Results
In this study, Terbium-161 [161Tb]Tb was radiolabeled with PSMA-617 to yield [161Tb]-PSMA-617 and the therapeutic efficacy of the radiolabeled compound investigated in vitro and in vivo. The radiochemical yield of [161Tb]-PSMA-617 was determined using radio-TLC and HPLC. Based on the radio-TLC chromatograms presented in Fig. 1, the Rf (Relative Front) values of [161Tb]Tb, [161Tb]Tb+ 3, and [161Tb]-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, [161Tb]Tb, and [161Tb]-PSMA-617 were 2.663, 3.373, and 3.043 minutes, respectively. The radiochemical yield of [161Tb]-PSMA-617 was 97.98% ± 2.01 (n = 6) based on these measurements. Figure 3 shows that the [161Tb]-PSMA-617 molecule maintained its stability for 72 hours with a yield over 95%. In terms of lipophilicity, the logP value of [161Tb]-PSMA-617 was − 2.15 ± 0.31, with the negative logP value indicating that the [161Tb]-PSMA-617 molecule is hydrophilic.
The cytotoxicity graph based on LNCaP and PC3 viability values is given in Fig. 4. It was observed that [161Tb]-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 [161Tb]-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 [161Tb]TbCl3 and [161Tb]-PSMA-617 in LNCaP and PC3 cells.
Scintigraphy imaging visualized the accumulation of [161Tb]-PSMA-617 in the prostate, kidneys, and the bladder. Static images of [161Tb]-PSMA-617 in rats demonstrated that substantial tracer accumulation was present in the kidneys at 30 min, as seen in Fig. 6. In addition, [161Tb]-PSMA-617 activity in the abdominal and chest region also increased with time. [161Tb]-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 [161Tb]-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 [161Tb]-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 [161Tb]-radiolabeled compounds for radionuclide therapy (Al-Ibraheem et al., 2023; Cassells et al., 2021; Müller, Umbricht, et al., 2019). In this study, Terbium-161 [161Tb]Tb was radiolabeled with PSMA-617 to yield [161Tb]-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 [161Tb]-PSMA-617 was 97.98% ± 2.01 (n = 6).
[161Tb]-PSMA-617 molecule maintained its stability for 72 hours with a yield over 95%. [161Tb]Tb and [177Lu]Lu are both radiolanthanides with similar chemical properties, allowing them to form stable radiometal complexes through chelation with DOTA chelator. This means that [161Tb]Tb can be used with the same DOTA-functionalized biomolecules currently employed with [177Lu]Lu. The convenience of [161Tb]Tb being commercially available in dilute hydrochloric acid solution, like [177Lu]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 [161Tb]Tb or [177Lu]Lu (Borgna, Barritt, et al., 2021; Gracheva et al., 2019; Müller et al., 2014). The stability of the radioligand [161Tb]-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 [177Lu]-PSMA-617.
Our result in this study that [161Tb]-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 [161Tb]Tb ≥ 98% radiochemical purity and specific activities up to 100 MBq/nmol. While [161Tb]-PSMA-617 remained stable (> 98%) for 1 hour during incubation, radiolytic degradation occurred after. To avoid degradation, [161Tb]-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 [161Tb]Tb, optimized based on the existing literature, the use of L-ascorbic acid was also essential to ensure the stability of [161Tb]-PSMA-617. According to our results, [161Tb]-PSMA-617 was stable for 72 hours in the presence of L-Ascorbic acid. Of note, [161Tb]-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 [161Tb]-PSMA-617 was − 2.15 ± 0.31, with the negative logP value indicating that the [161Tb]-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, [161Tb]-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 [161Tb]-PSMA-617. Further, Müller et al. found that [161Tb]-PSMA-617 was significantly more effective than [177Lu]-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 [161Tb]-PSMA-617 than [177Lu]-PSMA-617 in their MTT experiments.
According to the graph of cell incorporation, the uptake rate of [161Tb]-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 [161Tb]TbCl3 and [161Tb]-PSMA-617 in LNCaP and PC3 cells. In vitro studies comparing [161Tb]-PSMA-617 and [177Lu]-PSMA-617 in the literature have noted that [161Tb]-PSMA-617 showed 3 times more uptake compared to [177Lu]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 [161Tb]Tb (Müller et al., 2014; Müller, Umbricht, et al., 2019).
Scintigraphy imaging visualized the accumulation of [161Tb]-PSMA-617 in the prostate, kidneys, and the bladder. Static images of [161Tb]-PSMA-617 in rats the substantial tracer accumulation was present in the kidneys at 30 min. In addition, [161Tb]-PSMA-617 activity in the abdominal and chest region also increased with time. [161Tb]-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 [161Tb]-PSMA-617. In that study, while [161Tb]-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 [161Tb]-PSMA-617 on the right side. They also reported that renal excretion of [161Tb]-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 [161Tb]-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 [161Tb]-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 [161Tb]-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.