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

The Radiolabeling of [161Tb]-PSMA-617 by a Novel Radiolabeling Method and Preclinical Evaluation by In Vitro/In Vivo Methods. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Radiolabeling of [161Tb]-PSMA-617 by a Novel Radiolabeling Method and Preclinical Evaluation by In Vitro/In Vivo Methods. EMRE UYGUR, Ceren Sezgin, Yasemin Parlak, Kadriye Busra Karatay, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3415703/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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, [ 161 Tb], a radiolanthanide introduced for treating micrometastatic foci, has shown great promise for treating prostate cancer. Results In this study, Terbium-161 [ 161 Tb]Tb was radiolabeled with prostate-specific membrane antigen (PSMA)-617 ([ 161 Tb]-PSMA-617) and the therapeutic efficacy of the radiolabeled compound investigated in vitro and in vivo . [ 161 Tb]-PSMA-617 was found to have a radiochemical yield of 97.99 ± 2.01% and was hydrophilic. [ 161 Tb]-PSMA-617 was also shown to have good stability, with a radiochemical yield of over 95% up to 72 hours. In vitro , [ 161 Tb]-PSMA-617 showed a cytotoxic effect on LNCaP cells but not on PC-3 cells. In vivo , scintigraphy imaging visualized the accumulation of [ 161 Tb]-PSMA-617 in the prostate, kidneys, and bladder. Conclusions The results suggest that [ 161 Tb]-PSMA-617 can be an effective radiolabeled agent for the treatment of PSMA positive foci in prostate cancer. Terbium-161 [161Tb]Tb PSMA-617 prostate cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Prostate cancer is the second most prevalent cancer among men and the fifth leading cause of cancer-related 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, defined 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 first-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, significant 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 field 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 specific 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 significantly 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 ). In terms of PSMA-targeted radionuclide therapy, various clinical studies have reported on the use of [ 177 Lu]-PSMA-617, [ 225 Ac]-PSMA-617, and [ 161 Tb]-PSMA-617 to treat metastatic castration resistant prostate cancer (Baum et al., 2016 ; Fendler et al., 2017 , 2019 ; Feuerecker et al., 2021 ; Gourni et al., 2017 ; Kessel et al., 2019 ; Kratochwil, Bruchertseifer, et al., 2016 ; Kratochwil et al., 2017 , 2018 ; Kratochwil, Giesel, et al., 2016 ; Rahbar et al., 2017 ; Sathekge et al., 2019 , 2020 ; Violet et al., 2020 ; Yadav et al., 2020 ). The use of [ 177 Lu]Lu as a theranostic agent has shown promising results (Baum et al., 2016 ; Fendler et al., 2017 ; Gourni et al., 2017 ). It is effective in prolonging the lives of patients, particularly in cases with larger lesions. However, it's important to note that the energy released by [ 177 Lu]Lu may not completely eliminate microscopic disease, highlighting the need for complementary treatments or therapies to address residual or smaller lesions (Kessel et al., 2019 ; Rahbar et al., 2017 ). As such, the use of [ 225 Ac]Ac has also been investigated (Feuerecker et al., 2021 ; Kratochwil, Bruchertseifer, et al., 2016 ; Kratochwil, Giesel, et al., 2016 ; Müller, Umbricht, et al., 2019 ). Of note, the first studies on the use of PSMA in radioligand-based therapy focused on its use for nuclear imaging and radioactive iodine therapy. PSMA ligands with various chelators were only later developed to enable their use with different radiometals for imaging and therapeutic purposes (Fendler et al., 2017 ). Currently, PSMA I&T (Imaging and Therapy) and PSMA-617 equipped with a DOTAGA and DOTA chelator, respectively, are used in the clinic for targeted radioligand therapy of metastatic castration resistant prostate cancer (Kratochwil, Giesel, et al., 2016 ; Sathekge et al., 2020 ; Violet et al., 2020 ; Yadav et al., 2020 ). For end-stage patients without other treatment options, PSMA ligands radiolabeled with [ 177 Lu]Lu (T 1/2 = 6.65 d; Eβ͞ av = 134 keV; E γ = 113 keV, I = 6.117%, E γ = 208 keV, I = 10.36%) are used [5], and [ 225 Ac]-PSMA-617 has been used in some special cases (Feuerecker et al., 2021 ; Kratochwil, Bruchertseifer, et al., 2016 ; Sathekge et al., 2019 ; Yadav et al., 2020 ). More recently, the radiolanthanide [ 161 Tb]Tb has been introduced for therapeutic applications because it emits β¯particles (E β͞av = 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 (Müller, Umbricht, et al., 2019 ). [ 161 Tb]Tb decays to the stable 161 Dy with a half-life of 6.89 days (Collins et al., 2022 ). Also, [ 161 Tb]Tb is very similar to [ 177 Lu]Lu in terms of radiochemical properties, although the γ-radiation emitted by [ 161 Tb]Tb is of a lower energy. In addition, the most important advantage of [ 161 Tb]Tb is that it emits a significant number of low energy conversions and auger electrons comparison with [ 177 Lu]Lu. This holds great promise for the treatment of prostate cancer that has progressed to disease with multiple metastases of various sizes (Borgna, Barritt, et al., 2021 ; Grünberg et al., 2014 ). In Hindié et al.’s study (Hindie et al., 2016 ), Monte Carlo simulations comparing [ 177 Lu]Lu with [ 161 Tb]Tb showed that the effect of [ 161 Tb]Tb was 3.6 and 1.8 times that of [ 177 Lu]Lu in a 10-µm cell and 1.8 times 100-µm micrometastasis, respectively. Some studies already indicate that [ 161 Tb]Tb outperforms other clinically used ([ 177 Lu]Lu, [ 90 Y]Y) and non-standard therapeutic radionuclides ([ 47 Sc]Sc, [ 67 Cu]Cu) in terms of dose delivery to small lesions (Fendler et al., 2017 ; Gourni et al., 2017 ; Hindie et al., 2016 ). In this study, the radiopharmaceutical potential of [ 161 Tb]-PSMA-617 radiolabeled with new method (Patent Id: TP23-1225) was investigated for the first time in Turkey through in vitro and in vivo methods. Methods Chemicals and Materials PSMA-617 was purchased from EDH Health Co (İstanbul, Turkey). Thin-layer chromatography paper (ITLC-silica), ammonium acetate, n-octanol, methanol and acetonitrile were purchased from Merck Chemical (İstanbul, Turkey). Minimum Essential Medium (MEM) non-essential amino acid, Dulbecco’s Modified Eagle Medium (DMEM), MEM Eagle, Roswell Park Memorial Institute (RPMI) 1640 medium, sodium bicarbonate, sodium pyruvate, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypan blue, phosphate buffer solution, and trypsin ethylenediaminetetraacetic acid (EDTA) were purchased from Biological Industries (Ankara, Turkey). [ 161 Tb]TbCl 3 was supplied by Terthera (Breda, Netherlands). PC-3 and LNCaP cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) Radiolabeling and Quality Control 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 ). Specifically, 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. Quality control studies were carried out using radio-TLC, with silica gel TLC strips and 3 mobile phases (Solvent 1: ammonium acetate (1M): methanol (1:1 v/v); Solvent 2: 100% ACN; and Solvent 3: 65 mL of solution A (2.94 g trisodium-citrate-dihydrate solved in 100 mL water) + 35 mL of solution B (2.10 g citric acid-monohydrate solved in 100 mL water)). Radio-TLC measurements were accomplished using a Perkin Elmer Cyclone Storage System (Massachusetts, USA) and a TLC scanner (Bioscan AR-2000 Scanner, Berlin, Germany). A low-pressure gradient high-performance liquid chromatography (HPLC) system [quaternary pump (LC-10ATvp), an NaI(Tl) radioactivity detector (Gabi Star, Raytest Angleur, Belgium), an autosampler (SIL-20A HT), a diode array detector (DAD; SPD-M20A), a fraction collector (FRC-10A), and a column (RP-C18; 5 lm, 250 4.6 mm I.D., ODS GL Sciences, Tokyo, Japan)] was also used, with methanol/dH 2 O (v/v, 80:20) as the mobile phase and a flow rate 1 mL/min. Radioactivity of the radiolabeled compound ([ 161 Tb]-PSMA-617) was confirmed using an NaI (Tl) detector (Gabi Star, Raytest, Belgium) at 210−254 nm wavelengths in the HPLC system. Stability Studies [ 161 Tb]-PSMA-617, i.e., PSMA-617 radiolabeled with [ 161 Tb]Tb under optimum conditions as confirmed 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. Sufficiently 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 96-well 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 efficiency 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 efficiency (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. Scintigraphy Imaging Scintigraphy imaging studies were performed on male Wistar Albino rats (n = 3). Rats were administered 2 mL/kg of the anesthetic agent [2.5 mL of ketamine (80 mg/kg) + 0.5 mL of SF + 2 mL xylazine (4 mg/kg)]. Anaesthetized rats were intravenously injected with [ 161 Tb]-PSMA-617 (~ 33.3 MBq) via the tail vein. Scintigraphy images were obtained with a dual-head gamma camera (Infinia, GE, Tirat Hacermel, Israel), with a low-energy, high-resolution collimator, imaging the whole body. After the injection of [ 161 Tb]-PSMA-617, static images were obtained at different time intervals (0.5, 1, 2, 4, 24 hours after injection) with a 256 × 256 matrix. CT images were also obtained. 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 sacrificed 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. Significance testing was conducted at a confidence level of 95% (p < 0.05) to determine if there was a significant difference between the intake and uptake values. Results and Discussion Results In this study, Terbium-161 [ 161 Tb]Tb was radiolabeled with PSMA-617 to yield [ 161 Tb]-PSMA-617 and the therapeutic efficacy 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 [ 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 significant 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. The findings 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 specific 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 significant elevation was noted in specific anatomical sites, including the pancreas, musculature, adipose tissue, salivary glands, and thyroid. Discussion There is increasing interest worldwide in the use of Tb radioisotopes in nuclear medicine applications for cancer therapy and diagnosis (Al-Ibraheem et al., 2023 ; Borgna, Barritt, et al., 2021 ; Borgna, Haller, et al., 2021 ; Cassells et al., 2021 ; De Jong et al., 1995 ; Favaretto et al., 2021 ; Grünberg et al., 2014 ; Hindie et al., 2016 ; Müller et al., 2014 ; Müller, Singh, et al., 2019 ; Müller, Umbricht, et al., 2019 ). Particularly, promising results have been reported concerning the potential of [ 161 Tb]-radiolabeled compounds for radionuclide therapy (Al-Ibraheem et al., 2023 ; Cassells et al., 2021 ; Müller, Umbricht, et al., 2019 ). In this study, Terbium-161 [ 161 Tb]Tb was radiolabeled with PSMA-617 to yield [ 161 Tb]-PSMA-617 and the therapeutic efficacy 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). [ 161 Tb]-PSMA-617 molecule maintained its stability for 72 hours with a yield over 95%. [ 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 (Borgna, Barritt, et al., 2021 ; Gracheva et al., 2019 ; Müller et al., 2014 ). The stability of the radioligand [ 161 Tb]-PSMA-617 is not significantly affected by the emitted conversion and Auger electrons, since its radiolytic decay is due to its behavior, similar to that of [ 177 Lu]-PSMA-617. 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 ). In Müller et al.’s study, PSMA-617 labeled with [ 161 Tb]Tb ≥ 98% radiochemical purity and specific 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 L-ascorbic 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. 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 affinity 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 ) 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 significantly 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 0.05–5.0 MBq/ML (P < 0.05 for both). Also, the average energy absorbed by tumor cells was also 3.2–4.2 times higher for [ 161 Tb]-PSMA-617 than [ 177 Lu]-PSMA-617 in their MTT experiments. 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 confirm these results. According to 2-way ANOVA for the optimum time of cell retention, a significant difference was found between [ 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/flu 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 flu tumor on the left side. Like LNCaP cells in our study, PC3-PIP cells are androgen receptor cells for which PSMA-617 exhibits higher affinity, 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. 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 specific 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 identifiable 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 blood-tumor 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 significant elevation was noted in specific 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. Conclusions While there have only been a few studies on [ 161 Tb]-PSMA-617 for the treatment of prostate cancer in the literature, the remarkable results obtained thus far in this study and in the literature may encourage more interest in [ 161 Tb]-PSMA-617. Specifically, this preclinical study will pave the way for further preclinical research activities on [ 161 Tb]-PSMA-617 by our research team, with the aim of clinical applications in the near future to benefit patients with metastatic metastatic castration resistant prostate cancer. Abbreviations Cd(Te) Cadmium telluride DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTAGA 2,2′,2”-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid HPLC High Performance Liquid chromatography PARP poly ADP ribose polymerase PC Prostate Cancer PSMA Prostate specific membran antigen ROI Region of interest SPECT Single Photon Emission Computed Tomography TLC Thin liquid chromatography Declarations Acknowledgements The patents for the radiolabeling method are supported by Ege University and Manisa Celal Bayar University. We also thank Ahmet Mutlu for his contribution to the in vivo biodistribution studies. Author contributions All authors contributed to the design of the study. E.U, C.S, Y.P, F.G.G, K.B.K, B.A, U.A, O.A and F.Z.B.M were responsible for acquiring and collecting the data. T.T, S.B and C.H performed the data analysis. E.U completed the frst draft of the manuscript. T.S.S, S.M, H.S, O.A, F.G.G, and F.Z.B.M reviewed and approved the manuscript. All authors read and approved the final manuscript. Funding This study is supported by EDH Health Co., and Terthera Co. Howard Scher and Omer Aras were partially supported by the NIH/NCI Cancer Center Support Grant P30 CA008748. Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Code availability Not applicable. Ethics approval and consent to participate 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). All methods were carried out in accordance with Manisa Celal Bayar University Local Animal Experiments Ethics Committee and ARRIVE guidelines and regulations. Consent for publication Not applicable. Competing interests None of the authors have a conflict of interest. Author details 1 Biomedical Device Technologies, Manisa Celal Bayar University, Şehit Prof. Dr. İlhan Varank Campus, Yunusemre, Manisa, 45140, Turkey, 2 Department of Nuclear Medicine, Manisa City Hospital, Adnan Menderes Neighborhood. 132nd Street, Şehzadeler, Manisa, 45140, Turkey, 3 Department of Nuclear Medicine, Manisa Celal Bayar University, Uncubozköy Campus, Mimar Sinan Street, Manisa, 45140, Turkey, 4 Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Erzene Street, Bornova, Izmir, 35100, Turkey, 5 Department of Chemistry, Faculty of Art and Science, Manisa Celal Bayar University, Şehit Prof. Dr. İlhan Varank Campus, Yunusemre, Manisa, 45140, Turkey, 6 Institute of Health Sciences, Yeditepe University, 26 Ağustos Campus, Ataşehir, İstanbul,34755 Turkey, 7 Department of Internal Medicine, Faculty of Medicine, Aydın Adnan Menderes University, Zafer Street, Efeler, Aydın, 09010, Turkey, 8 Ege Higher Vocational School, Ege University Erzene Street, Bornova, Izmir, 35100, Turkey, 9 Faculty of Medicine, Department of Urology, Gazi University, Emniyet Street, Yenimahalle, Ankara, 06500, Turkey, 10 Department of Nuclear Medicine, Saarland University-Medical Center, Kirrbergerstrasse, D-66421 Homburg, Germany, 11 Department of Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, United States, 12 Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, United States References Al-Ibraheem, A., Doudeen, R. 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3","display":"","copyAsset":false,"role":"figure","size":45133,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3415703/v1/7dffb9ee160a42bd092de635.jpg"},{"id":45482745,"identity":"2c48f792-c868-496c-9ae5-331672265bb0","added_by":"auto","created_at":"2023-10-30 20:51:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56727,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3415703/v1/c5b0911921bc20ae8419c947.jpg"},{"id":45482747,"identity":"747d8d7e-c75c-4fcc-8ded-4cdec06a039c","added_by":"auto","created_at":"2023-10-30 20:51:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76515,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3415703/v1/0cf3d1d285f37c7b2c953053.jpg"},{"id":45482748,"identity":"b807f95d-6edd-4850-9e39-4772227e772a","added_by":"auto","created_at":"2023-10-30 20:51:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86223,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3415703/v1/253467b8632b48fc81629c99.jpg"},{"id":45482746,"identity":"37ea2d4c-7d1c-41d2-86ab-2e743d129b61","added_by":"auto","created_at":"2023-10-30 20:51:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82067,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3415703/v1/3c4521b3ea7a86346f08d2ee.jpg"},{"id":47407570,"identity":"310fa863-23e5-4f60-8f31-8aa599bdc637","added_by":"auto","created_at":"2023-12-01 01:45:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":676414,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3415703/v1/d1ea4400-ce95-421f-bd7d-13bd6dedfc8f.pdf"}],"financialInterests":"","formattedTitle":"The Radiolabeling of [161Tb]-PSMA-617 by a Novel Radiolabeling Method and Preclinical Evaluation by In Vitro/In Vivo Methods.","fulltext":[{"header":"Background","content":"\u003cp\u003eProstate cancer is the second most prevalent cancer among men and the fifth leading cause of cancer-related deaths in males globally (Arnold et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; He et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The management of prostate cancer at disease presentation is based on disease extent, defined by states(Scher \u0026amp; Heller, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) ranging from clinically localized disease to clinical metastases in need of or having been treated with androgen deprivation therapy. Androgen deprivation therapy remains the first-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, significant 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., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The field 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., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); further, prostate specific membrane antigen (PSMA)-directed approaches are emerging as an especially potent treatment strategy (Kratochwil, Giesel, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCollectively, 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., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; M\u0026uuml;ller, Singh, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). PSMA is a glycoprotein found on the surface of cells. While it is naturally expressed in normal prostate tissue, it is significantly 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., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn terms of PSMA-targeted radionuclide therapy, various clinical studies have reported on the use of [\u003csup\u003e177\u003c/sup\u003eLu]-PSMA-617, [\u003csup\u003e225\u003c/sup\u003eAc]-PSMA-617, and [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 to treat metastatic castration resistant prostate cancer (Baum et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fendler et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Feuerecker et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gourni et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kessel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kratochwil, Bruchertseifer, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kratochwil et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kratochwil, Giesel, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rahbar et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sathekge et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Violet et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The use of [\u003csup\u003e177\u003c/sup\u003eLu]Lu as a theranostic agent has shown promising results (Baum et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fendler et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gourni et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is effective in prolonging the lives of patients, particularly in cases with larger lesions. However, it's important to note that the energy released by [\u003csup\u003e177\u003c/sup\u003eLu]Lu may not completely eliminate microscopic disease, highlighting the need for complementary treatments or therapies to address residual or smaller lesions (Kessel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rahbar et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As such, the use of [\u003csup\u003e225\u003c/sup\u003eAc]Ac has also been investigated (Feuerecker et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kratochwil, Bruchertseifer, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kratochwil, Giesel, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Of note, the first studies on the use of PSMA in radioligand-based therapy focused on its use for nuclear imaging and radioactive iodine therapy. PSMA ligands with various chelators were only later developed to enable their use with different radiometals for imaging and therapeutic purposes (Fendler et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Currently, PSMA I\u0026amp;T (Imaging and Therapy) and PSMA-617 equipped with a DOTAGA and DOTA chelator, respectively, are used in the clinic for targeted radioligand therapy of metastatic castration resistant prostate cancer (Kratochwil, Giesel, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sathekge et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Violet et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For end-stage patients without other treatment options, PSMA ligands radiolabeled with [\u003csup\u003e177\u003c/sup\u003eLu]Lu (T\u003csub\u003e1/2\u003c/sub\u003e = 6.65 d; Eβ͞\u003csub\u003eav\u003c/sub\u003e = 134 keV; E\u003csub\u003eγ\u003c/sub\u003e = 113 keV, I\u0026thinsp;=\u0026thinsp;6.117%, E\u003csub\u003eγ\u003c/sub\u003e = 208 keV, I\u0026thinsp;=\u0026thinsp;10.36%) are used [5], and [\u003csup\u003e225\u003c/sup\u003eAc]-PSMA-617 has been used in some special cases (Feuerecker et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kratochwil, Bruchertseifer, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sathekge et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMore recently, the radiolanthanide [\u003csup\u003e161\u003c/sup\u003eTb]Tb has been introduced for therapeutic applications because it emits β\u0026macr;particles (E\u003csub\u003eβ͞av\u003c/sub\u003e = 154 keV) as well as γ-radiation (E\u003csub\u003eγ\u003c/sub\u003e = 49 keV, I\u0026thinsp;=\u0026thinsp;17.0%; E\u003csub\u003eγ\u003c/sub\u003e = 75 keV, I\u0026thinsp;=\u0026thinsp;10.0%) that are suitable for therapeutic purposes and single-photon emission computed tomography (SPECT), respectively (M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). [\u003csup\u003e161\u003c/sup\u003eTb]Tb decays to the stable \u003csup\u003e161\u003c/sup\u003eDy with a half-life of 6.89 days (Collins et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Also, [\u003csup\u003e161\u003c/sup\u003eTb]Tb is very similar to [\u003csup\u003e177\u003c/sup\u003eLu]Lu in terms of radiochemical properties, although the γ-radiation emitted by [\u003csup\u003e161\u003c/sup\u003eTb]Tb is of a lower energy. In addition, the most important advantage of [\u003csup\u003e161\u003c/sup\u003eTb]Tb is that it emits a significant number of low energy conversions and auger electrons comparison with [\u003csup\u003e177\u003c/sup\u003eLu]Lu. This holds great promise for the treatment of prostate cancer that has progressed to disease with multiple metastases of various sizes (Borgna, Barritt, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gr\u0026uuml;nberg et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In Hindi\u0026eacute; et al.\u0026rsquo;s study (Hindie et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), Monte Carlo simulations comparing [\u003csup\u003e177\u003c/sup\u003eLu]Lu with [\u003csup\u003e161\u003c/sup\u003eTb]Tb showed that the effect of [\u003csup\u003e161\u003c/sup\u003eTb]Tb was 3.6 and 1.8 times that of [\u003csup\u003e177\u003c/sup\u003eLu]Lu in a 10-\u0026micro;m cell and 1.8 times 100-\u0026micro;m micrometastasis, respectively. Some studies already indicate that [\u003csup\u003e161\u003c/sup\u003eTb]Tb outperforms other clinically used ([\u003csup\u003e177\u003c/sup\u003eLu]Lu, [\u003csup\u003e90\u003c/sup\u003eY]Y) and non-standard therapeutic radionuclides ([\u003csup\u003e47\u003c/sup\u003eSc]Sc, [\u003csup\u003e67\u003c/sup\u003eCu]Cu) in terms of dose delivery to small lesions (Fendler et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gourni et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hindie et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the radiopharmaceutical potential of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 radiolabeled with new method (Patent Id: TP23-1225) was investigated for the first time in Turkey through \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e methods.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Materials\u003c/h2\u003e \u003cp\u003ePSMA-617 was purchased from EDH Health Co (İstanbul, Turkey). Thin-layer chromatography paper (ITLC-silica), ammonium acetate, n-octanol, methanol and acetonitrile were purchased from Merck Chemical (İstanbul, Turkey). Minimum Essential Medium (MEM) non-essential amino acid, Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), MEM Eagle, Roswell Park Memorial Institute (RPMI) 1640 medium, sodium bicarbonate, sodium pyruvate, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypan blue, phosphate buffer solution, and trypsin ethylenediaminetetraacetic acid (EDTA) were purchased from Biological Industries (Ankara, Turkey). [\u003csup\u003e161\u003c/sup\u003eTb]TbCl\u003csub\u003e3\u003c/sub\u003e was supplied by Terthera (Breda, Netherlands). PC-3 and LNCaP cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRadiolabeling and Quality Control\u003c/h2\u003e \u003cp\u003eA new radiolabeling method was developed by optimizing the radiolabeling of PSMA-617 with [\u003csup\u003e161\u003c/sup\u003eTb]Tb according to the literature (Al-Ibraheem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Specifically, 1 mL sodium acetate buffer (labelling buffer) and 185 MBq [\u003csup\u003e161\u003c/sup\u003eTb]TbCl\u003csub\u003e3\u003c/sub\u003e were added into a tube containing 50 \u0026micro;L ascorbic acid and the reaction mixture (pH 4.5) was incubated at 95\u0026deg;C for 10 min. Then, 25 \u0026micro;L of PSMA-617 was added to the mixture. The mixture was incubated in a hot pot at 95\u0026deg;C for ~\u0026thinsp;25 min and subsequently cooled at room temperature.\u003c/p\u003e \u003cp\u003eQuality control studies were carried out using radio-TLC, with silica gel TLC strips and 3 mobile phases (Solvent 1: ammonium acetate (1M): methanol (1:1 v/v); Solvent 2: 100% ACN; and Solvent 3: 65 mL of solution A (2.94 g trisodium-citrate-dihydrate solved in 100 mL water)\u0026thinsp;+\u0026thinsp;35 mL of solution B (2.10 g citric acid-monohydrate solved in 100 mL water)). Radio-TLC measurements were accomplished using a Perkin Elmer Cyclone Storage System (Massachusetts, USA) and a TLC scanner (Bioscan AR-2000 Scanner, Berlin, Germany). A low-pressure gradient high-performance liquid chromatography (HPLC) system [quaternary pump (LC-10ATvp), an NaI(Tl) radioactivity detector (Gabi Star, Raytest Angleur, Belgium), an autosampler (SIL-20A HT), a diode array detector (DAD; SPD-M20A), a fraction collector (FRC-10A), and a column (RP-C18; 5 lm, 250 4.6 mm I.D., ODS GL Sciences, Tokyo, Japan)] was also used, with methanol/dH\u003csub\u003e2\u003c/sub\u003eO (v/v, 80:20) as the mobile phase and a flow rate 1 mL/min. Radioactivity of the radiolabeled compound ([\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617) was confirmed using an NaI (Tl) detector (Gabi Star, Raytest, Belgium) at 210\u0026minus;254 nm wavelengths in the HPLC system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStability Studies\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617, i.e., PSMA-617 radiolabeled with [\u003csup\u003e161\u003c/sup\u003eTb]Tb under optimum conditions as confirmed by quality control studies, was dropped (2.5 \u0026micro;L) 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLipophilicity Studies\u003c/h2\u003e \u003cp\u003e300 \u0026micro;L of n-octanol and 300 \u0026micro;L of ultrapure water were placed in a centrifuge tube, 150 \u0026micro;L of [\u003csup\u003e161\u003c/sup\u003eTb]-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 \u0026micro;L 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).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eCell Culture Studies\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePC3 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. Sufficiently proliferated cells were removed using trypsin-EDTA solution and seeded in 24- or 96-well plates and kept at 37 \u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e until use in further studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMTT Tests\u003c/h2\u003e \u003cp\u003eSolutions containing [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA at different concentrations corresponding to 1, 2, 4, and 8 \u0026micro;g 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 96-well plate was incubated at 37 \u003csup\u003eo\u003c/sup\u003eC in 5% CO\u003csub\u003e2\u003c/sub\u003e environment for 24 hours. At the 24th hour, 10 \u0026micro;L 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) \u0026times; 100. The absorbance of the negative control was accepted as zero.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIncorporation\u003c/h2\u003e \u003cp\u003eIn order to determine the uptake efficiency of [\u003csup\u003e161\u003c/sup\u003eTb]-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 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e 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 [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 (4.625 MBq / 0.625 \u0026micro;g PSMA) were added to each well. In the experimental study, each plate's culture medium containing 4.625 MBq [\u003csup\u003e161\u003c/sup\u003eTb]TbCl\u003csub\u003e3\u003c/sub\u003e was added as a control group. At 1, 2, 4, 8, and 24 hours, the initial amount of radioactivity (A\u003csub\u003e0\u003c/sub\u003e) 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 \u0026micro;L of PBS was added to each well and radioactivity counting (A\u003csub\u003e1\u003c/sub\u003e) was performed again. The A1 and A0 values detected for the radiolabeled compound and free [\u003csup\u003e161\u003c/sup\u003eTb] were ratioed to determine the % binding efficiency (A\u003csub\u003e1\u003c/sub\u003e/A\u003csub\u003e0\u003c/sub\u003e * 100). In each cell line, all time parameters were performed in 3 replicates to reach enough repetitions of the study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eStudies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale Wistar Albino rats were used for scintigraphy imaging (n\u0026thinsp;=\u0026thinsp;3) and for biodistribution studies (n\u0026thinsp;=\u0026thinsp;12) of [\u003csup\u003e161\u003c/sup\u003eTb]PSMA-617 within the scope of \u003cem\u003ein vivo\u003c/em\u003e studies. Ethics committee approval for \u003cem\u003ein vivo\u003c/em\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eScintigraphy Imaging\u003c/h2\u003e \u003cp\u003eScintigraphy imaging studies were performed on male Wistar Albino rats (n\u0026thinsp;=\u0026thinsp;3). Rats were administered 2 mL/kg of the anesthetic agent [2.5 mL of ketamine (80 mg/kg)\u0026thinsp;+\u0026thinsp;0.5 mL of SF\u0026thinsp;+\u0026thinsp;2 mL xylazine (4 mg/kg)]. Anaesthetized rats were intravenously injected with [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 (~\u0026thinsp;33.3 MBq) via the tail vein. Scintigraphy images were obtained with a dual-head gamma camera (Infinia, GE, Tirat Hacermel, Israel), with a low-energy, high-resolution collimator, imaging the whole body. After the injection of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617, static images were obtained at different time intervals (0.5, 1, 2, 4, 24 hours after injection) with a 256 \u0026times; 256 matrix. CT images were also obtained.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eBiodistribution Studies\u003c/h2\u003e \u003cp\u003eBiodistribution studies were performed in 12 rats at the 1st, 4th, 24th, and 48th hour (n\u0026thinsp;=\u0026thinsp;3 rats for each time point) after the injection of [\u003csup\u003e161\u003c/sup\u003eTb]-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 sacrificed 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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eMean radiochemical yields and standard deviations were calculated, with three replicates conducted for each parameter. For \u003cem\u003ein vitro\u003c/em\u003e cell culture studies, the Graph Pad program was utilized to conduct one-way analysis of variance (ANOVA) and Pearson correlation statistics. Significance testing was conducted at a confidence level of 95% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to determine if there was a significant difference between the intake and uptake values.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\n\u003ch3\u003eResults\u003c/h3\u003e\n\u003cp\u003eIn this study, Terbium-161 [\u003csup\u003e161\u003c/sup\u003eTb]Tb was radiolabeled with PSMA-617 to yield [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 and the therapeutic efficacy of the radiolabeled compound investigated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The radiochemical yield of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was determined using radio-TLC and HPLC. Based on the radio-TLC chromatograms presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the R\u003csub\u003ef\u003c/sub\u003e (Relative Front) values of [\u003csup\u003e161\u003c/sup\u003eTb]Tb, [\u003csup\u003e161\u003c/sup\u003eTb]Tb\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e, and [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 were 0.053, 0.043, and 0.073, respectively. Conversely, according to the HPLRC chromatograms seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the retention times of PSMA-617, [\u003csup\u003e161\u003c/sup\u003eTb]Tb, and [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 were 2.663, 3.373, and 3.043 minutes, respectively. The radiochemical yield of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was 97.98% \u0026plusmn; 2.01 (n\u0026thinsp;=\u0026thinsp;6) based on these measurements. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 molecule maintained its stability for 72 hours with a yield over 95%. In terms of lipophilicity, the logP value of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was \u0026minus;\u0026thinsp;2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, with the negative logP value indicating that the [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 molecule is hydrophilic.\u003c/p\u003e \u003cp\u003eThe cytotoxicity graph based on LNCaP and PC3 viability values is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It was observed that [\u003csup\u003e161\u003c/sup\u003eTb]-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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, showing that the uptake rate of [\u003csup\u003e161\u003c/sup\u003eTb]-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 significant difference was found between [\u003csup\u003e161\u003c/sup\u003eTb]TbCl\u003csub\u003e3\u003c/sub\u003e and [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in LNCaP and PC3 cells.\u003c/p\u003e \u003cp\u003eScintigraphy imaging visualized the accumulation of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in the prostate, kidneys, and the bladder. Static images of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in rats demonstrated that substantial tracer accumulation was present in the kidneys at 30 min, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. In addition, [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 activity in the abdominal and chest region also increased with time. [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 activity was almost entirely excreted after 4 h by renal excretion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe findings stemming from our investigation of the biodistribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) patterns of [\u003csup\u003e161\u003c/sup\u003eTb]-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 specific inclination toward renal tissues underscores the dominant route of excretion for [\u003csup\u003e161\u003c/sup\u003eTb]-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 significant elevation was noted in specific anatomical sites, including the pancreas, musculature, adipose tissue, salivary glands, and thyroid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDiscussion\u003c/h3\u003e\n\u003cp\u003eThere is increasing interest worldwide in the use of Tb radioisotopes in nuclear medicine applications for cancer therapy and diagnosis (Al-Ibraheem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Borgna, Barritt, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Borgna, Haller, et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cassells et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; De Jong et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Favaretto et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gr\u0026uuml;nberg et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hindie et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; M\u0026uuml;ller et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; M\u0026uuml;ller, Singh, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Particularly, promising results have been reported concerning the potential of [\u003csup\u003e161\u003c/sup\u003eTb]-radiolabeled compounds for radionuclide therapy (Al-Ibraheem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cassells et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, Terbium-161 [\u003csup\u003e161\u003c/sup\u003eTb]Tb was radiolabeled with PSMA-617 to yield [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 and the therapeutic efficacy of the radiolabeled compound investigated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The radiochemical yield is an important parameter for radiopharmaceuticals and is expected to be over 95%. In this study, the radiochemical yield of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was 97.98% \u0026plusmn; 2.01 (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003cp\u003e[\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 molecule maintained its stability for 72 hours with a yield over 95%. [\u003csup\u003e161\u003c/sup\u003eTb]Tb and [\u003csup\u003e177\u003c/sup\u003eLu]Lu are both radiolanthanides with similar chemical properties, allowing them to form stable radiometal complexes through chelation with DOTA chelator. This means that [\u003csup\u003e161\u003c/sup\u003eTb]Tb can be used with the same DOTA-functionalized biomolecules currently employed with [\u003csup\u003e177\u003c/sup\u003eLu]Lu. The convenience of [\u003csup\u003e161\u003c/sup\u003eTb]Tb being commercially available in dilute hydrochloric acid solution, like [\u003csup\u003e177\u003c/sup\u003eLu]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 [\u003csup\u003e161\u003c/sup\u003eTb]Tb or [\u003csup\u003e177\u003c/sup\u003eLu]Lu (Borgna, Barritt, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gracheva et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; M\u0026uuml;ller et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The stability of the radioligand [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 is not significantly affected by the emitted conversion and Auger electrons, since its radiolytic decay is due to its behavior, similar to that of [\u003csup\u003e177\u003c/sup\u003eLu]-PSMA-617.\u003c/p\u003e \u003cp\u003eOur result in this study that [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 has a radiochemical yield of 97.98% \u0026plusmn; 2.01 is similar to the radiochemical yield of 98% reported in M\u0026uuml;ller et al\u0026rsquo;s study (M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In M\u0026uuml;ller et al.\u0026rsquo;s study, PSMA-617 labeled with [\u003csup\u003e161\u003c/sup\u003eTb]Tb\u0026thinsp;\u0026ge;\u0026thinsp;98% radiochemical purity and specific activities up to 100 MBq/nmol. While [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 remained stable (\u0026gt;\u0026thinsp;98%) for 1 hour during incubation, radiolytic degradation occurred after. To avoid degradation, [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was maintained in the presence of L-ascorbic acid, where it showed stability (\u0026ge;\u0026thinsp;98%) for up to 24 hours without degradation. In our study which used a new method to radiolabel PSMA-617 with [\u003csup\u003e161\u003c/sup\u003eTb]Tb, optimized based on the existing literature, the use of L-ascorbic acid was also essential to ensure the stability of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617. According to our results, [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was stable for 72 hours in the presence of L-Ascorbic acid. Of note, [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in Al-Ibraheem et al.\u0026rsquo;s study also required the use of L-ascorbic acid to ensure stability (Al-Ibraheem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the logP value of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was \u0026minus;\u0026thinsp;2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, with the negative logP value indicating that the [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 molecule is hydrophilic. Meanwhile, a lipophilicity value of \u0026minus;\u0026thinsp;3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 was reported in M\u0026uuml;ller et al.\u0026rsquo;s study (M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The difference in lipophilicity values is thought to be due to the equipment used; whereas Cd(Te) detector was used in this study, M\u0026uuml;ller et al. obtained measurements with a Perkin Elmer, Wallac Wizard 1480 Gamma Counter.\u003c/p\u003e \u003cp\u003eAccording to the cytotoxicity graph, [\u003csup\u003e161\u003c/sup\u003eTb]-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 affinity 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 M\u0026uuml;ller et al.\u0026rsquo;s study (M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) which demonstrated \u003cem\u003ein vitro\u003c/em\u003e that the viability and survival of PSMA-positive PC-3 PIP tumor cells decreased corresponding to the administered activity concentration of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617. Further, M\u0026uuml;ller et al. found that [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was significantly more effective than [\u003csup\u003e177\u003c/sup\u003eLu]-PSMA-617 in decreasing tumor cell viability (at an activity concentration of 0.1\u0026ndash;10 MBq/mL) and survival (at an activity concentration of 0.05\u0026ndash;5.0 MBq/ML (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both). Also, the average energy absorbed by tumor cells was also 3.2\u0026ndash;4.2 times higher for [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 than [\u003csup\u003e177\u003c/sup\u003eLu]-PSMA-617 in their MTT experiments.\u003c/p\u003e \u003cp\u003eAccording to the graph of cell incorporation, the uptake rate of [\u003csup\u003e161\u003c/sup\u003eTb]-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 confirm these results. According to 2-way ANOVA for the optimum time of cell retention, a significant difference was found between [\u003csup\u003e161\u003c/sup\u003eTb]TbCl\u003csub\u003e3\u003c/sub\u003e and [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in LNCaP and PC3 cells. \u003cem\u003eIn vitro\u003c/em\u003e studies comparing [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 and [\u003csup\u003e177\u003c/sup\u003eLu]-PSMA-617 in the literature have noted that [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 showed 3 times more uptake compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-PSMA-617 in the PC3-PIP cell line (Gracheva et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), probably due to the incorporation of the PSMA-617 peptide by the cells and the Auger electrons emitted by [\u003csup\u003e161\u003c/sup\u003eTb]Tb (M\u0026uuml;ller et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eScintigraphy imaging visualized the accumulation of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in the prostate, kidneys, and the bladder. Static images of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in rats the substantial tracer accumulation was present in the kidneys at 30 min. In addition, [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 activity in the abdominal and chest region also increased with time. [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 activity was almost entirely excreted after 4 h by renal excretion. Our results here are similar to M\u0026uuml;ller et al.\u0026rsquo;s study (M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where SPECT/CT images were obtained of PC-3 PIP/flu tumor-bearing mice at 1 h, 4 h, and 24 h after being injected with ~\u0026thinsp;25 MBq [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617. In that study, while [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 accumulated in the PIP-3 tumor xenograft on the right side, there was only negligible uptake in the PSMA-negative PC-3 flu tumor on the left side. Like LNCaP cells in our study, PC3-PIP cells are androgen receptor cells for which PSMA-617 exhibits higher affinity, explaining the accumulation of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 on the right side. They also reported that renal excretion of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was rapid, with almost the entire activity excreted within 4 hours.\u003c/p\u003e \u003cp\u003eBiodistribution results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) were compatible with the imaging results. Biodistribution of [\u003csup\u003e161\u003c/sup\u003eTb]-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 specific inclination toward renal tissues underscores the dominant route of excretion for [\u003csup\u003e161\u003c/sup\u003eTb]-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 identifiable cause for this trend lies in the noticeable absence of an established tumor model within the experimental group of Albino Wistar rats. In contrast, M\u0026uuml;ller et al.\u0026rsquo;s study (M\u0026uuml;ller, Umbricht, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) which involved well-established tumor models consistently demonstrated a declining trajectory in systemic [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 levels, as evidenced by the blood-tumor 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 significant elevation was noted in specific 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.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWhile there have only been a few studies on [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 for the treatment of prostate cancer in the literature, the remarkable results obtained thus far in this study and in the literature may encourage more interest in [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617. Specifically, this preclinical study will pave the way for further preclinical research activities on [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 by our research team, with the aim of clinical applications in the near future to benefit patients with metastatic metastatic castration resistant prostate cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCd(Te)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCadmium telluride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDOTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDOTAGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,2\u0026prime;,2\u0026rdquo;-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePARP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epoly ADP ribose polymerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstate Cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstate specific membran antigen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRegion of interest\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPECT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle Photon Emission Computed Tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThin liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe patents for the radiolabeling method are supported by Ege University and Manisa Celal Bayar University. We also thank Ahmet Mutlu for his contribution to the \u003cem\u003ein vivo\u003c/em\u003e biodistribution studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the design of the study. E.U, C.S, Y.P, F.G.G, K.B.K, B.A, U.A, O.A and F.Z.B.M were responsible for acquiring and collecting the data. T.T, S.B and C.H performed the data analysis. E.U completed the frst draft of the manuscript. T.S.S, S.M, H.S, O.A, F.G.G, and F.Z.B.M reviewed and approved the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study is supported by EDH Health Co., and Terthera Co. Howard Scher and Omer Aras were partially supported by the NIH/NCI Cancer Center Support Grant P30 CA008748.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics committee approval for \u003cem\u003ein vivo\u003c/em\u003e 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). All methods were carried out in accordance with Manisa Celal Bayar University Local Animal Experiments Ethics Committee and ARRIVE guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors have a conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eBiomedical Device Technologies, Manisa Celal Bayar University, Şehit Prof. Dr. İlhan Varank Campus, Yunusemre, Manisa, 45140, Turkey, \u003csup\u003e2\u003c/sup\u003eDepartment of Nuclear Medicine, Manisa City Hospital, Adnan Menderes Neighborhood. 132nd Street, Şehzadeler, Manisa, 45140, Turkey, \u003csup\u003e3\u003c/sup\u003eDepartment of Nuclear Medicine, Manisa Celal Bayar University, Uncubozk\u0026ouml;y Campus, Mimar Sinan Street, Manisa, 45140, Turkey, \u003csup\u003e4\u003c/sup\u003eDepartment of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Erzene Street, Bornova, Izmir, 35100, Turkey, \u003csup\u003e5\u003c/sup\u003eDepartment of Chemistry, Faculty of Art and Science, Manisa Celal Bayar University, Şehit Prof. Dr. İlhan Varank Campus, Yunusemre, Manisa, 45140, Turkey, \u003csup\u003e6\u003c/sup\u003eInstitute of Health Sciences, Yeditepe University, 26 Ağustos Campus, Ataşehir, İstanbul,34755 Turkey,\u003csup\u003e7\u003c/sup\u003eDepartment of Internal Medicine, Faculty of Medicine, Aydın Adnan Menderes University, Zafer Street, Efeler, Aydın, 09010, Turkey, \u003csup\u003e8\u003c/sup\u003eEge Higher Vocational School, Ege University Erzene Street, Bornova, Izmir, 35100, Turkey, \u003csup\u003e9\u003c/sup\u003eFaculty of Medicine, Department of Urology, Gazi University, Emniyet Street, Yenimahalle, Ankara, 06500, Turkey,\u003csup\u003e10\u003c/sup\u003eDepartment of Nuclear Medicine, Saarland University-Medical Center, Kirrbergerstrasse, D-66421 Homburg, Germany, \u003csup\u003e11\u003c/sup\u003eDepartment of Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, United States, \u003csup\u003e12\u003c/sup\u003eDepartment of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, United States\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl-Ibraheem, A., Doudeen, R. 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R., K\u0026ouml;ster, U., Schibli, R., \u0026amp; van der Meulen, N. P. (2019). Terbium-161 for PSMA-targeted radionuclide therapy of prostate cancer. \u003cem\u003eEuropean Journal of Nuclear Medicine and Molecular Imaging\u003c/em\u003e, \u003cem\u003e46\u003c/em\u003e(9), 1919\u0026ndash;1930. https://doi.org/10.1007/s00259-019-04345-0\u003c/li\u003e\n\u003cli\u003eRahbar, K., Ahmadzadehfar, H., Kratochwil, C., Haberkorn, U., Schafers, M., Essler, M., Baum, R. P., Kulkarni, H. R., Schmidt, M., Drzezga, A., Bartenstein, P., Pfestroff, A., Luster, M., Lutzen, U., Marx, M., Prasad, V., Brenner, W., Heinzel, A., Mottaghy, F. M., \u0026hellip; Krause, B. J. (2017). 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Efficacy and safety of 225Ac-PSMA-617 targeted alpha therapy in metastatic castration-resistant prostate cancer patients. \u003cem\u003eTheranostics\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(20), 9364\u0026ndash;9377. https://doi.org/10.7150/THNO.48107\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Terbium-161 [161Tb]Tb, PSMA-617, prostate cancer","lastPublishedDoi":"10.21203/rs.3.rs-3415703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3415703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eProstate 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, [\u003csup\u003e161\u003c/sup\u003eTb], a radiolanthanide introduced for treating micrometastatic foci, has shown great promise for treating prostate cancer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, Terbium-161 [\u003csup\u003e161\u003c/sup\u003eTb]Tb was radiolabeled with prostate-specific membrane antigen (PSMA)-617 ([\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617) and the therapeutic efficacy of the radiolabeled compound investigated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was found to have a radiochemical yield of 97.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01% and was hydrophilic. [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 was also shown to have good stability, with a radiochemical yield of over 95% up to 72 hours. \u003cem\u003eIn vitro\u003c/em\u003e, [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 showed a cytotoxic effect on LNCaP cells but not on PC-3 cells. \u003cem\u003eIn vivo\u003c/em\u003e, scintigraphy imaging visualized the accumulation of [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 in the prostate, kidneys, and bladder.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results suggest that [\u003csup\u003e161\u003c/sup\u003eTb]-PSMA-617 can be an effective radiolabeled agent for the treatment of PSMA positive foci in prostate cancer.\u003c/p\u003e","manuscriptTitle":"The Radiolabeling of [161Tb]-PSMA-617 by a Novel Radiolabeling Method and Preclinical Evaluation by In Vitro/In Vivo Methods.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-30 20:51:50","doi":"10.21203/rs.3.rs-3415703/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"160dfe8e-d8f3-4ed5-9510-52d356f3c671","owner":[],"postedDate":"October 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-13T09:30:54+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-30 20:51:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3415703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3415703","identity":"rs-3415703","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}