[99mTc]Tc-PSMA-T4—Novel SPECT Tracer for Metastatic PCa: From Bench to Clinic

Despite significant advances in nuclear medicine for diagnosing and treating prostate cancer (PCa), research into new ligands with increasingly better biological properties is still ongoing. Prostate-specific membrane antigen (PSMA) ligands show great potential as radioisotope carriers for the diagnosis and therapy of patients with metastatic PCa. PSMA is expressed in most types of prostate cancer, and its expression is increased in poorly differentiated, metastatic, and hormone-refractory cancers; therefore, it may be a valuable target for the development of radiopharmaceuticals and radioligands, such as urea PSMA inhibitors, for the precise diagnosis, staging, and treatment of prostate cancer. Four developed PSMA-HYNIC inhibitors for technetium-99m labeling and subsequent diagnosis were subjected to preclinical in vitro and in vivo studies to evaluate and compare their diagnostic properties. Among the studied compounds, the PSMA-T4 (Glu-CO-Lys-L-Trp-4-Amc-HYNIC) inhibitor showed the best biological properties for the diagnosis of PCa metastases. [99mTc]Tc-PSMA-T4 also showed effectiveness in single-photon emission computed tomography (SPECT) studies in humans, and soon, its usefulness will be extensively evaluated in phase 2/3 clinical trials.


Introduction
Prostate cancer is the second most common malignancy in men, and its prognosis depends on the stage of the disease [1,2]. Many cases of PCa are curable if they are detected early. However, a number of patients will still progress to metastatic cancers that evolve towards hormone resistance or metastatic castration-resistance prostate cancer (mCRPCa) [2]. Therefore, early detection of metastases or recurrent prostate cancer is of great clinical importance in terms of clinical evaluation, prognosis, and treatment [3][4][5].
Prostate cancer has been initially diagnosed by testing the prostate-specific antigen (PSA) level in the blood, through digital rectal examination (DRE) or prostate gland biopsy tests. However, measurement of PSA level is related to false-positive results caused by diseases other than prostate cancer, such as benign prostatic hyperplasia (BPH), urological manipulations, or prostatitis, and the biopsy is associated with numerous complications such as risks of bleeding and infection [5,6]. Furthermore, currently, morphological imaging such as ultrasound (US), computerized tomography (CT), magnetic resonance imaging (MRI), and metabolic positron emission tomography-computed tomography (PET/CT) imaging with [ 11 C]-choline have lower sensitivity to nodal metastasis and small tumor recurrence [7][8][9].
Despite significant advances in nuclear medicine for the diagnosis and treatment of prostate cancer, research into new ligands with increasingly better biological properties is still ongoing. Prostate-specific membrane antigen is an attractive and promising target for mPCa therapy. Similar conclusions about the significant effect of linker design on the in vitro and in vivo affinity for PSMA and the change in biodistribution profile were drawn by Wirtz et al. [41], subjecting the PSMA inhibitor PSMA-I&T to a modification towards increased lipophilicity.
Therefore, in the development process of the new PSMA ligand, we focused on using the most appropriate linker system to improve the pharmacokinetics of the final 99m Tc-labeled preparation. The undertaken studies showed that the presence of L-Trp in PSMA-T4, as one of the linkers, instead of naphthylalanine (L-2NaI), has led to a significant improvement in the biodistribution of the labeled molecule (reducing kidney accumulation) and its increased affinity to PSMA in vivo.
This work presents the results of comparative preclinical studies for four developed small-molecule inhibitors of PSMA with modified linker systems in order to select the one with the most favorable in vitro and in vivo biological properties.

Synthesis
The designed PSMA-HYNIC compounds, Figure 1, were synthesized on the solid phase followed by C18 reversed phase high-performance liquid chromatography (HPLC) purification, resulting peptides of >97% HPLC purity. The identity of synthesized compounds was confirmed by mass spectrometry (MS) and, in the case of PSMA-T4, additionally by the nuclear magnetic resonance (NMR), Figure S1.
modified. It was presented by Benesova et al. [26,40] that the pharmacokinetics properties, including PSMA inhibition potencies, cellular internalization, and biodistribution behavior of the PSMA inhibitors, can be significantly influenced by modification of the linker. Thus, the linker moiety's chemical constitution significantly impacts the in vivo tumor-targeting and pharmacokinetics of PSMA-targeting radioligands. Their research resulted in developing the PSMA-617 inhibitor, which had favorable biological properties and became the gold standard as a carrier for lutetium-177 for mPCa therapy. Similar conclusions about the significant effect of linker design on the in vitro and in vivo affinity for PSMA and the change in biodistribution profile were drawn by Wirtz et al. [41], subjecting the PSMA inhibitor PSMA-I&T to a modification towards increased lipophilicity.
Therefore, in the development process of the new PSMA ligand, we focused on using the most appropriate linker system to improve the pharmacokinetics of the final 99m Tclabeled preparation. The undertaken studies showed that the presence of L-Trp in PSMA-T4, as one of the linkers, instead of naphthylalanine (L-2NaI), has led to a significant improvement in the biodistribution of the labeled molecule (reducing kidney accumulation) and its increased affinity to PSMA in vivo.
This work presents the results of comparative preclinical studies for four developed small-molecule inhibitors of PSMA with modified linker systems in order to select the one with the most favorable in vitro and in vivo biological properties.

Synthesis
The designed PSMA-HYNIC compounds, Figure 1, were synthesized on the solid phase followed by C18 reversed phase high-performance liquid chromatography (HPLC) purification, resulting peptides of >97% HPLC purity. The identity of synthesized compounds was confirmed by mass spectrometry (MS) and, in the case of PSMA-T4, additionally by the nuclear magnetic resonance (NMR), Figure S1.

Radiolabeling, QC, Stability
Synthesized PSMA-HYNIC compounds were radiolabeled with 99m Tc in presence of tricine and N,N-diacetic acid (EDDA) as co-ligands, Table S1. The radiochemical purity was >95% for all labeled compounds, determined via radio HPLC and thin layer chromatography (TLC) methods.  In the radiochromatograms of all labeled PSMA-HYNIC analogs, a minor signal before the main peak was observed. The identity of this species was assessed for PSMA-T4 radiocomplex by liquid chromatography-mass spectrometry (LC-MS) method using the long-lived technetium-99. The sample was ionized using the electro-spray method and the mass was determined in positive ionization mode. The main peak on the radio-chromatogram with retention time of 10.2 min and a small peak with retention time of 9.9 min had the same mass of the molecular ion found in LC-MS at m/z 613.6 ([M+2H] 2+ ) and m/z 1,226.3 ([M+H] + ). The masses observed were consistent with the addition of 1 technetium and 2 EDDA molecules to the peptide, with the concomitant displacement of 5 protons, Table 1. The presence of labeled species containing one EDDA molecule, mixed tricine/EDDA ternary complex, and an oxo or halide group in complexes was excluded. The binding of a technetium atom to PSMA-T4 and co-ligands is accompanied by the displacement of five hydrogen atoms, implicating the presence of technetium in oxidation state (V). The presence of two peaks with the same mass of ions found in the LC-MS studies, which corresponded to [ 99m Tc]Tc-(EDDA)2-PSMA-T4 complex, could be the result of isomerism in the technetium coordination sphere. The presence of two peaks with the same mass of ions found in the LC-MS studies, which corresponded to [ 99m Tc]Tc-(EDDA)2-PSMA-T4 complex (hereafter abbreviated as [ 99m Tc]Tc-PSMA-T4), could be the result of isomerism in the technetium coordination sphere. HYNIC is capable of coordinating technetium in monodentate and bidentate modes. Several possible structures can be proposed for technetium-HYNIC complexes, according to the interpretation of the structure of model technetium and rhenium complexes with hydrazinopyridine [42]. The resulting interpretation favors a bidentate chelating role for HYNIC, in which both the hydrazine and pyridine groups are coordinated with losing all free hydrazinic hydrogens and two In the radiochromatograms of all labeled PSMA-HYNIC analogs, a minor signal before the main peak was observed. The identity of this species was assessed for PSMA-T4 radiocomplex by liquid chromatography-mass spectrometry (LC-MS) method using the long-lived technetium-99. The sample was ionized using the electro-spray method and the mass was determined in positive ionization mode. The main peak on the radiochromatogram with retention time of 10.2 min and a small peak with retention time of 9.9 min had the same mass of the molecular ion found in LC-MS at m/z 613.6 ([M+2H] 2+ ) and m/z 1226.3 ([M+H] + ). The masses observed were consistent with the addition of 1 technetium and 2 EDDA molecules to the peptide, with the concomitant displacement of 5 protons, Table 1. The presence of labeled species containing one EDDA molecule, mixed tricine/EDDA ternary complex, and an oxo or halide group in complexes was excluded. The binding of a technetium atom to PSMA-T4 and co-ligands is accompanied by the displacement of five hydrogen atoms, implicating the presence of technetium in oxidation state (V). The presence of two peaks with the same mass of ions found in the LC-MS studies, which corresponded to [ 99m Tc]Tc-(EDDA) 2 -PSMA-T4 complex, could be the result of isomerism in the technetium coordination sphere. The presence of two peaks with the same mass of ions found in the LC-MS studies, which corresponded to [ 99m Tc]Tc-(EDDA) 2 -PSMA-T4 complex (hereafter abbreviated as [ 99m Tc]Tc-PSMA-T4), could be the result of isomerism in the technetium coordination sphere. HYNIC is capable of coordinating technetium in monodentate and bidentate modes. Several possible structures can be proposed for technetium-HYNIC complexes, according to the interpretation of the structure of model technetium and rhenium complexes with hydrazinopyridine [42]. The resulting interpretation favors a bidentate chelating role for HYNIC, in which both the hydrazine and pyridine groups are coordinated with losing all free hydrazinic hydrogens and two hydrogens from the co-ligand molecules. There is no direct possibility to identify which EDDA donor atoms bind to technetium. The stability studies in phosphate buffer saline (PBS) and human serum at 37 • C revealed that the radiolabeled compounds tested were stable for at least 4 h, Figure 3, with no statistically significant differences between compounds. The stability studies in phosphate buffer saline (PBS) and human serum at 37 °C revealed that the radiolabeled compounds tested were stable for at least 4 h, Figure 3, with no statistically significant differences between compounds. The study on the lipophilicity of the technetium-99m labeled PSMA ligands showed that due to use of tryptophan (L-Trp) in the structure of the linker instead of naphthylalanine (L-2NaI), PSMA-T3 and -T4 are more hydrophilic than PSMA-T1 and -T2 analogues, which logD values are comparable with reference [ 99m Tc]Tc-iPSMA [35], Table 2.
The study showed that all the PSMA-HYNIC analogs had a higher binding affinity to the LNCaP cells than PSMA-11 used as a reference substance (Table 5, Figure 4). The most promising of the four studied were PSMA-T3 and -T4 with IC50 in the 70-80 nM range.   11 11.4 ± 7.1 [15] Binding affinities (IC50) values for cold ligands PSMA-T1, PSM PSMA-T4 were determined in the competitive binding assay using L cell membranes and [ 131 I]I-MIP1095 as a competitive radioligand. A widely used PSMA-11 inhibitor was also tested.
The study showed that all the PSMA-HYNIC analogs had a h to the LNCaP cells than PSMA-11 used as a reference substance ( most promising of the four studied were PSMA-T3 and -T4 with range.

In Vivo Study
The selection of the best of the synthesized PSMA-HYNIC compounds for pharmacokinetics and toxicity studies was based on a single-point (4 h i.v.) physiological distribution study of the four radioligands compared to the reference [ 99m Tc]Tc-iPSMA complex. The obtained results are presented in Table 6. The ordinary two-way ANOVA with Tukey's multiple comparisons test (GraphPad Prism 9.4 for Windows [43]) was used for statistical analysis. As a result, we found out that the statistically significant differences (p < 0.05) were for kidneys uptake (p < 0.0001) and urine excretion (p < 0.0001) for all of the developed PSMA ligands comparing to [ 99m Tc]Tc-iPSMA, Table S5. The uptake of the analyzed radioligands in other of the examined organs was non-significant (p > 0.05) compared to the [ 99m Tc]Tc-iPSMA uptake. In our comparative biodistribution study, we noted the lowest renal uptake (15.9 ± 2.09 %ID/g) and highest urinary excretion of radioactivity (91.23 ± 1.09 %ID) for [ 99m Tc]Tc-PSMA-T4.
Therefore, among the studied compounds, PSMA-T4 showed the best physiological distribution thanks to low kidney accumulation and was selected for further in vivo examination.
The accumulation profile of [ 99m Tc]Tc-PSMA-T4 in kidneys is shown in Figure 6. This profile of renal clearance with a clear peak in radioactivity accumulation 2-4 h after administration may indicate that glomerular filtration is not the only mechanism in the excretion of [ 99m Tc]Tc-PSMA-T4. This process is probably accompanied by the reabsorption of the radiopharmaceutical.
Several research groups [44][45][46][47] have shown that variously labeled urea-based molecules have an unusually high uptake in mouse kidneys. This uptake is mainly specific as a consequence of physiologically expressed PSMA in mouse kidneys [44] but is also associated with renally localized glutamate carboxypeptidase II (NAALADase) belonging to the metallopeptidase family [48]. NAALADase has been located in both neural and non-neural tissues, such as the kidney, prostate, and small intestine [49][50][51]. Luthi-Carter et al. cloned, characterized, and found the NAALADase to be homologous to the prostate cancer marker PSMA [52]. Therefore, it is highly likely that such a specific accumulation of [ 99m Tc]Tc-PSMA-T4 in the kidneys is due to the presence of binding targets in the animals' kidneys. Their concentration may vary depending on the type and species of rodent.
Relatively high tracer concentrations were observed in the spleen, in which the %ID/g was more than 1% up to 6 h (4.1 ± 0.9 %ID/g and 1.5 ± 0.5 %ID/g at 2 h and 6 h, respectively).
Specific binding of PSMA-T4 to PSMA receptors in the LNCaP cells was confirmed by the in vivo study using tumor-bearing mice. The saturation of the receptor protein was determined by adding the respective 100-fold excess of unlabeled PSMA-T4 (22 µg) to a bolus of [ 99m Tc]Tc-PSMA-T4 (100 µL, 0.22 µg, 10 MBq). The obtained results are presented in Figure 7 and Table S7.
Using a 100-fold excess of unlabeled PSMA-T4 as a targeting tracer, the biodistribution data at 4 h showed a minimal (0.83 ± 0.53 %ID/g) non-specific tumor uptake, a low renal uptake (1.17 ± 0.58 %ID/g) and rapid excretion with urine.

Kit formulation
Pre-formulation studies were performed using PSMA-T4, which was selected bas on the promising in vitro and in vivo evaluation. The influence of quantity and numb of co-ligands were verified in the initial step. The radiochemical purity of [ 99m Tc]Tc-PSM T4 in the presence of one co-ligand ethylenediamine N,N-diacetic acid (EDDA) or trici is presented in Table S1.
The use of two co-ligand systems such as EDDA/tricine, and the selection of the a propriate quantity of the corresponding co-ligand to obtain radiochemical purity PSMA-T4 over 90% is shown in Table S2. Radiolabeling of these formulations show radiochemical purity (RCP) >90% for 50 mg of tricine and 5 mg of EDDA as the small possible quantity of co-ligands necessary for properly binding technetium-99m to HYN chelator.
The stannous chloride dihydrate reduces technetium-99m in the form of pertechn tate to lower oxidation state. The content of stannous chloride dihydrate was tested fro 10 to 150 µg/vial. It was concluded that the use of 10 µg/vial of the reducing agent result in satisfactory results in the labeling yield. Stannous chloride dihydrate is easily oxidiz during the lyophilization process, so it is necessary to use more tin in the initial stage the process. On the other hand, applying a higher content of stannous chloride dehydr than 50 µg/vial increases the probability of forming undesirable impurities in the form reduced, colloidal forms of technetium-99m. For further evaluation, the formulation co taining 50 µg SnCl2, 2H2O was chosen, Table S3.
PSMA-T4 amount should be low to avoid possible receptor saturation and undesi ble pharmacological effect.
It was shown that even 23 µg of PSMA-T4 in the labeling solutions would enable t efficient preparation of [ 99m Tc]Tc-PSMA-T4. This amount of PSMA-T4 assures repeata achievement of the [ 99m Tc]Tc-PSMA-T4 of the highest quality and stability even using radiolabeling technetium-99m of radioactivity as high as 1.5 GBq .
The composition of the developed PSMA-T4 kit, which allows obtaining the techn

Kit formulation
Pre-formulation studies were performed using PSMA-T4, which was selected based on the promising in vitro and in vivo evaluation. The influence of quantity and number of co-ligands were verified in the initial step. The radiochemical purity of [ 99m Tc]Tc-PSMA-T4 in the presence of one co-ligand ethylenediamine N,N-diacetic acid (EDDA) or tricine is presented in Table S1.
The use of two co-ligand systems such as EDDA/tricine, and the selection of the appropriate quantity of the corresponding co-ligand to obtain radiochemical purity of PSMA-T4 over 90% is shown in Table S2. Radiolabeling of these formulations showed radiochemical purity (RCP) >90% for 50 mg of tricine and 5 mg of EDDA as the smallest possible quantity of co-ligands necessary for properly binding technetium-99m to HYNIC chelator.
The stannous chloride dihydrate reduces technetium-99m in the form of pertechnetate to lower oxidation state. The content of stannous chloride dihydrate was tested from 10 to 150 µg/vial. It was concluded that the use of 10 µg/vial of the reducing agent resulted in satisfactory results in the labeling yield. Stannous chloride dihydrate is easily oxidized during the lyophilization process, so it is necessary to use more tin in the initial stage of the process. On the other hand, applying a higher content of stannous chloride dehydrate than 50 µg/vial increases the probability of forming undesirable impurities in the form of reduced, colloidal forms of technetium-99m. For further evaluation, the formulation containing 50 µg SnCl 2 , 2H 2 O was chosen, Table S3.
PSMA-T4 amount should be low to avoid possible receptor saturation and undesirable pharmacological effect.
It was shown that even 23 µg of PSMA-T4 in the labeling solutions would enable the efficient preparation of [ 99m Tc]Tc-PSMA-T4. This amount of PSMA-T4 assures repeatable achievement of the [ 99m Tc]Tc-PSMA-T4 of the highest quality and stability even using for radiolabeling technetium-99m of radioactivity as high as 1.5 GBq.
The composition of the developed PSMA-T4 kit, which allows obtaining the technetium-99m labeled PSMA inhibitor with high radiochemical purity without the additional purification, is presented in Table 9.

Synthesis of PSMA-HYNIC Derivatives
The synthesis of PSMA-HYNIC compounds (including iPSMA) and PSMA-11 were started from the solid phase synthesis of the Glu(tBu)-urea-Lys-NH2 by the method described previously [29]. Further steps of synthesis where performed by standard solid phase synthesis procedures with use of 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminemorpholino)]uronium hexafluorophosphate (COMU) as a coupling reagent and N,N-diisopropylethylamine as a base. The final PSMA-HYNIC ligands were purified by the preparative HPLC method on the C18 reversed phase column. The identity of the compounds was confirmed by LCMS-IT-TOF and 1 H, 13 C, 15 N NMR, and purity were analyzed by the HPLC method.  [53] was used in the in vitro study as a reference substance. It was synthesized and radiolabeled, with minor modifications, according to method described by Maresca et al. [16]. The organostannanyl intermediate was isolated and purified before radiolabeling with 131 I. For radiolabeling 1 GBq (~45 µL) of 131 I was used, resulting 470 MBq of final purified [ 131 I]I-MIP-1095 (specific activity 156 GBq/mg) of >90% HPLC purity.

[ 99 Tc]Tc-PSMA-T4 Complex
The radiocomplexes with long-lived technetium-99 were obtained with 2 molar excess of 99 Tc to peptide starting from PSMA-T4 kit and using standardized labeling conditions. The kit vial was reconstituted with 385 µL of water. To this solution, 100 µL of Na 99m TcO 4 (100 MBq) eluate, and 15 µL of NH 4 [ 99 Tc]TcO 4 solution in water (400 µg/mL) were added. The mixture was incubated for 15 min at 100 • C. The labeled species were analyzed by LC-MS using electrospray ionization mass spectroscopy with positive ionization mode.
LC-MS analysis was run on Shimadzu HPLC Prominence system equipped with mass spectrometer LCMS-IT-TOF system consisting of electrospray source (ESI), ion trap (IT), time of flight analyzer (TOF), and the LCMS Solution software. The samples were analyzed using the same LC analytical method for the radiochemical purity determination with a lower TFA addition of 0.05% to the elution solvents.

Lipophilicity Determination
The partition coefficient logD of the radioligands were determined by shake-flask method [54,55]. The solution of 10 to 50 MBq of technetium-99m radiolabeled PSMA ligands (T1-T4) and iPSMA in 1.0 mL of phosphate-buffered saline (PBS, pH 7.4) was added to 1.0 mL of pre-saturated n-octanol solution (n = 3). Vials were shaken vigorously for 10 min. To achieve quantitative phase separation, the vials were centrifuged at 1600 rpm for 5 min. The radioactivity concentration in a defined volume of both the aqueous and the organic phase in six replicates each was measured in a γ-counter (Wallac Wizard 1470, PerkinElmer, USA). The partition coefficient was calculated as the logarithm of the ratio between counts per minute (cpm) measured in the n-octanol phase to the PBS phase counts.

Cell Uptake
The LNCaP cells in concentration 5 × 10 5 per well were cultured on 12 wells plates to confluence. After 48 h of incubation, the wells were washed with un-supplemented RPMI 1640. The cells were incubated with one concentration of radiolabeled compounds for 2 h. After incubation, cells were washed with PBS. The binding of tested radiocomplexes to PSMA antigen was evaluated by measurement of the radioactivity of 1 mL of glycine buffer (50 mM in 0.1 M NaCl, pH 2.8) used for rinsing the cells. The internalization was determined by radioactivity of 1 mL 1 M NaOH used for the lysis. Total binding was calculated as a sum of membrane and internalized fraction.

Cell Membrane Isolation
Cells from prostate cancer carcinomas: LNCaP and PC3, were isolated like previously described [56]. The cells were harvested using 1 mL/flask of Trypsin-EDTA solution and centrifuged (1500 rpm for 15 min (LNCaP) and 1300 rpm for 6 min (PC3)). Received cell sediment was resuspended in 9.7 mM NaHCO 3 containing 0.3 mM NaH 2 PO 4 and incubated for 30 min at 4 • C. The solution was dispensed on 1.5 mL Eppendorf and centrifuged (13,500 rpm for 30 min). The supernatant was removed, the pellet was resuspended in 50 mM Tris-HCl Buffer (pH 7.4) and homogenized using a syringe with a thin needle. The solution was centrifuged (13,500 rpm for 30 min), and the final pellet was suspended in 50 mM Tris-HCl buffer (pH 7.4) and transferred to −70 • C.

Saturation Binding
The K d value for 99m Tc-labeled PSMA ligands were determined by saturation binding analysis on membranes isolated from LNCaP (PSMA+). Unspecific binding was evaluated using PC3 (PSMA−) cell membrane, and specific binding was evaluated as a difference of total binding (LNCaP) and unspecific binding to the human prostate cancer cell line (PC3). Cells were incubated with 41-131,000 pM radiolabeled PSMA inhibitors for 2 h at 37 • C on special MultiScreen™ 96 well assay plates (Merck). The supernatant solution was filtered under vacuum, and the membranes were washed twice using phosphate buffer saline (PBS, IITD Wroclaw). The filters containing membranes with the attached radiotracer were extruded into tubes using MultiScreen Multiple Punch (Merck). The number of associated inhibitors was determined by measuring the radioactivity of the filters using Wallac Wizard 1470 γ counter. The K d was analyzed and calculated by nonlinear regression using GraphPad Prism 9.4 statistic program.

Competitive Binding
The LNCaP cell membranes were distributed (in four replicates) in the constant amount on special MultiScreen™ 96 well assay plates. 100 µL of un-supplemented RPMI 1640 medium and 50 µL of increasing concentration of the unlabeled PSMA inhibitors was added to each well. As a control, 50 µL of PBS in 100 µL of media was used. After 15 min incubation at 37 • C, 50 µL of [ 131 I]I-MIP-1095 was added. Plates were incubated for 2 h at 37 • C. After an appropriate time, the supernatant solution was filtered under vacuum, and the membranes were washed twice with PBS solution. Filters and membranes were extruded to the tubes using MultiScreen Multiple Punch. The radioactivity of the filters was measured using a Wallac Wizard 1470 γ counter. The IC50 was evaluated and determined using GraphPad Prism 9.4 statistic program. As a control, PSMA-11 was used.
3.11. In Vivo Study 3.11.1. Animal Models BALB/c NUDE male mice (5-6 weeks old, mean body mass of 20 g) were purchased from the Charles River Laboratories (Sulzfeld, Germany). Wistar male rats and BALB/c mice (both 5-7 weeks old) were purchased from the M. Mossakowski Institute of Experimental and Clinical Medicine, Polish Academy of Sciences in Warsaw (Poland).
On arrival, animals were housed for 5 days in groups of five in standard cages (Wistar rats and BALB/c mice) and IVS cages (BALB/c NUDE) in the animal facility of the Radioisotope Centre POLATOM (Otwock, Poland). They were housed in a quiet room under constant conditions (22 • C, 50% relative humidity, 12-h light/dark cycles with dark periods from 7 p.m. to 7 a.m.) with free access to standard food and water. Veterinarian staff and investigators observed the rodents daily to ensure animal welfare and determine if humane endpoints were reached (e.g., hunched and ruffled appearance, apathy, ulceration, severe weight loss, tumor burden). Experimental procedures were carried out in conformity with the National Legislation and the Council Directive of the European Communities on the Protection of Animals Used for Experimental and Other Scientific Purposes (2010/63/UE) and the "ARRIVE guidelines for reporting animal research" [57]. The POLATOM protocol was approved by the Ist Local Animal Ethics Committee in Warsaw (authorization 681/2018, approval date 4 July 2018 for both type of mice, and authorization 400/2017, approval date 21 November 2017 for rats).
The standard protocol involved animals randomized into fixed groups (five per group). Before injection, the labeled compounds were diluted in 0.9% NaCl and then intravenously injected (0.1 mL per mice and 0.2 mL per rat). At established time points after-injection, the animals were euthanized by cervical dislocation and dissected. Selected organs and tissues were weighed and their radioactivity was measured in a gamma counter equipped with a NaI(Tl) crystal. The physiological distribution was calculated and expressed in terms of the percentage of administrated radioactivity found in each of the selected organs or tissues per gram (%ID/g) with the aid of suitable standards of the injected dose. The xenografted mice were prepared according to the methods previously given in the literature [29]. The animals were randomized into two groups (five mice per group): Group 1, treated with a single intravenous injection of [ 99m Tc]Tc-PSMA-T4 (0.1 mL, 0.2 µg, 9.5 MBq, SA 16.7 GBq/mg = 17.2 GBq/µmol) and Group 2, where the PSMA receptors were locally blocked with a single intravenous co-injection of PSMA-T4 (dose range: 100× mass equivalent of the radioactive [ 99m Tc]Tc-PSMA-T4 dose). The mice were euthanized after 4 h p.i.v.

Statistics
Results are provided as mean ± SD. The results of physiological distribution expressed as a percentage of the dose administered per gram of tissue (%ID/g) were presented in the form of an average with standard deviation (mean ± standard deviation (SD)), with n representing the number of animals per group. Data were statistically analyzed using GraphPad Prism version 9.4 for Windows and tested for normal distribution with the Kolmogorov-Smirnov test. In case of normal distribution, results were assessed by two-tailed, unpaired Student's t-tests. Otherwise, results were assessed by two-way ANOVA. A p value of <0.05 with two-tailed testing was considered statistically significant.
For blood activity data in healthy Wistar rats, a mono-exponential decay model (Equation (1)) was used to describe the percentage of remaining activity (%ID/g) as a function of time post-injection (t): where: • Span is the difference between %ID/g (0) and Plateau • %ID/g (0) is the %ID/g value when t (time) is zero • Plateau is the %ID/g value at infinite times • K is the rate constants For renal excretion of [ 99m Tc]Tc-PSMA-T4, the two-phases model was applied (Equation (2)).

Conclusions
This work presents the development and preclinical evaluation of a novel PSMA-T4 ligand (Glu-CO-Lys-L-Trp-4-Amc-HYNIC), which showed excellent radiolabeling characteristics, high selectivity towards PSMA receptors in vitro and favorable tumor accumulation in LNCaP tumor-bearing mice. As a final result, we prepared a single vial lyophilized kit to prepare [ 99m Tc]Tc-PSMA-T4 with desired radiochemical and pharmaceutical purity. This kit allows for rapid and reproducible preparation of radiopharmaceuticals in a hospital environment [58]. Preclinical studies in the tumor-bearing mice indicated a high tracer accumulation in the PSMA-positive tumor and led to steadily increasing tumor to muscle ratios (T/M) over time (e.g., T/M: 78, 174, 262 after 2 h, 6 h, and 24 h p.i.v, respectively). Such properties mean that [ 99m Tc]Tc-PSMA-T4 can be an effective tracer for SPECT imaging, even after a long time after administration. It may therefore also prove useful for the radioguided surgery (RGS) of patients with mPCa. Furthermore, [ 99m Tc]Tc-PSMA-T4 showed effectiveness in SPECT studies in humans, and soon, its usefulness will be extensively evaluated in phase 2/3 clinical trials (EudraCT No. 2021-005113-14).