Despite progress in the treatment of MTC, current 10-year survival rates demand improved therapeutic approaches. Recent studies suggest that targeting the CCK-2R by radiolabeled compounds could improve treatment outcomes for MTC. Moreover, such compounds could also be used in other cancer types expressing CCK-2R, such as stromal ovarian cancer, astrocytoma and small cell lung cancer, which indicates a promising clinical value for CCK-2R ligands. Particularly the latter could be of high interest for RLT, as small cell lung cancer is very aggressive and conventional treatment options are scarce, thus resulting in a 5-year survival rate of less than 7% [27]. In a previous study, we introduced DOTA-CCK-66, a metabolically more stable CCK-2R-targeted peptide with comparable as well as improved in vivo characteristics to currently clinically tested minigastrin analogs such as DOTA-MGS5 and DOTA-PP-F11N. Initial PET/CT images of patients suffering from MTC confirmed the clinical value of [68Ga]Ga-DOTA-CCK-66 and warrants the further development of a therapeutic analog [24, 25]. Therefore, our presented study goal was to elucidate the therapeutic efficacy of DOTA-CCK-66 labeled either with 225Ac for TAT or 177Lu for RLT.

The logD7.4 value of [225Ac]Ac-DOTA-CCK-66 (logD7.4 = − 3.04 ± 0.05) presented to be in a similar and slightly lower range than that of its previously published [67Ga]Ga- (logD7.4 = − 3.01 ± 0.07; [24]) and [177Lu]Lu-labeled (logD7.4 = − 2.60 ± 0.07; [24]) analogs, respectively, which indicates favorable hydrophilic properties of the compound. In vitro stability of [225Ac]Ac-DOTA-CCK-66 in human serum was high over a time span of 10 d, confirming sufficient complex stability of [225Ac]Ac-DOTA. In addition, a similar stability was found for [225Ac]Ac-DOTA-CCK-66 and [177Lu]Lu-DOTA-CCK-66 when incubated in human serum at 37 °C for 3 d (91.3 ± 0.3% versus 90.8 ± 0.9%, p = 0.35) [24]. A limitation of this study is, that the stability data collected only depict the stability of the 225Ac-DOTA complex, not that of its daughter nuclides.

[68Ga]Ga-DOTA-CCK-66 PET/CT imaging studies of AR42J tumor-bearing animals assigned to the control group cohort underlined previously published data [24], displaying high tumor uptake while activity uptake in off-target tissue remained low.

Both single-dose administration of [225Ac]Ac-DOTA-CCK-66 (37 kBq) and [177Lu]Lu-DOTA-CCK-66 (37 MBq) revealed a significant impact on tumor growth inhibition, when compared to the control. In addition, the mean survival of animals treated with [177Lu]Lu-DOTA-CCK-66 and [225Ac]Ac-DOTA-CCK-66, respectively, was observed to be increased by 3- and 4.5-fold, respectively, when compared to the control group animals, indicating a substantial therapeutic efficacy of both compounds. As expected, the higher linear energy transfer in combination with a higher particle energy and lower particle path length of α-emitters (e.g. 225Ac), when compared to β–-emitters (e.g. 177Lu) [28,29,30], led to a 1.5-fold increase in mean survival rate of animals treated with [225Ac]Ac-DOTA-CCK-66 compared to [177Lu]Lu-DOTA-CCK-66. Despite noticeable effects observed after [177Lu]Lu-DOTA-CCK-66 and [225Ac]Ac-DOTA-CCK-66 RLT, the small animal cohorts tested are a limitation of this preliminary study.

Our study indicates the theranostic potential of DOTA-CCK-66 labeled with either 68Ga/177Lu or 68Ga/225Ac as theranostic pairs. This finding is a great advantage of our compound DOTA-CCK-66 compared to currently clinical applied diagnostic tools for MTC, such as the gold standard [18F]F-DOPA, which have no therapeutic analog available [31, 32]. Radiotheranostics, in our case DOTA-CCK-66, can provide patients with personalized health care options. CCK-2R positive lesions identified by [68Ga]Ga-DOTA-CCK-66 PET/CT in MTC patients could make them eligible for RLT or TAT with 177Lu or 225Ac-labeled DOTA-CCK-66, respectively.

Previously published studies by Qin et al. as well as Grzmil et al. revealed that treatment with 177Lu- (60 MBq) and 225Ac-labeled DOTA-PP-F11N (60 kBq), respectively, led to an increase in mean survival of 1.4- and 2-fold, respectively, when compared to the control group cohort [6, 7]. It has to be mentioned that a different animal model (CD-1 nude mice) and cell line (A431/CCKBR+) was used in their studies, which is why comparisons between our study and their studies have to be drawn with caution. Nevertheless, previously performed biodistribution studies of [177Lu]Lu-DOTA-PP-F11N and [177Lu]Lu-DOTA-CCK-66 using the same parameters (AR42J-tumor bearing CB17-SCID mice, same activity and precursor amounts) revealed significantly higher activity levels in the tumor at 24 h p.i. for the latter (1.88 ± 0.82%ID/g versus 8.56 ± 1.08). This supports our hypothesis of an enhanced therapeutic efficacy of DOTA-CCK-66 compared to DOTA-PP-F11N [24, 33].

Higher activity levels for [177Lu]Lu-DOTA-CCK-66 (compared to [177Lu]Lu-DOTA-PP-F11N) were also observed in the stomach, endogenously expressing the CCK-2R [34], which can lead to adverse effects when using 177Lu-based and 225Ac-based treatment, respectively. Therefore, the weight of all tumor-bearing animals was monitored regularly, and none of the mice reached a continuous or critical weight loss of more than 20%. Mean weight slowly increased for all animals from the three cohorts evaluated, which did not indicate any signs of malnutrition. However, it has to be mentioned that the increasing weight of the tumor also contributes to the mean body weight of the animals.

Furthermore, blood samples of 177Lu- and 225Ac-treated naïve mice in contrast to tumor-bearing animals, as well as the non-treated control cohort, were analyzed to elucidate potential side-effects. Both ALB as well as TP blood levels, considered nutritional laboratory markers for malnutrition [35, 36], were found to be within a similar range (TP: 41–55 g/L; ALB: 31–44 g/L) for all cohorts evaluated, which supports a healthy nutrition of all animals from the experiment. In addition, laboratory markers for renal function, namely BUN, K+, Na+, and Ca2+ blood levels [37,38,39,40], were found to be in a similar range within the five different animal cohorts examined (BUN: 5.8–7.9 mmol/L; K+: 5.4–6.2 mmol/L; Na+: 150–157 mmol/L; Ca2+: 2.4–3.3 mmol/L), which does not indicate any signs of renal malfunction at this point. It needs to be mentioned, that nephrotoxicity is known to develop late (> 6 months) after treatment, thus a long-term toxicity study is required to fully asses treatment safety [41].

In order to investigate potential liver toxicity, serum markers (ALT, AST, ALP, and TBIL) were analyzed [42]. While mean AST and TBIL concentrations of tumor-bearing 225Ac-treated (AST: 1713 ± 264 U/L; TBIL: 11.8 ± 3.9 µmol/L) and control group animals (AST: 516 ± 68 U/L; TBIL: 8.8 ± 2.5 µmol/L) were found to be noticeably elevated, those of 225Ac-treated naïve mice (AST: 298 ± 187 U/L; TBIL: 5.4 ± 0.5 µmol/L) and 177Lu-treated naïve (AST: 115 ± 77 U/L; TBIL: 5.3 ± 0.4 µmol/L) as well as 177Lu-treated tumor-bearing animals (AST: 269 ± 69 U/L; TBIL: 5.4 ± 0.5 µmol/L) presented to be lower. Elevated AST and TBIL levels can be mainly attributed to tumor burden, as tumor-bearing cohorts, in general, displayed similar or higher serum concentrations than naïve animals. However, further experiments and larger animal groups would be required to confirm this assumption. For both ALT (225Ac-treated tumor-bearing animals: 345 ± 166 U/L; all other cohorts: 35–87 U/L) and ALP (225Ac-treated naïve animals: 76 ± 14 U/L; all other cohorts: 19 to 32 U/L) values, one out of five cohorts presented elevated serum concentrations, whereas all other cohorts displayed mean values within a comparable range. Further experiments are required to elucidate potential explanations for this observation. However, in general, blood toxicity studies did not indicate an impeded liver function upon [177Lu]Lu- or [225Ac]Ac-DOTA-CCK-66 treatment.

IHC imaging of animals assigned to the control group (n = 3) as well as to the [225Ac]Ac-DOTA-CCK-66 treated tumor-bearing cohort (n = 3) confirmed endogenous CCK-2R expression of the stomach. However, only a weak CCK-2R signal in tumor tissues of 225Ac-treated animals was detected. This observation can be attributed to the necrotic tumor volume of the animals at the end-point of the study in combination with potentially reduced receptor expression levels after 225Ac-treatment. Flow cytometry analysis of the AR42J cells used for inoculation confirmed CCK-2R expression of the cell line. In addition, PET/CT imaging of tumor-bearing control group animals verified a CCK-2R-specific uptake of [68Ga]Ga-DOTA-CCK-66. Not surprisingly, no CCK-2R expression was observed in the liver, the muscle, or the kidneys, as these organs do not overexpress the CCK-2R. In addition, HE stains of all tissues evaluated displayed no structural difference between the control group and the 225Ac-treated animals. Thus, no toxcicity delivered by 225Ac-directed TAT to healthy organs could be found. The main focus of this study was to evaluate the possibilities and challenges of 225Ac-TAT for the treatment of MTC. Thus, no histological analysis of tissues from 177Lu-treated animals was performed, which is a limitation of this study.

The promising data of this preliminary study, which reflects the high therapeutic potential of CCK-2R directed 177Lu-RLT and 225Ac-TAT, should encourage future comparative preclinical evaluation of our novel compounds [177Lu]Lu-DOTA-CCK-66 and [225Ac]Ac-DOTA-CCK-66 in regard to biodistribution, dosimetry, dose-dependant response and long term-toxicity studies, to further pave the way for first in-human clinical applications in MTC patients.