The Radiolabeling of [161Tb]-PSMA-617 by a Novel Radiolabeling Method and Preclinical Evaluation by In Vitro/In Vivo Methods

Background Prostate cancer (PC) is the most common type of cancer in elderly men, with a positive correlation with age. As resistance to treatment has developed, particularly in the progressive stage of the disease and in the presence of microfocal multiple bone metastases, new generation radionuclide therapies have emerged. Recently, [161Tb], a radiolanthanide introduced for treating micrometastatic foci, has shown great promise for treating prostate cancer. Results In this study, Terbium-161 [161Tb]Tb was radiolabeled with prostate-specific membrane antigen (PSMA)-617 ([161Tb]-PSMA-617) and the therapeutic efficacy of the radiolabeled compound investigated in vitro and in vivo. [161Tb]-PSMA-617 was found to have a radiochemical yield of 97.99 ± 2.01% and was hydrophilic. [161Tb]-PSMA-617 was also shown to have good stability, with a radiochemical yield of over 95% up to 72 hours. In vitro, [161Tb]-PSMA-617 showed a cytotoxic effect on LNCaP cells but not on PC-3 cells. In vivo, scintigraphy imaging visualized the accumulation of [161Tb]-PSMA-617 in the prostate, kidneys, and bladder. Conclusions The results suggest that [161Tb]-PSMA-617 can be an effective radiolabeled agent for the treatment of PSMA positive foci in prostate cancer.


Background
Prostate cancer is the second most prevalent cancer among men and the fth leading cause of cancerrelated deaths in males globally (Arnold et al., 2015;He et al., 2021).The management of prostate cancer at disease presentation is based on disease extent, de ned by states (Scher & Heller, 2000) ranging from clinically localized disease to clinical metastases in need of or having been treated with androgen deprivation therapy.Androgen deprivation therapy remains the rst-line standard systemic approach for tumors at a high risk of metastasizing or that have already spread to distant sites and can be given in the form of monotherapy or in combination with recently approved next-generation inhibitors of androgen signaling to produce a dramatic response.However, androgen deprivation therapy is not curative and virtually all cancers treated with this therapy progress to a metastatic castration resistant state which is lethal for most patients.Hence, in the ever-evolving landscape of prostate cancer treatment, signi cant strides have been made to further improve patient outcomes, including the development of approved agents like taxanes and radium which have been pivotal in managing this complex disease since their introduction (Corn et al., 2019).The eld has now further transitioned into the era of precision medicine, marked by the approval of poly ADP ribose polymerase (PARP) inhibitors and the recognition of microsatellite instability alterations as promising therapeutic targets (Fujimoto et al., 2021); further, prostate speci c membrane antigen (PSMA)-directed approaches are emerging as an especially potent treatment strategy (Kratochwil, Giesel, et al., 2016).
Collectively, advancements to date in the management of prostate cancer have laid the foundation for the next generation of theranostic PSMA-directed approaches, with terbium (Tb) poised to play a central role (Al-Ibraheem et al., 2023;Müller, Singh, et al., 2019).PSMA is a glycoprotein found on the surface of cells.While it is naturally expressed in normal prostate tissue, it is signi cantly upregulated or overexpressed in cases prostate cancer.Studies report that PSMA expression level is associated with disease stage and the risk of progression (Kratochwil, Giesel, et al., 2016).
More recently, the radiolanthanide [ 161 Tb]Tb has been introduced for therapeutic applications because it emits β¯particles (E β av = 154 keV) as well as γ-radiation (E γ = 49 keV, I = 17.0%;E γ = 75 keV, I = 10.0%) that are suitable for therapeutic purposes and single-photon emission computed tomography (SPECT), respectively ( In this study, the radiopharmaceutical potential of [ 161 Tb]-PSMA-617 radiolabeled with new method (Patent Id: TP23-1225) was investigated for the rst time in Turkey through in vitro and in vivo methods.

Stability Studies
[ 161 Tb]-PSMA-617, i.e., PSMA-617 radiolabeled with [ 161 Tb]Tb under optimum conditions as con rmed by quality control studies, was dropped (2.5 µL) onto TLC plates at 1, 2, 4, 24, 48 and 72 hours, respectively.TLC silica gel strips were run in the optimum bath, i.e., Solvent 1, and additional quality control studies were carried out using radio-TLC.In addition, the variation of the % radiochemical yield versus time was analyzed.
Lipophilicity Studies 300 µL of n-octanol and 300 µL of ultrapure water were placed in a centrifuge tube, 150 µL of [ 161 Tb]-PSMA-617 was added, and the whole mixture was vortexed for 1 min.Then, the upper and lower phases were separated by centrifugation at 1000 rpm for 30 min.150 µL of these phases were sampled and a Cd(Te) (RAD-501, Isın Electronics, Izmir, Turkey) detector was used to measure the radioactivity between phases.LogP, i.e., lipophilicity, values were then calculated using the formula log (CPS n-octanol phase/ CPS phosphate buffer phase).
In Vitro Cell Culture Studies PC3 cells were grown in DMEM, 2 mM of glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM of non-essential amino acids, 1 mM of sodium pyruvate, and 10% of FBS.Meanwhile, LNCaP cells were grown in RPMI 1640 medium, 2 mM of glutamine, 1.5 g/L of sodium bicarbonate, 0.1 mM of non-essential amino acids, 1 mM of sodium pyruvate, and 20% of FBS.Cryotubes in a nitrogen tank were opened and cells were grown in appropriate media and passaged to reach the number of cells required.Su ciently proliferated cells were removed using trypsin-EDTA solution and seeded in 24-or 96-well plates and kept at 37 o C and 5% CO 2 until use in further studies.

MTT Tests
Solutions containing [ 161 Tb]-PSMA at different concentrations corresponding to 1, 2, 4, and 8 µg of PSMA per well and 0.2, 0.4, 0.8, and 1.6 mCi activity were added to PC3 and LNCaP cells seeded in 96-well plate (104 cells per well).As a negative control, cell-free medium was added to the wells.Subsequently, the 96well plate was incubated at 37 o C in 5% CO 2 environment for 24 hours.At the 24th hour, 10 µL of MTT solution was added to each well and the 96-well plate was kept under the same conditions for another 4 hours.At the end of those 4 hours, the 96-well plate was read by a spectrophotometer at 570 nm wavelength and the absorbance value for each well was determined.Viability (%) values were calculated using the following formula: viability = (measured absorbance value/control value) × 100.The absorbance of the negative control was accepted as zero.

Incorporation
In order to determine the uptake e ciency of [ 161 Tb]-PSMA-617 on cell lines, cells belonging to both cell lines in the experimental and study groups were seeded in 24-well culture dishes with 5 × 10 3 cells and 0.5 mL of medium in each well.The time parameters to be examined in the study were determined as 1, 2, 4, 8, and 24 h.Media containing [ 161 Tb]-PSMA-617 (4.625 MBq / 0.625 µg PSMA) were added to each well.In the experimental study, each plate's culture medium containing 4.625 MBq [ 161 Tb]TbCl 3 was added as a control group.At 1, 2, 4, 8, and 24 hours, the initial amount of radioactivity (A 0 ) per well was determined by counting the activity of the labeled medium on the cells in each well using a Cd(Te) detector.When the planned incubation periods were completed, the labeled media in the wells were removed and the cells were washed with sterile PBS.500 µL of PBS was added to each well and radioactivity counting (A 1 ) was performed again.The A1 and A0 values detected for the radiolabeled compound and free [ 161 Tb] were ratioed to determine the % binding e ciency (A 1 /A 0 * 100).In each cell line, all time parameters were performed in 3 replicates to reach enough repetitions of the study.

In Vivo Studies
Male Wistar Albino rats were used for scintigraphy imaging (n = 3) and for biodistribution studies (n = 12) of [ 161 Tb]PSMA-617 within the scope of in vivo studies.Ethics committee approval for in vivo studies were obtained from the Manisa Celal Bayar University Local Animal Experiments Ethics Committee (approval date, February 28, 2023; protocol number 77.637.435-254).The male Wistar Albino rats were obtained from Manisa Celal Bayar University Experimental Animal Center.

Biodistribution Studies
Biodistribution studies were performed in 12 rats at the 1st, 4th, 24th, and 48th hour (n = 3 rats for each time point) after the injection of [ 161 Tb]-PSMA-617 into the tail vein.The activity of the injectors in the full state just before injection and the activity of the injectors in the empty state after injection was measured using a dose calibrator (CRC-55t, Capintec, New Jersey, USA) and the net mean injection activity was determined to be 37 MBq (1 mCi).After injection, the rats were sacri ced under anesthesia and the blood, the heart, the lung, the liver, the kidney, the small intestine, the large intestine, the stomach, the spleen, the pancreas, the muscle, the testis, the prostate, the fat, the bladder, the brain, the salivary glands, the thyroid, the skin, and the stool parts were removed.Extracted samples were placed in pre-tightened containers and weighed with a precision balance, and then activity counts were obtained using a Cd(Te) detector.Activity values for each organ/tissue were calculated in Microsoft Excel, accounting for time corrections, and the % ID/g-time graph of each organ/tissue was drawn.

Statistical Analysis
Mean radiochemical yields and standard deviations were calculated, with three replicates conducted for each parameter.For in vitro cell culture studies, the Graph Pad program was utilized to conduct one-way analysis of variance (ANOVA) and Pearson correlation statistics.Signi cance testing was conducted at a con dence level of 95% (p < 0.05) to determine if there was a signi cant difference between the intake and uptake values.

Results
In this study, Terbium-161 [ 161 Tb]Tb was radiolabeled with PSMA-617 to yield [ 161 Tb]-PSMA-617 and the therapeutic e cacy of the radiolabeled compound investigated in vitro and in vivo.The radiochemical yield of [ 161 Tb]-PSMA-617 was determined using radio-TLC and HPLC.Based on the radio-TLC chromatograms presented in Fig. 1, the R f (Relative Front) values of [ 161 Tb]Tb, [ 161 Tb]Tb + 3 , and [ 161 Tb]-PSMA-617 were 0.053, 0.043, and 0.073, respectively.Conversely, according to the HPLRC chromatograms seen in Fig. 2, the retention times of PSMA-617, [ 161 Tb]Tb, and [ 161 Tb]-PSMA-617 were 2.663, 3.373, and 3.043 minutes, respectively.The radiochemical yield of [ 161 Tb]-PSMA-617 was 97.98% ± 2.01 (n = 6) based on these measurements.Figure 3 shows that the [ 161 Tb]-PSMA-617 molecule maintained its stability for 72 hours with a yield over 95%.In terms of lipophilicity, the logP value of The ndings stemming from our investigation of the biodistribution (Fig. 7) patterns of [ 161 Tb]-PSMA-617 in Albino Wistar rats revealed a notable concentration of the compound within a 24-hour timeframe in the renal, vesicular, and urinary compartments.This speci c inclination toward renal tissues underscores the dominant route of excretion for [ 161 Tb]-PSMA-617 being through the kidneys as noted above.Hematological dynamics displayed an initial surge over a 24-hour period, followed by a subsequent reduction at the 48-hour mark.At the 24-hour time point, a marked increase in fecal content was observed, while at the subsequent 48-hour time point, a statistically signi cant elevation was noted in speci c anatomical sites, including the pancreas, musculature, adipose tissue, salivary glands, and thyroid.

Discussion
There is increasing interest worldwide in the use of  Tb]-PSMA-617 and the therapeutic e cacy of the radiolabeled compound investigated in vitro and in vivo.The radiochemical yield is an important parameter for radiopharmaceuticals and is expected to be over 95%.In this study, the radiochemical yield of [ 161 Tb]-PSMA-617 was 97.98% ± 2.01 (n = 6).
Our result in this study that [ 161 Tb]-PSMA-617 has a radiochemical yield of 97.98% ± 2.01 is similar to the radiochemical yield of 98% reported in Müller et al's study (Müller, Umbricht, et al., 2019) According to the cytotoxicity graph, [ 161 Tb]-PSMA-617 at increased concentrations showed a cytotoxic effect on LNCaP cells, while no cytotoxic effect was observed on PC-3 cells.This can be attributed to the fact that LNCaP cells are androgen receptor cells, and PSMA-617 exhibits higher a nity towards these cells.On the other hand, PC-3 cell lines are androgen receptor-negative cells, which may explain their relatively lower survival compared to LNCaP cells.In terms of cytotoxicity, the results obtained in our study were also similar to those in Müller et al.'s study (Müller, Umbricht, et al., 2019)  According to the graph of cell incorporation, the uptake rate of [ 161 Tb]-PSMA in LNCaP and PC3 cells was approximately 40% for 4 hours.However, since PC3 is an androgen receptor-negative cell line, PSMA uptake was not expected.For this reason, further studies are planned to con rm these results.According to 2-way ANOVA for the optimum time of cell retention, a signi cant difference was found between Biodistribution results (Fig. 7) were compatible with the imaging results.Biodistribution of [ 161 Tb]-PSMA-617 in Albino Wistar rats revealed a notable concentration of the compound within a 24-hour timeframe in the renal, vesicular, and urinary compartments.This speci c inclination toward renal tissues underscores the dominant route of excretion for [ 161 Tb]-PSMA-617 being through the kidneys.Hematological dynamics displayed an initial surge over a 24-hour period, followed by a subsequent reduction at the 48-hour mark.The identi able cause for this trend lies in the noticeable absence of an established tumor model within the experimental group of Albino Wistar rats.In contrast, Müller et al.'s study (Müller, Umbricht, et al., 2019) which involved well-established tumor models consistently demonstrated a declining trajectory in systemic [ 161 Tb]-PSMA-617 levels, as evidenced by the bloodtumor ratio.In our study, at the 24-hour time point, a marked increase in fecal content was observed, while at the subsequent 48-hour time point, a statistically signi cant elevation was noted in speci c anatomical sites, including the pancreas, musculature, adipose tissue, salivary glands, and thyroid.The presence of PSMA accumulation in salivary glands is a known phenomenon in PSMA-related research, justifying the routine clinical application of cold compress therapy during the course of treatment.Similarly, the upsurge in fecal levels is interpreted as an indicative outcome of PSMA excretion via the fecal route.Furthermore, a gradual increase in prostatic tissue uptake was distinctly observed over the initial 24-hour window.In contrast, minimal alterations were observed across other tissue types.

A
new radiolabeling method was developed by optimizing the radiolabeling of PSMA-617 with [ 161 Tb]Tb according to the literature (Al-Ibraheem et al., 2023; Müller, Umbricht, et al., 2019).Speci cally, 1 mL sodium acetate buffer (labelling buffer) and 185 MBq [ 161 Tb]TbCl 3 were added into a tube containing 50 µL ascorbic acid and the reaction mixture (pH 4.5) was incubated at 95°C for 10 min.Then, 25 µL of PSMA-617 was added to the mixture.The mixture was incubated in a hot pot at 95°C for ~ 25 min and subsequently cooled at room temperature.

[
161 Tb]-PSMA-617 was − 2.15 ± 0.31, with the negative logP value indicating that the [ 161 Tb]-PSMA-617 molecule is hydrophilic.The cytotoxicity graph based on LNCaP and PC3 viability values is given in Fig.4.It was observed that [ 161 Tb]-PSMA-617 at increased concentrations showed a cytotoxic effect on LNCaP cells, while no cytotoxic effect was observed on PC-3 cells.The graph of cell incorporation results is given in Fig.5, showing that the uptake rate of [ 161 Tb]-PSMA in LNCaP and PC3 cells was approximately 40% for 4 hours.According to 2-way ANOVA for the optimum time of cell retention, a signi cant difference was found between [ 161 Tb]TbCl 3 and [ 161 Tb]-PSMA-617 in LNCaP and PC3 cells.Scintigraphy imaging visualized the accumulation of [ 161 Tb]-PSMA-617 in the prostate, kidneys, and the bladder.Static images of [ 161 Tb]-PSMA-617 in rats demonstrated that substantial tracer accumulation was present in the kidneys at 30 min, as seen in Fig. 6.In addition, [ 161 Tb]-PSMA-617 activity in the abdominal and chest region also increased with time.[ 161 Tb]-PSMA-617 activity was almost entirely excreted after 4 h by renal excretion.
[ 161 Tb]Tb and [ 177 Lu]Lu are both radiolanthanides with similar chemical properties, allowing them to form stable radiometal complexes through chelation with DOTA chelator.This means that [ 161 Tb]Tb can be used with the same DOTA-functionalized biomolecules currently employed with [ 177 Lu]Lu.The convenience of [ 161 Tb]Tb being commercially available in dilute hydrochloric acid solution, like [ 177 Lu]Lu, enables the utilization of identical labeling protocols for both radionuclides.Preliminary investigations have also shown comparable stability of radioligands, regardless of whether they are labeled with [ 161 Tb]Tb or [ 177 Lu]Lu . In Müller et al.'s study, PSMA-617 labeled with [ 161 Tb]Tb ≥ 98% radiochemical purity and speci c activities up to 100 MBq/nmol.While [ 161 Tb]-PSMA-617 remained stable (> 98%) for 1 hour during incubation, radiolytic degradation occurred after.To avoid degradation, [ 161 Tb]-PSMA-617 was maintained in the presence of Lascorbic acid, where it showed stability (≥ 98%) for up to 24 hours without degradation.In our study which used a new method to radiolabel PSMA-617 with [ 161 Tb]Tb, optimized based on the existing literature, the use of L-ascorbic acid was also essential to ensure the stability of [ 161 Tb]-PSMA-617.According to our results, [ 161 Tb]-PSMA-617 was stable for 72 hours in the presence of L-Ascorbic acid.Of note, [ 161 Tb]-PSMA-617 in Al-Ibraheem et al.'s study also required the use of L-ascorbic acid to ensure stability (Al-Ibraheem et al., 2023).In this study, the logP value of [ 161 Tb]-PSMA-617 was − 2.15 ± 0.31, with the negative logP value indicating that the [ 161 Tb]-PSMA-617 molecule is hydrophilic.Meanwhile, a lipophilicity value of − 3.90 ± 0.1 was reported in Müller et al.'s study (Müller, Umbricht, et al., 2019).The difference in lipophilicity values is thought to be due to the equipment used; whereas Cd(Te) detector was used in this study, Müller et al. obtained measurements with a Perkin Elmer, Wallac Wizard 1480 Gamma Counter.
which demonstrated in vitro that the viability and survival of PSMA-positive PC-3 PIP tumor cells decreased corresponding to the administered activity concentration of [ 161 Tb]-PSMA-617.Further, Müller et al. found that [ 161 Tb]-PSMA-617 was signi cantly more effective than [ 177 Lu]-PSMA-617 in decreasing tumor cell viability (at an activity concentration of 0.1-10 MBq/mL) and survival (at an activity concentration of 0.05-5.0MBq/ML (P < 0.05 for both).Also, the average energy absorbed by tumor cells was also 3.2-4.2times higher for [ 161 Tb]-PSMA-617 than [ 177 Lu]-PSMA-617 in their MTT experiments.

[
161 Tb]TbCl 3 and [ 161 Tb]-PSMA-617 in LNCaP and PC3 cells.In vitro studies comparing [ 161 Tb]-PSMA-617 and [ 177 Lu]-PSMA-617 in the literature have noted that [ 161 Tb]-PSMA-617 showed 3 times more uptake compared to [ 177 Lu]Lu-PSMA-617 in the PC3-PIP cell line (Gracheva et al., 2019), probably due to the incorporation of the PSMA-617 peptide by the cells and the Auger electrons emitted by [ 161 Tb]Tb (Müller et al., 2014; Müller, Umbricht, et al., 2019).Scintigraphy imaging visualized the accumulation of [ 161 Tb]-PSMA-617 in the prostate, kidneys, and the bladder.Static images of [ 161 Tb]-PSMA-617 in rats the substantial tracer accumulation was present in the kidneys at 30 min.In addition, [ 161 Tb]-PSMA-617 activity in the abdominal and chest region also increased with time.[ 161 Tb]-PSMA-617 activity was almost entirely excreted after 4 h by renal excretion.Our results here are similar to Müller et al.'s study (Müller, Umbricht, et al., 2019), where SPECT/CT images were obtained of PC-3 PIP/ u tumor-bearing mice at 1 h, 4 h, and 24 h after being injected with ~ 25 MBq [ 161 Tb]-PSMA-617.In that study, while [ 161 Tb]-PSMA-617 accumulated in the PIP-3 tumor xenograft on the right side, there was only negligible uptake in the PSMA-negative PC-3 u tumor on the left side.Like LNCaP cells in our study, PC3-PIP cells are androgen receptor cells for which PSMA-617 exhibits higher a nity, explaining the accumulation of [ 161 Tb]-PSMA-617 on the right side.They also reported that renal excretion of [ 161 Tb]-PSMA-617 was rapid, with almost the entire activity excreted within 4 hours.