Synthesis of [211At]PSMA1, [211At]PSMA5, and [211At]PSMA6

Precursor molecules of PSMA1, PSMA5, and PSMA6 were synthesized based on solid-phase peptide synthesis by Peptide Institute, Inc. (Osaka, Japan). 211At was produced by a nuclear reaction of 209Bi(α, 2n)211At using a cyclotron and purified by a dry distillation method, providing the aqueous solution of 211At (0.1–1 MBq/μL) [20].

211At-labeled PSMA1, PSMA5, and PSMA6 were synthesized by the substitution reaction of 211At with the dihydroxyboryl groups introduced to the corresponding precursor molecules, as described in a previous paper [20]. Twenty microliters of 0.1 mg/mL PSMA1, PSMA5, or PSMA6 solution (containing 7% (w/v) sodium hydrogen carbonate was mixed with 1–40 MBq (1–100 μL) of aqueous solution of 211At. Subsequently, 20–40 μL of 0.1 mol/L KI solution was added to the mixture, which was allowed to react for 45 min at 80 °C. The molecular structures of [211At]PSMA1, [211At]PSMA5, and [211At]PSMA6 are shown in Fig. 1.

Fig. 1

Molecular structures of [18F]PSMA-1007, [211At]PSMA1, [211At]PSMA5, and [211At]PSMA6

The crude reaction mixture of [211At]PSMA1, [211At]PSMA5, or [211At]PSMA6 was purified by solid-phase extraction (SPE). The mixture was loaded onto an Oasis HLB cartridge (Waters, Milford, US), and the cartridge was rinsed with 1 mL of aqueous solution of sodium hydrogen carbonate (7% (w/v)). [211At]PSMA1, [211At]PSMA5, and [211At]PSMA6 trapped in the cartridge were eluted with 1 mL of 20% (v/v) ethanol. The radiochemical yields of [211At]PSMA1, [211At]PSMA5, and [211At]PSMA6 were not less than 60% (radioactivity decay corrected), and the radiochemical purity of the products was greater than 96% after the SPE purification. Molar activities of the radioligands were 175–190 MBq/nmol. In the treatment experiments, the mass was approximately 0.002 μg per animal for 0.40 MBq of [211At]PSMA1 or [211At]PSMA5 solution. In a biodistribution study, the mass was approximately 0.0005–0.0006 μg when [211At]PSMA1, 5 or 6 was administered at 0.10–0.12 MBq/mouse.

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 (FUJIFILM 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 × 104/well) and cultured for 2 days. After washing twice with phosphate-buffered saline (PBS) (−), the culture medium was changed to Hanks’ balanced salt solution (+). After treatment with [211At]PSMA1 or [211At]PSMA5 (approximately 30–50 kBq/well), cells were washed twice with PBS (–). After washing, all cells were lysed with 0.1 N sodium hydroxide, and the radioactivity of the cells was calculated using a 2480 Wizard2 γ counter (Perkin Elmer, MA, USA). Protein levels were measured using a plate reader (MultiScan FC, Thermo Fisher) and the BCA Protein Assay Kit (FUJIFILM Wako Pure Chemical). Uptake (%uptake/mg protein) was compared between PC-3 and LNCaP cells at 30 min after incubation with [211At]PSMA1 or [211At]PSMA5.

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 × 106 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.

Biodistribution of [211At]PSMA1, [211At]PSMA5, and [211At]PSMA6

LNCaP xenograft mice (body weight = 18.8 ± 3.0 g, n = 25) and normal ICR mice (body weight = 33.0 ± 1.3 g, n = 12) were used to evaluate biodistribution after the administration of [211At]PSMA solutions ([211At]PSMA1, 0.12 ± 0.10 MBq; [211At]PSMA5, 0.11 ± 0.04 MBq; and [211At]PSMA6, 0.11 ± 0.02 MBq, 0.0005–0.0006 μg). 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 Wizard2 γ counter. The detection efficiency for 211At with the γ counter was calibrated by measurement of the 211At 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 211Po (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).

Evaluation of treatment effect of [211At]PSMA5 and [211At]PSMA1

LNCaP xenograft mice (body weight = 21.8 ± 5.98 g) were administered [211At]PSMA5 (0.39 ± 0.03 MBq, 0.002 μg, n = 12), [211At]PSMA1 (0.40 ± 0.07 MBq, 0.002 μg, n = 5), or saline (n = 10). The treatment dose was based on that used in our previous study, in which 0.4MBq of [211At]NaAt showed sufficient therapeutic effect without significant toxicity [16, 23]. Non-radiolabeled PSMA5 (high mass, 0.02 μg; low mass, 0.002 μg) and PSMA1 (high mass, 0.02 μg; low mass, 0.002 μg) were also administered to LNCaP xenograft mice (each n = 3) and compared with saline-injected mice (n = 3) to assess the absence of antitumor effect by the PSMA compound itself. Tumor sizes (mm3) were measured using a caliper, calculated using the following elliptical sphere model equation, and compared between injected mice and controls. The body weight (g) was also monitored.

Evaluation of side effects

After euthanasia, the thyroid, salivary gland, stomach, small intestine, spleen, and kidney were excised from normal ICR mice (body weight = 32.0 ± 2.0 g, n = 10) 8 weeks after the administration of [211At]PSMA5 (0.33 ± 0.003 MBq) or [211At]PSMA6 (0.35 ± 0.023 MBq). The thyroid, salivary gland, and kidney were excised from normal ICR mice (body weight = 33.5 ± 1.6 g, n = 4) 2 weeks after the administration of [211At]PSMA5 (high-dose: 1.06 ± 0.06 MBq). The salivary gland, stomach, and kidney were excised from LNCaP xenograft mice (n = 14, body weight = 21.8 ± 5.98 g) 3 and 6 weeks after the administration of [211At]PSMA5 (0.37 ± 0.02 MBq). The excised organs and tissues were fixed in a 10% neutral buffered formalin solution. After fixation, paraffin sections were prepared and stained with hematoxylin and eosin. Specimens were evaluated using an integrated microscope (BZ-X810; Keyence Corporation, Osaka, Japan). Histological evaluation was performed by a toxicopathology specialist with the support of KAC Co. Ltd. (Kyoto, Japan).

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 (SPOTCHEM 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 [211At]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.