The Precursor peptides for labeling with ⁶⁸Ga (PSMA-11) and ¹⁷⁷Lu (PSMA-I&T) were purchased from ABX (Advanced Biochemical Compounds, Germany) and CMR (Center of Molecular Research, Russia), respectively. A 37% solution of Suprapur^® hydrochloric acid was purchased from Merck, Germany, with purity of 99.9%. Sodium acetate was purchased from Merck, Germany. All other reagents and solvents were of analytical grade and were purchased from Sigma-Aldrich, USA. All materials and media for cell culture, including RPMI (Roswell Park Memorial Institute medium), were purchased from Gibco (USA). Isoflurane was purchased from Cristália Laboratory, Brazil, and Matrigel^® from Corning^®, USA.

The ⁶⁸GaCl₃ eluate was obtained from GMP-grade generators provided by two different manufacturers: Isotopen Technologies Garching (ITG) and Eckert & Ziegler (both based in Germany).

The ⁶⁸Ge/⁶⁸Ga generator from ITG was eluted with 4 mL of sterile 0.05 M hydrochloric acid solution and the ⁶⁸Ge/⁶⁸Ga generator from Eckert & Ziegler was eluted with 7 mL of sterile 0.1 M hydrochloric acid solution. The hydrochloric acid solution was prepared using 37% ultrapure HCl (99.99%, Merck, Germany) and water for injection as sterile solution. The eluate from both generators was analyzed and met the quality criteria specified by the manufacturers.

¹⁷⁷LuCl₃ was obtained from INM (Russia) with a specific activity of around 1332 GBq/mg.

Pre-formulation experiments were conducted to select sodium acetate buffer concentration, pH, and volume to achieve the desired pH range (4.0 - 5.0) for the optimal complexation of ⁶⁸Ga after the addition of eluates from two ⁶⁸Ge/⁶⁸Ga generators (GMP grade, ITG and Eckert & Ziegler) [21] [22] [23] . The maintenance of the desired pH range (4.0 - 5.0) of the buffer after lyophilization was evaluated following lyophilization of the vials by adding an acid-base indicator (Congo Red). The amount of mannitol to be used in the formulation was also evaluated in the freeze-drying process. Labeling experiments were conducted using the intended formulation with the aim of optimizing the reaction pH, assessing the radiolabeling temperature, and investigating the influence of adding two different concentrations of mannitol to the formulation.

The thermal analysis of the formulation containing mannitol and the PSMA-11 peptide was conducted using a differential scanning calorimeter (DSC 4000 Perkin Elmer, USA). The primary objective of this analysis was to investigate the thermal properties of the formulation, including phase transitions and thermal behavior during cooling and heating. Initially, a 30 µL sample of the formulation was carefully pipetted into a hermetically sealed aluminum container. The analyses were conducted using a cooling program, starting from room temperature to −60˚C at a rate of 5˚C/minute, followed by heating to 20˚C at a rate of 10˚C /minute, with the insertion of hold times to stabilize the temperature at specific points.

The results obtained from the thermal analysis were used to guide the lyophilization process designed to ensure the stability and integrity of the final product. The lyophilization test cycle took place in a benchtop lyophilizer model Advantage Pro with Intellitronics controller from SP Scientific, Pennsylvania (USA).

The PSMA-11 kit was radiolabeled by adding 1 mL of sodium acetate buffer (1.5 M, pH 5.0) and 4 mL of Ga-68 eluted in 0.05 M HCl from the ITG generator (~223.8 MBq) or 3.5 mL of ⁶⁸GaCl₃ eluted in 0.1M HCl from Eckert & Ziegler generator, at room temperature for 10 minutes.

The radiopharmaceutical ¹⁷⁷Lu-PSMA-I&T for in vivo studies was prepared by incubating 20 µg (13.3 nmols) of PSMA-I&T with 429.2 MBq of ¹⁷⁷LuCl₃ at 95˚C and 450 rpm for 30 minutes in a 0.52 M sodium ascorbate buffer (200 µL, pH 4.7).

Two chromatographic systems determined the radiochemical purity of PSMA-11 kit with Ga-68. High Pressure Liquid Chromatography (radio-HPLC) (Shimadzu, Japan) analysis was conducted using a C-18 column (Waters model Symmetry^® C18, 5 µm, 250 mm × 4.6 mm), eluted at a flow rate of 0.6 mL/min by (A) water 0.1% TFA and (B) acetonitrile 0.1% TFA, in the following gradient mode: 0 - 0.5 min 5% B; 0.5 - 10.0 min 40% B; 10.0 - 16.0 min 5% B. Thin Layer Chromatography (TLC-SG) was performed on silica gel 60 plate (Merck, Germany) and ammonium acetate 77 g/L: methanol (1:1) RF, ⁶⁸Ga-colloidal = 0.0 - 0.1; RF, ⁶⁸Ga-peptide = 0.7 - 1.0. The TLC-SG strips were quantified with a radio TLC-scanner (AR-2000 Eckert & Ziegler Radiopharma Inc., USA) [24] .

The HPLC analysis method for determining the radiochemical purity of ¹⁷⁷Lu-PSMA-I&T was conducted under the following conditions: Symmetry^® C18 column, 5 µm, Waters 250 × 4.6 mm; mobile phase: (A) water/0.1% TFA and (B) Acetonitrile/0.1% TFA; gradient 10% to 45% of B over 20 minutes at a flow rate of 1 mL/minute [25] .

The in vitro and in vivo competition assays were performed with the radiotracer ¹⁷⁷Lu-PSMA-I&T and lyophilized PSMA-11 (employed as a competitor). The cell line used was human androgen-dependent prostate carcinoma (LNCaP-Lymph Node Carcinoma of the Prostate) PSMA-positive (ATCC^® CRL-1740^TM). LNCaP cells were cultured in RPMI 1640 medium (Life Technologies, MD, USA) supplemented with 10% Fetal Bovine Serum (FBS), 300 µg/mL streptomycin and 100 U/mL penicillin. Cultures were maintained at 37˚C under an atmosphere of 5% CO₂/humidified air. One day before the experiments, cells were harvested using Trypsin/EDTA (0.05% and 0.02%), centrifuged and suspended in culture medium, counted and 2 × 10⁵ LNCaP cells were seeded per well in 6-well plates. The plates were maintained at 37˚C under a humidified atmosphere of 5% CO₂/air in an incubator.

In vivo competition study was performed in SCID-BALB/c mice with LNCap cell prostate tumor model. To induce tumor growth, LNCaP cells (5 × 10⁶ cells/100 µL) were suspended in PBS supplemented with 20% Matrigel (Corning^® Matrigel^®, USA) and inoculated subcutaneously into the dorsal area of SCID mice (4 - 5 weeks old; IPEN Animal Facility). The mice were previously anesthetized with isoflurane.

In the competition experiments, the final concentration of the ¹⁷⁷Lu-PSMA-I&T radiotracer in each tube was 5 nM/mL, and the concentration of PSMA-11 competitor was 75 nM/mL, in RPMI medium supplemented with 10% FBS [26] . The PSMA-11 was reconstituted from a lyophilized vial obtained with the parameters developed in the present study, aiming to assess the effect of freeze-drying on the ability of the peptide to recognize the PSMA receptor. The experiments were performed in triplicate as two independent experiments. LNCaP cells (2 × 10⁵ per well) were incubated with the radiotracer ¹⁷⁷Lu-PSMA-I&T with and without the PSMA-11 competitor at 4˚C for 60 minutes. The incubation was stopped by removing the radiotracer and competitor and washing the wells with 1 mL of PBS. The cells were then lysed using 1 mL of 1M NaOH solution. The lysate was transferred to counting tubes. The quantification of radioactivity in CPM was performed using an automatic gamma counter with NaI (TI) crystals (D5002 Cobra II, Packard Canberra), and the values were converted to fmol of ¹⁷⁷Lu-PSMA-I&T. Finally, the data were analyzed using GraphPad Prism software (version 5.00^®). Quantitative data were expressed as mean ± standard deviation. Significant difference was assessed using the unpaired t-test (two-tailed) with Welch’s correction. P values below 0.05 were considered statistically significant.

The in vivo competition study was conducted by injecting the radiotracer ¹⁷⁷Lu-PSMA-I&T into two groups of tumor-bearing mice (n = 5 for each group), one of the groups receiving an excess of PSMA-11 (100 µg/animal), prepared by reconstituting lyophilized PSMA-11 with 0.9% sodium chloride solution, 30 minutes before the administration of the ¹⁷⁷Lu-PSMA-I&T. After 60 minutes of the radiopharmaceutical administration, the animals were euthanized, and samples of blood and organs of interest were collected as well as the tumor. The percentages of administered activity per gram of organ or tumor were determined relative to a standard of administered dose using an automatic gamma counter with NaI (TI) crystals (D5002 Cobra II, Packard Canberra). Quantitative data are expressed as mean ± standard deviation. Significant differences were assessed using two-way ANOVA followed by Bonferroni post-test for in vivo statistical analysis. P values below 0.05 were considered statistically significant.

All graphs and statistical analyses were performed using GraphPad Prism software (version 5.00^®). Quantitative data are expressed as mean ± standard deviation. The tools used to analyze significant differences were described for each competition assay. P values below 0.05 were considered statistically significant.

A volume of 1 mL of sodium acetate buffer at a concentration of 1.5 M and pH 5.0 maintained the desired pH between 4 and 5 when adding volumes of 4 mL and 3.5 mL of 0.05 M HCl and 0.1 M HCl, respectively. The peptide should be lyophilized without the presence of the labeling buffer. The high concentration of salts in the labeling buffer resulted in the collapse of the cakes in initial lyophilization cycle tests. Furthermore, buffer freeze-drying tests employing Red of Congo indicator revealed the change in buffer pH during the freeze-drying cycle. In this way, the formulation of the PSMA-11 kit was developed in two vials: vial 1, containing the peptide (20 µg) and the bulking agent mannitol (20 mg), in lyophilized form and vial 2, containing the radiolabeling sodium acetate buffer solution at pH 5.0.

Studies on variation of mannitol mass made it possible to conclude that the use of 20 mg of mannitol instead 5 mg per vial resulted in an elegant and consistent lyophilized cake.

The thermal analysis of the formulation containing the PSMA-11 peptide and mannitol showed a glass transition temperature ( ${\text{T}}_{\text{g}}^{\text{'}}$) of −42.90˚C and a eutectic temperature (T_eu) of 5.10˚C (data shown in “ Figure 1 ”).

Based on the critical temperature characterization studies, the freezing temperature of the lyophilization cycle was defined and primary drying was conducted at 2˚C. A freezing rate of 0.5˚C/minute was utilized, along with a holding time before the freezing step, to form larger pores and crystals, aiming to facilitate sublimation.

The direct labeling of the ready-to-use PSMA-11 kit with the ⁶⁸GaCl₃ eluted from the ITG generator was evaluated by HPLC immediately after lyophilization and after 40 days of kit storage at −20˚C. The radiolabeling took place at room temperature for 10 minutes and the final pH of the reaction mixture was 4.5. The radiochemical purity was > 95% at HPLC analyses “ Figure 2 ”.

The radiolabeling of ready-to-use PSMA-11 kit with gallium-68 obtained from Eckert & Ziegler generator, was evaluated by radio-TLC scanner, after approximately 45 days of storage at −20˚C. All labelings were performed at room temperature for 10 minutes and the final pH of the reaction mixture was 4.0 in this case. The radiochemical purity was > 95% evaluated by TLC “ Table 1 ”.

Radiolabeling of PSMA-I&T with lutetium-177 resulted in radiochemical purity > 95% without purification. The SA was 718.9 GBq/mg. The retention time

[1] Lyophilized kit—PSMA-11/ mannitol; *TLC—Thin layer chromatography.

was similar to the cold peptide determined by HPLC with identification of a single peak corresponding to the product of interest.

The integrity and preservation of PSMA-11 affinity post-lyophilization were evaluated in a competitive binding assay with the human prostate carcinoma cell line LNCaP-PSMA + (2 × 10⁵ cells/well), 1-hour incubation at 4˚C with ¹⁷⁷Lu-PSMA-I&T (5 nM) as the radioligand and lyophilized, unlabeled PSMA-11 (75 nM) as the competitor. The experiment demonstrated the binding of ¹⁷⁷Lu-PSMA-I&T, being 1712.08 ± 63.03 fmol without competitor and 1010.54 ± 243.49 fmol with competitor “ Figure 3 ”.

A significant difference was observed between the uptake of ¹⁷⁷Lu-PSMA-I&T without the competitor and with the competitor (P = 0.0320, P < 0.05), which confirms the preservation of PSMA-11 affinity for the receptor after one month of lyophilization procedure.

In the in vivo competition experiment using ¹⁷⁷Lu-PSMA-I&T and lyophilized PSMA-11 as competitor, a reduction in tumor uptake was observed from 2.18 ± 0.27 %ID/g to 0.62 ± 0.17 %ID/g, representing a decrease in the tumor uptake rate by 71.56% “ Table 2 ”.

% IA/g = percentage of activity injected per gram or per mL of blood.

Additionally, renal uptake was strongly reduced by the presence of the lyophilized PSMA-11 competitor, with the reduction in renal uptake from 97.68 ± 19.24 %ID/g to 7.99 ± 1.59 %ID/g (***P ≤ 0.001) “ Figure 4 ”. Conversely, the percentage of the radiopharmaceutical in circulation increased in the blocking group, a fact that also reflects the competition established between the lutetium-177 radiopharmaceutical and the competitor (lyophilized PSMA-11) for PSMA receptors on tumor cells.