinuria that was later resolved. In our study, we did not observe proteinuria or increased BUN and Cre levels 8 weeks after the administration of [ 211 At]PSMA5, although uptake in the kidneys was similar in ICR mice compared with the report (60-70%ID/g at 1-18 h after administration) (Supplementary Fig. S2). However, chronic long-term kidney toxicity requires further evaluation from the perspective of future translation [29]. We aim to perform an extended single-dose toxicity study with three doses, including the evaluation of hematological toxicity (acute and recovery phases) and longterm chronic kidney toxicity (additional group) based on our previous report [23].
Xerostomia is the most common side effect of clinical targeted α-therapy using [ 225 Ac]PSMA-617, since PSMA expression was also observed in the salivary gland [12,13]. However, we did not observe any histological abnormalities in salivary glands. There might be a species difference, since the uptake in the salivary glands was not significantly higher in mice than in humans. We need to carefully monitor the toxicity in salivary glands in future clinical applications.
In clinical translation, species differences are sometimes observed. In [ 18 F]PSMA-1007 PET, high urine excretion was observed in mice, but its excretion was minimal in humans [21,30]. Diagnostic PET for evaluating prostate cancer recurrence has better detectability without excretion in the urinary tract [9]. However, for therapeutic applications, rapid urine excretion is ideal for reducing the absorbed dose in the kidneys. Although a continuous high uptake of [ 211 At]PSMA5 was observed in the kidneys, no serious toxicity was observed in this study. The radioactivity in the kidneys was presumably from the intact molecule since most of the radioactivity observed in the blood and urine was from intact molecules at 3 h post injection of the agent. Kidney retention may not be a significant problem for targeted α-therapy using 211 At because of its short physical half-life (7.2 h). However, in humans, it has been hypothesized that renal function tends to decline due to past cancer treatments, and the initial clinical dose of [ 211 At]PSMA5 should be determined carefully.
This study had some limitations. First, we evaluated the treatment effect in the LNCaP model using a single-dose administration. Repeated administration or dose escalation should be evaluated in future studies to mimic clinical situations and to define a minimum or maximum effective dose with a longer observation period. Second, we evaluated toxicity mainly using histological analysis. Hematological toxicity, including myelosuppression, should be evaluated in greater detail. Third, a detailed evaluation of whole-body distribution at multiple time points is essential for a precise dosimetric approach. Pharmacokinetic studies should be conducted in the future to perform precise estimation of absorbed doses and comparison with histological abnormalities.

Conclusion
[ 211 At]PSMA5 exhibited excellent tumor growth suppression in xenograft models of prostate cancer, with minimal side effects. [ 211 At]PSMA5 could be a new possible targeted α-therapy for prostate cancer, specifically metastatic CRPC, and future translational prospective trials are warranted.