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Abstract
To elucidate potential benefits of the Auger-electron–emitting radionuclide 161Tb, we compared the preclinical performance of the gastrin-releasing peptide receptor antagonists RM2 (DOTA-Pip5-d-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) and AMTG (α-Me-Trp8-RM2), each labeled with both 177Lu and 161Tb. Methods: 161Tb/177Lu labeling (90°C, 5 min) and cell-based experiments (PC-3 cells) were performed. In vivo stability (30 min after injection) and biodistribution studies (1–72 h after injection) were performed on PC-3 tumor–bearing CB17-SCID mice. Results: Gastrin-releasing peptide receptor affinity was high for all compounds (half-maximal inhibitory concentration [nM]: [161Tb]Tb-RM2, 2.46 ± 0.16; [161Tb]Tb-AMTG, 2.16 ± 0.09; [177Lu]Lu-RM2, 3.45 ± 0.18; [177Lu]Lu-AMTG, 3.04 ± 0.08), and 75%–84% of cell-associated activity was receptor-bound. In vivo, both AMTG analogs displayed distinctly higher stability (30 min after injection) and noticeably higher tumor retention than their RM2 counterparts. Conclusion: On the basis of preclinical results, [161Tb]Tb-/[177Lu]Lu-AMTG might reveal a higher therapeutic efficacy than [161Tb]Tb-/[177Lu]Lu-RM2, particularly [161Tb]Tb-AMTG because of additional Auger-electron emissions at the cell membrane level.
In nuclear medicine, 161Tb is a promising radionuclide because it has physical properties similar to those of the clinically established 177Lu (half-life, 6.9 vs. 6.7 d; average electron energy, ∼0.15 vs. ∼0.14 MeV) and it additionally emits Auger electrons, which provide a higher linear energy transfer than β− particles (1). In general, the short-ranged Auger electrons must be close to the cell nucleus to inflict damage, limiting their usability to agonists. However, a recently reported study showed that a noninternalizing 161Tb-labeled somatostatin-2 receptor antagonist demonstrated therapeutic efficacy superior to a 161Tb-labeled somatostatin-2 receptor agonist, suggesting that Auger emissions at the cell membrane (“membrane effect”) may be therapeutic even if they do not reach the nucleus (2). This membrane effect could thus pave the way for an extended use of antagonists.
Gastrin-releasing peptide receptor (GRPR) antagonists represent an alternative for detection and treatment of prostate-specific membrane antigen–negative prostate cancer lesions (3,4), as shown using [177Lu]Lu-RM2 (DOTA-Pip5-d-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) (5). To further improve the therapeutic efficacy of radiolabeled GRPR ligands, we recently developed [177Lu]Lu-AMTG (α-Me-Trp8-RM2), a RM2 derivative (Fig. 1) demonstrating favorable biodistribution and noticeably increased in vivo stability, which resulted in higher tumor retention and, thus, increased tumor-to-background ratios in all organs (6). Because most of the cell-associated activity of these GRPR antagonists was membrane-bound (<20% internalized) (6), a combination with 161Tb might result in improved therapeutic efficacy.
Chemical structures of RM2- and AMTG-based radiopharmaceuticals used in this study.
To elucidate whether 161Tb would be a suitable or better alternative to 177Lu in these GRPR ligands, we completed a comparative preclinical evaluation on [161Tb]Tb-/[177Lu]Lu-AMTG and [161Tb]Tb-/[177Lu]Lu-RM2 with regard to GRPR affinity (half-maximal inhibitory concentration), membrane-bound activity, lipophilicity (distribution coefficient at pH 7.4), in vivo stability, and biodistribution studies in PC-3 tumor–bearing mice.
MATERIALS AND METHODS
Synthesis and Labeling
Precursor synthesis and 161Tb/177Lu labeling were performed according to a published procedure (6). [161Tb]TbCl3 was provided by Paul Scherrer Institute and Belgian Nuclear Research Centre. [177Lu]LuCl3 was acquired from ITM Isotope Technologies Munich SE. 3-[125I]I-tyr6-MJ9 (Supplemental Fig. 1; supplemental materials are available at http://jnm.snmjournals.org) was prepared according to reported procedures (6,7). Characterization of all GRPR ligands is provided in Supplemental Figure 2.
In Vitro Experiments
All in vitro experiments (half-maximal inhibitory concentration and internalization studies, n-octanol/phosphate-buffered saline solution distribution coefficient at pH 7.4) were performed in analogy to a previously published procedure (supplemental materials) (6).
In Vivo Experiments
All animal experiments were approved by the General Administration of Upper Bavaria (ROB-55.2-1-2532.Vet_02-18-109), were completed according to a previously published protocol (6), and complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (supplemental materials).
Statistics
Acquired data were statistically analyzed by a Student t-test via Excel (Microsoft Corp.) and OriginPro software (version 9.7; OriginLab Corp.). Acquired P values of less than 0.05 were considered statistically significant.
RESULTS
Synthesis and Radiolabeling
Complexation with a 2.5-fold excess of TbCl3 and LuCl3 resulted in quantitative yields. 161Tb and 177Lu labeling resulted in radiochemical yields and purities of more than 98% and molar activities of 65 ± 5 GBq/μmol. Labeling with another batch of [161Tb]TbCl3 resulted in radiochemical purities of more than 95%, and 2 minor impurities were observed (Supplemental Figs. 3A and 3B). Although one impurity could be attributed to free 161Tb3+ (Supplemental Fig. 3C), the other was not identified. All 161Tb- and 177Lu-labeled compounds were used without further purification.
In Vitro Characterization
nat/161Tb- and nat/177Lu-labeled AMTG and RM2 revealed comparably high GRPR affinity (half-maximal inhibitory concentration, 2.2–3.5 nM; Supplemental Fig. 4), low internalization (74%–84% of cell-associated activity membrane-bound), and favorable lipophilicity (distribution coefficient at pH 7.4, −2.6 to −2.3) (Figs. 2A–2C; Supplemental Table 1). Significant differences are depicted in Figure 2.
In vitro data of 161Tb- and 177Lu-labeled GRPR ligands. Data are expressed as mean ± SD. (A) Affinity data (n = 3) on PC-3 cells (1.5 × 105 cells/mL/well) using 3-[125I]I-tyr6-MJ9 (0.2 nM/well) as radiolabeled reference (2 h, room temperature). (B) Receptor-bound fraction (n = 6) on PC-3 (1.0 nM/well) cells as percentage of cell-associated activity (37°C, 1 h, 1.5 × 105 cells/mL/well). Data are corrected for nonspecific binding (10−3 M Lu-RM2). (C) Lipophilicity depicted as distribution coefficients at pH 7.4 (logD7.4). (D) In vivo stability in murine serum and urine at 30 min after injection (n = 3). IC50 = half-maximal inhibitory concentration. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.
In Vivo Characterization
A significantly higher stability was determined for the AMTG than for the RM2 derivatives (Fig. 2D; Supplemental Fig. 5). Biodistribution studies demonstrated high activity levels in the tumor for all ligands at all time points, with [161Tb]Tb-AMTG exhibiting the highest levels (Fig. 3A). Estimates of area under the curve (AUC) 1–72 h after injection revealed 14% higher tumor levels for [161Tb]Tb-AMTG than for [177Lu]Lu-AMTG and 30%–45% higher tumor levels than for either RM2 analog (Supplemental Table 2).
Activity accumulation of 161Tb- and 177Lu-labeled GRPR ligands in tumor (A), pancreas (B), kidneys (C), and blood (D) at 1, 4, 24, and 72 h after injection (n = 4 each), as well as 168 h after injection (n = 1 each) in PC-3 tumor–bearing CB17-SCID mice (100 pmol each). Data are expressed as mean ± SD. %ID = percentage injected dose.
Activity accumulation in the pancreas was high for all GRPR ligands. However, more than 95% of the activity was cleared from the pancreas within the first 24 h for all compounds (Fig. 3B). [161Tb]Tb-AMTG displayed a 63%, 172%, and 423% higher AUC (1–72 h after injection) for the pancreas than did [161Tb]Tb-RM2, [177Lu]Lu-AMTG, and [177Lu]Lu-RM2, respectively. Apart from that, low off-target accumulation was observed for all organs (Supplemental Fig. 6; Supplemental Tables 3–6). Activity levels in the kidneys and the blood were less than 4% injected dose/g at all time points for all analogs (Figs. 3C and 3D). Activity levels in the liver and the spleen were slightly elevated for both 161Tb-labeled GRPR ligands at all time points, except at 4 h after injection.
Further imaging studies at 1, 4, 24, 72, and 168 h after injection in PC-3 tumor–bearing mice (n = 1), applying [161Tb]Tb-AMTG and [161Tb]Tb-RM2, confirmed the favorable tumor uptake and biodistribution profiles (Fig. 4).
Maximum-intensity projection of 1 CB17-SCID mouse bearing PC-3 tumor (arrows) and injected with [161Tb]Tb-AMTG (A) and [161Tb]Tb-RM2 (B) (100 pmol each). Images were acquired at 1, 4, 24, 72, and 168 h after injection. %ID = percentage injected dose; p.i. = after injection.
DISCUSSION
The recent observation that antagonists do not internalize but are bound to the cell membrane revealed an even improved therapeutic efficacy when labeled with Auger-electron–emitting radionuclides. This type of result will ensure that Auger-emitting radionuclides continue to gain attention in the field of nuclear medicine. In view of our promising data on 177Lu-labeled GRPR ligands (6) and the similar physical properties of 177Lu and 161Tb (similar half-lives and β− energies, whereas the latter additionally emits more Auger electrons per decay (1)), we completed a comparative preclinical study on AMTG and RM2 labeled with both radionuclides.
Although most 177Lu/161Tb labelings resulted in radiochemical purities of more than 98%, labeling with 1 batch of [161Tb]TbCl3 resulted in approximately only 95% radiochemical purity, and 2 minor impurities were observed (used for biodistribution studies at 1, 24, and 72 h after injection). Because we consider radiochemical purity of more than 95% sufficient for preclinical experiments, no further investigation was conducted. The 161Tb- and 177Lu-labeled GRPR ligands revealed comparable in vitro properties (GRPR affinity, lipophilicity, and membrane-bound activity; Fig. 2). In vivo, although biodistribution profiles similar to those of the 177Lu-labeled analogs were observed (Supplemental Fig. 6), higher uptake and retention were observed for RM2 and AMTG labeled with this [161Tb]TbCl3 batch (at 1, 24, and 72 h after injection), particularly in the liver and the spleen, likely because of the aforementioned impurities. Notably, the compounds used for the studies at 4 h after injection (labeled with a different [161Tb]TbCl3 batch, free of impurities) did not show any enhanced uptake in these organs, which is why this elevated uptake was likely not caused by the compound itself.
High initial tumor and pancreas uptake was observed for all derivatives. However, whereas activity was retained in the tumor for several days, more than 95% of the initial activity (1 h after injection) was cleared from the pancreas within the first 24 h after injection, as was also shown for the human situation (5). Elevated pancreas uptake observed for the 161Tb-labeled ligands could be due to their slightly enhanced GRPR affinity. In general, higher activity levels were found for the AMTG derivatives in the tumor (except at 72 h after injection), which can be attributed to their increased in vivo stability. This led to noticeably increased AUCs (1–72 h after injection) for the tumor for the AMTG than for the RM2 analogs. On the basis of the high therapeutic efficacy observed for a noninternalizing, 161Tb-labeled somatostatin-2 receptor antagonist due to yet unknown damage by Auger electrons at the cell membrane (2), and the high percentage of membrane-bound [161Tb]Tb-AMTG (Fig. 2B), an improved therapeutic efficacy might be predicted for this compound. Favorable biodistribution profiles for [161Tb]Tb-AMTG over time were confirmed by imaging studies (Fig. 4) and were in agreement with previously reported profiles for [177Lu]Lu-AMTG (6).
Nevertheless, AUCs (1–72 h after injection) for the pancreas were also elevated for the AMTG analogs compared with their RM2 correlates, which is why a higher dose to the pancreas is expected. However, AUCs (1–72 h after injection) for the tumor were 2- to 8-fold higher than those for the pancreas for all these compounds. Moreover, other than estimates of the dose limit for the pancreas based on external-beam radiation therapy, only limited evidence is currently available that the pancreas is a radiation-sensitive organ (8,9). Further studies on animals and humans must be conducted to elucidate tumor and pancreas dose, as well as the potential damage caused.
Overall, this study delivered further evidence of the potential therapeutic usability of [161Tb]Tb- or [177Lu]Lu-AMTG. Moreover, because of the similar physical properties of 177Lu and 161Tb but additional emission of Auger and conversion electrons by the latter, 161Tb could become a valuable addition to the armamentarium of nuclear medicine, once its clinical availability improves. Provided the membrane effect is accessible for noninternalizing GRPR antagonists, a combination with short-range Auger- and α-emitters might be applicable. A limitation of this study was the use of 161Tb-labeled RM2 and AMTG batches that contained 2 minor impurities, which likely caused increased activity retention in the liver and spleen and affected the overall tumor-to-background ratios.
CONCLUSION
The data from this study indicate that both [161Tb]Tb-AMTG and [177Lu]Lu-AMTG might improve radioligand therapy because of their high tumor retention. Ongoing treatment studies in our laboratory will enable conclusions to be drawn on the potentially increased therapeutic efficacy of AMTG over RM2 (due to in vivo stability) and of 161Tb over 177Lu (due to Auger-electron emission) and whether there are detrimental effects on the pancreas.
DISCLOSURE
This work was supported by the European Union’s Horizon 2020 research and innovation program as a user project of PRISMAP—the European medical radionuclides program (GA 101008571). Thomas Günther acknowledges the 2023 Sanjiv Sam Gambhir–Philips and the 2023 Translational Research and Applied Medicine fellowships for support at Stanford University. A patent application on modified GRPR-targeted ligands, including AMTG, with Thomas Günther as the inventor has been filed (WO2021121734A1). No other potential conflict of interest relevant to this article was reported.
KEY POINTS
QUESTION: Is it possible to improve GRPR-based radioligand therapy (currently performed with [177Lu]Lu-RM2) using the metabolically more stable AMTG peptide and alternative radionuclides such as 161Tb?
PERTINENT FINDINGS: Compared with [161Tb]Tb-/[177Lu]Lu-RM2, [161Tb]Tb-/[177Lu]Lu-AMTG revealed noticeably increased tumor AUCs, which might be beneficial for future clinical use.
IMPLICATIONS FOR PATIENT CARE: Although the clinical value of [161Tb]Tb-/[177Lu]Lu-AMTG and a potential dose-limiting toxicity to the pancreas have to be elucidated, improved therapeutic efficacy on the tumor and, thus, improved patient care are anticipated.
ACKNOWLEDGMENTS
We thank the members of the PRISMAP consortium and the PRISMAP user selection panel, coordination and management team, for their advice and support.
Footnotes
Published online Dec. 14, 2023.
- © 2024 by the Society of Nuclear Medicine and Molecular Imaging.
REFERENCES
- Received for publication June 24, 2023.
- Accepted for publication November 9, 2023.
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