Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [99m Tc]Tc-maSSS-PEG 2-RM26 (based on [D-Phe 6, Sta 13, Leu 14-NH 2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 °C was monitored for 18 months. The biological properties of [99m Tc]Tc-maSSS-PEG 2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG 2-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16–24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics.
Prostate cancer is one of the most commonly diagnosed cancers and is the cause of many cancer-related deaths. Among the explored prostate cancer cell markers are the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPR), which are also expressed in other malignancies, including breast cancer.
Numerous PSMA-targeting radiotracers have been developed for positron emission tomography (PET) and single-photon emission computed tomography (SPECT), resulting in several approved radiotracers both for diagnosis and therapy of PSMA-expressing prostate cancer. These radiotracers rely on the high expression of PSMA in the majority of prostate cancer cells, especially in the late stages of the disease, providing an improved diagnosis and monitoring of therapeutic response in comparison with the other available diagnostic and therapeutic options. There are several limitations of targeting PSMA, which include PSMA-negative prostate tumors. Thus, it is necessary to develop and use radiotracers that target other markers, which include GRPR.
GRPR expression is found in 63–100% of primary prostate tumors and in the majority of lymph node and bone metastases. Additionally, its expression in the prostate is mainly limited to malignant cells and has been found to be higher in the earlier stages of prostate cancer. Therefore, we have selected and evaluated GRPR as a potential target for diagnostics and therapeutics of prostate cancer.
Radiolabeled GRPR antagonists have been proposed to have advantages over GRPR agonists in that they evade GRPR activation, which can stimulate cell proliferation and growth and lead to GRPR downregulation, among other unwanted effects. In addition to this, radiolabeled GRPR antagonists were found to perform similarly to GRPR agonists, in imaging GRPR expression in vivo, if not better. Various GRPR antagonists have been developed and evaluated in preclinical and early-phase clinical studies, showing great promise for imaging of GRPR-expressing prostate cancer.
Technetium-99m is one of the most commonly used radionuclides in nuclear medicine, which is attributed to its optimal energy of emitted photons, sufficiently long half-life, and to its feasibility of being acquired by elution from a 99 Mo/99m Tc generator, enabling the acquisition of technetium-99m at a low cost. SPECT cameras, with their advantages and pitfalls, are still more widely available than PET cameras. Therefore, radiotracers suitable for SPECT imaging will have a wider outreach and thereby benefit more patients in the near future; hence, several GRPR antagonists labelled with technetium-99m were developed.
For the aforementioned reasons, our group recently reported the development and preclinical evaluation of the GRPR antagonist maSSS-PEG 2-RM26, consisting of a mercaptoacetyl-triserine amino acid sequence linked to the GRPR antagonist RM26 [D-Phe 6, Sta 13, Leu 14-NH 2]BBN(6–14) via a diethylene glycol linker. We used an amino acid-based N 3 S chelator formed by mercaptoacetyl-triserine for the labelling of peptide with freshly eluted pertechnetate (99m TcO 4−) that enabled single-step labeling. [99m Tc]Tc-maSSS-PEG 2-RM26 demonstrated strong affinity for GRPR along with favorable biodistribution and dosimetry, showing promise for SPECT imaging of GRPR-positive prostate cancer.
Following our recent report on the development of [99m Tc]Tc-maSSS-PEG 2-RM26, we aimed herein to prepare kits for single-step technetium-99m labeling of maSSS-PEG 2-RM26 to be used in an early-phase clinical study. We also aimed to test the radiolabeling and target binding of [99m Tc]Tc-maSSS-PEG 2-RM26 after kit preparation in addition to testing the sterility and toxicity of the kit constituents.
Furthermore, we wanted to study the effect of long-term (18 months) storage of the kit formulation on the radiolabeling of maSSS-PEG 2-RM26 and evaluate its in vitro and in vivo binding to GRPR after storage.
Gluconic acid sodium salt and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO, USA), stannous chloride was purchased from Fluka Chemika (Buchs, Switzerland), and PC-3 cells were purchased from ATCC (Rockville, MD, USA). Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with L-Glutamine was purchased from Biowest (Nuaillé, France), fetal bovine serum was purchased from Sigma-Aldrich (St. Louis, MO, USA), and penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin) and trypsin-EDTA were purchased from Biochrom AG (Berlin, Germany).
The glass vials used for kit preparation were adaptiQ® vials, purchased from Schott AG (Mainz, Germany), the stoppers used were 20 mm Serum NovaPure® stoppers and the seals were 20 mm Flip-Off® CCS seals, both purchased from West Pharmaceutical Services (West Whiteland Township, PA, USA). Technetium-99m pertechnetate (99m TcO 4−) was eluted from an Ultra-TechneKow® FM 99 Mo/99m Tc generator (Curium, Petten, The Netherlands). Radioactivity measurements for cell experiments were performed using a 2480 Wizard 2® automatic gamma counter from PerkinElmer (Waltham, MA, USA). Instant thin-layer chromatography (ITLC) strips (Agilent Technologies, Santa Clara, CA, USA) were used to estimate the radiochemical yield using a Cyclone® Plus Storage Phosphor System from PerkinElmer (Waltham, MA, USA). The reversed-phase high-performance liquid chromatography (RP-HPLC) system we used was from Hitachi High-Tech (Tokyo, Japan), using a Luna C18 column (5 μm, 100 A°, 150 × 4.6 mm, Phenomenex, Værløse, Denmark) with a gradient from 5 to 70% acetonitrile (0.1% v/v trifluoroacetic acid) in water over 15 min. Sep-Pak® C8 1 cc Vac cartridges (Waters Corp, Milford, MA, USA) were used for purification of the radiolabeled GRPR antagonist when needed.
The animal studies on biodistribution were approved by the Ethics Committee for Animal Research in Uppsala, Sweden following the national legislation on protection of laboratory animals (protocol code 5.8.18-00473/2021 approved on 26 February 2021). Acute toxicity experiments were approved by the Board of Medical Ethics, Tomsk National Research Medical Center of the Russian Academy of Sciences (protocol № 12 04.12.2020).
PC-3 cells were cultured in the RPMI-1640 medium supplemented with 20% fetal-bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine. The cells were incubated at 37 °C and 5% carbon dioxide.
The kit for the clinical study was formulated as follows: freshly dissolved gluconic acid sodium salt (5 mg, in H 2 O), EDTA (100 µg, in H 2 O) and stannous chloride (75 µg, in 0.01 M HCl) were added to glass vials prior to direct addition of maSSS-PEG 2-RM26 (40 µg, in H 2 O). The kits were prepared on ice and frozen at −20 °C directly after preparation. The kits were freeze-dried. Finally, the vials were sealed and sterilized by heat sterilization.
To radiolabel maSSS-PEG 2-RM26, freshly eluted technetium-99m pertechnetate (99m TcO 4−, 400–600 MBq) was added, in sterile saline, to the kit-containing vial followed by incubation at 90 °C for 60 min. The radiochemical yield was determined by ITLC and the ITLC strips were eluted with PBS (Rf = 0 for the radiolabeled peptide and Rf = 1 for 99m Tc-pertechnetate and 99m Tc-gluconate) and pyridine:acetic acid:water, 5:3:1.5 (Rf = 0 for hydrolyzed-reduced technetium colloid and Rf = 1 for the radiolabeled peptide, 99m Tc-pertechnetate and 99m Tc-gluconate). The reaction mixture was also analyzed using RP-HPLC.
The radiolabeled peptide binding to GRPR was tested in PC-3 cells (GRPR +) by the addition of 1 nM of [99m Tc]Tc-maSSS-PEG 2-RM26 to the cells (~10 6 cells, in triplicates), with and without pre-blocking of receptors, by the addition of 1 µM of NOTA-PEG 2-RM26 (10 min at room temperature). After incubation at 37 °C for 1 h, the cells were treated with trypsin-EDTA, collected, and measured for their radioactivity content.
The sterility of the lyophilized kit product was evaluated via growth promotion testing. Autoclaved growth medium, 30 g/L tryptic soy broth (TSB, Merck, Kenilworth, NJ, USA), was prepared in 200 mL flasks and incubated in 25 °C for 14 days either without or together with the kit sample. As reference samples, microorganisms starting at ~100 CFU were used. The reference microorganisms used were Candida albicans (C. albicans, ATCC® 10231), Asperigillus brasiliensis (A. brasiliensis, ATCC® 16404), and Bacillus subtilis (B. subtilis, ATCC® 6633) (Thermo Fisher Diagnostics, Uppsala, Sweden). Culture growth was continuously monitored by visual inspection. The samples were considered sterile if no visible growth developed over the duration of the experiment. The suitability of the method was investigated by comparing parallel growth rates in between cultures with and without the added kit sample. For the method suitability tests, a kit sample buffer,
