In the past few years, the rh concept was successfully implemented for prostate-specific membrane antigen targeted compounds, enabling the generation of chemically identical ligands that are either 18F- or 177Lu-labeled [19, 28]. These so called “true theranostics” allow for the design of chemical identical pairs, such as 18F/natLu (PET/CT) and 19F/177Lu (therapy), by combining a chelator as well as a SiFA moiety within the peptide structure. In May 2023, rhPSMA-7.3 (Posluma®) has been approved by the FDA for diagnosis of suspected metastatic as well as recurrent prostate cancer. In addition, clinical trials using rhPSMA-10.1 for therapeutic approaches are ongoing [29,30,31,32].

As currently applied CCK-2R-targeted compounds bear no option for 18F-labeling, we recently transferred the rh concept to minigastrin analogs via introduction of a D-Dap(p-SiFA) moiety into the peptide structure of DOTA-PP-F11N [8, 20]. The most promising rh-based minigastrin analog, [18/19F]F-[177/natLu]Lu-DOTA-rhCCK-18, displayed decelerated clearance kinetics accompanied by high activity levels in the tumor at 1 and 24 h p.i., rendering this compound a valuable asset for PET/CT imaging of MTC. However, unfavorably elevated renal activity retention of DOTA-rhCCK-18 [20] might be a limiting factor for radioligand therapy when 177Lu-labeled, particularly with regard to the kidney as a dose-limiting organ.

In this study, we wanted to reduce activity retention in the kidneys for our rhCCK ligands while maintaining high activity levels in the tumor. Therefore, we first examined the influence of PEG linkers of various length (4 to 11) on in vitro properties of rh-based minigastrin analogs. Moreover, the influence of differently charged SiFA moieties e.g., p-SiFA (neutral), SiFAlin (positively charged) and SiFA-ipa (negatively charged) on overall lipophilicity was evaluated. In addition, lipophilicity of compounds with and without a D-γ-Glu unit in direct proximity to the D-Dap(p-SiFA) unit were evaluated.

Displaying apparent IC50 values of 12 to 16 nM, no trend on CCK-2R affinity was observed for peptides comprising different PEG linker lengths (4 to 11; [natLu]Lu-DOTA-rhCCK-67, -68, -70, -73, -74 and -76). Compounds that contain an additional D-γ-Glu moiety in proximity to the SiFA building block ([natLu]Lu-68, -70 and -76) revealed similar apparent IC50 values (12 to 16 nM) to their counterparts lacking said entity ([natLu]Lu-67, -73 and -74). In contrast, substitution of p-SiFA by SiFA-ipa led to significantly elevated apparent IC50 values (p < 0.0001, [natLu]Lu-DOTA-rhCCK-69: 42.1 ± 1.7 nM and -72: 52.3 ± 6.3 nM), suggesting a low tolerability towards negative charges at the SiFA moiety. Replacing p-SiFA by a positively charged SiFAlin unit had a positive impact on CCK-2R affinity of compounds comprising a (PEG)4 chain ([natLu]Lu-DOTA-rhCCK -90 and -91: apparent IC50 = 8 to 10 nM), whereas no influence as well as a negative influence on apparent IC50 values of peptides containing a (PEG)7 and (PEG)11 chain ([natLu]Lu-DOTA-rhCCK-71 and -75: apparent IC50 = 13 to 19 nM), respectively, was observed.

Compared to the reference [natLu]Lu-DOTA-rhCCK-18 (apparent IC50 = 4.71 ± 0.62 nM, [20]), all compounds evaluated in this study revealed increased apparent IC50 values (8 to 53 nM), indicating a negative impact of the reduction of negative charges within the linker sequence on CCK-2R affinity. However, the previously published compound [177Lu]Lu-(R)-DOTAGA-rhCCK-16 ((R)-DOTAGA-D-Dap(p-SiFA)-(D-γ-Glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2), displaying an apparent IC50 value of 20.4 ± 2.7 nM, revealed high activity levels in the tumor (18.0 ± 0.7%ID/g) at 24 h p.i. in AR42J tumor-bearing CB17-SCID mice, which could be attributed to its increased HSA binding and circulation in the blood, thus increasing the window for the delivery of the radiolabeled compound [8]. As all compounds evaluated in this study also comprised a SiFA building block and revealed high HSA binding, we considered apparent IC50 values < 20 nM sufficient for further in vivo evaluation.

In line with previous findings, the additional negative charge of a D-γ-Glu moiety in proximity to the SiFA building block resulted in a lower lipophilicity for the respective peptides. Moreover, it could be observed that the impact of PEG linker length on overall lipophilicity of CCK2R-targeted compounds was low, which corroborates the data from Novak et al. [33]. Furthermore, peptides comprising a SiFA-ipa moiety displayed the most favorable lipophilicity (logD7.4: − 2.3 to − 2.1). Surprisingly, the additional positive charge of the SiFAlin moiety had no impact on lipophilicity for [177Lu]Lu-DOTA-rhCCK-71, [177Lu]Lu-DOTA-rhCCK-90 and [177Lu]Lu-DOTA-rhCCK-91, or even led to increased logD7.4 values ([177Lu]Lu-DOTA-rhCCK-75). We assume that this is attributed to the change of the overall charge of the peptides. Namely, introduction of a positive charge via a SiFAlin moiety into the mainly negatively charged minigastrin analog would lead to a decreased overall charge and thus, a less beneficial impact on lipophilicity. Except [177Lu]Lu-DOTA-rhCCK-69 and -72, each comprising a SiFA-ipa moiety, no other peptides evaluated within this study displayed logD7.4 values within a range of –3 to –2, which we usually consider ideal due to the favorable pharmacokinetic properties observed for several compounds in the field of nuclear medicine, among those [177Lu]Lu-DOTA-rhCCK-18 (logD7.4 = − 2.69 ± 0.06 [20]) and [177Lu]Lu-DOTA-MGS5 (logD7.4 = − 2.21 ± 0.08 [34]) in case of CCK-2R ligands. However, currently clinically applied radiotracers e.g., [177Lu]Lu-Pentixather (logD7.4 = − 1.8 ± 0.2 [35]) for C-X-C chemokine receptor type 4 targeting and [177Lu]Lu-NeoBOMB1 (logD7.4 = − 0.57 ± 0.03 [27]) addressing the gastrin releasing peptide receptor, also display elevated lipophilicity (logD7.4 values were evaluated in our group using a comparable experimental setup). Therefore, we decided to extend the range of suitable logD7.4 values from − 2 to − 1.5, bearing in mind that enhanced hepatic accumulation and thus, effects on the biodistribution profile could occur.

HSA binding was observed to be high (85–95%) for all rhCCK derivatives tested. In comparison, the reference compound [natLu]Lu-DOTA-rhCCK-18 (87%) displayed a similar HSA interaction. All compounds evaluated comprise a SiFA building block within their peptide structure, which was reported to increase HSA binding [29]. Elevated HSA binding is usually associated with a decelerated activity clearance and prolonged circulation of the compound in the blood stream, which can result in increased activity accumulation in the tumor [36,37,38]. This corroborates the observed tumor accumulation and retention for previous rhCCK derivatives, such as [177Lu]Lu-(R)-DOTAGA-rhCCK-16 and [177Lu]Lu-DOTA-rhCCK-18. Therefore, we anticipated a similarly beneficial effect on our novel rhCCK ligands.

In order to evaluate the influence of the different SiFA moieties paired with a reduced number of negative charges within the linker sequence of rhCCK derivatives on in vivo performance, particularly with regard to kidney retention, we decided to further investigate both [177Lu]Lu-DOTA-rhCCK-70 (apparent IC50 = 12.6 ± 2.0 nM, logD7.4 = − 1.67 ± 0.08) and [177Lu]Lu-DOTA-rhCCK-91 (apparent IC50 = 8.7 ± 0.7 nM, logD7.4 = − 1.66 ± 0.08) at 24 h p.i. in AR42J tumor-bearing mice, since both displayed acceptable CCK-2R affinity and lipophilicity. Compounds comprising a SiFA-ipa building block were excluded from further in vivo studies due to their insufficient CCK-2R affinity.

Biodistribution profiles of [177Lu]Lu-DOTA-rhCCK-70 and -91 confirmed our assumption that synergistic effects between the multiple negative charges of the poly-D-γ-glutamate linker section and the D-Dap(p-SiFA) building block led to elevated activity levels in the kidneys observed for earlier generations of rh-based minigastrin analogs, such as [177Lu]Lu-(R)-DOTAGA-rhCCK-16 and [177Lu]Lu-DOTA-rhCCK-18. In line with previously published data on minigastrin analogs [21, 22], the reduction of negative charges within the linker section from eight to two via substitution of D-γ-Glu moieties by PEGX chains led to significantly decreased activity uptake in the kidneys for both [177Lu]Lu-DOTA-rhCCK-70 and [177Lu]Lu-DOTA-rhCCK-91 compared to [177Lu]Lu-DOTA-rhCCK-18 (8.4 ± 0.8%ID/g and 6.6 ± 0.5%ID/g vs. 134 ± 18%ID/g, [20], p < 0.0001). Hence, distinctly improved tumor-to-kidney ratios were observed for [177Lu]Lu-DOTA-rhCCK-70 (1.45 ± 0.12) and [177Lu]Lu-DOTA-rhCCK-91 (1.14 ± 0.12) opposed to [177Lu]Lu-DOTA-rhCCK-18 (0.19 ± 0.01, [20]).

However, the increased lipophilicity of [177Lu]Lu-DOTA-rhCCK-70 and [177Lu]Lu-DOTA-rhCCK-91 led to lower overall tumor-to-background ratios, particularly in the liver (3.48 ± 1.66%ID/g and 1.96 ± 0.08%ID/g vs. 0.22 ± 0.01%ID/g) and spleen (1.92 ± 0.60%ID/g and 1.04 ± 0.26%ID/g vs. 0.34 ± 0.09%ID/g). In addition, increased activity levels in the bone were observed, especially for [177Lu]Lu-DOTA-rhCCK-70 (2.4 ± 1.5%ID/g) in comparison to our reference compound [177Lu]Lu-DOTA-rhCCK-18 (0.20 ± 0.03%ID/g). This can be partly attributed to the 177Lu-batch used for in vivo experiments performed within this study. While usually, radiolabeling with our rhCCK derivatives results in < 1% free lutetium-177 (e.g., [177Lu]Lu-DOTA-rhCCK-18), in the study presented herein up to 3% of free lutetium-177 was detected, depending on the 177Lu-batch used. Thereby, the amount of free lutetium-177 observed was found to correlate with activity levels in the bone, which was observed to be between 0.5 and 4.3%ID/g, resulting in a high standard deviation (± 1.5%).

Worth mentioning, tumor retention was also reduced noticeably for our novel compounds (12.0 ± 0.8%ID/g and 7.5 ± 1.0%ID/g vs. 25.4 ± 4.7%ID/g, 24 h p.i. [20]). We suggest that this is partly due to their reduced CCK-2R affinity. However, although both compounds revealed a higher CCK-2R affinity compared to [177Lu]Lu-(R)-DOTAGA-rhCCK-16 (IC50 = 20.4 ± 2.7 nM; activity levels in the tumor: 18.0 ± 0.7%ID/g [8]), significantly lower activity levels were found in the tumor. Therefore, a more detailed investigation on albumin binding has to be completed, as we expect a higher albumin binding and thus, slower activity clearance from the blood for [177Lu]Lu-(R)-DOTAGA-rhCCK-16 (containing more negative charges in proximity to the SiFA moiety than both [177Lu]Lu-DOTA-rhCCK-70 and -91), which results in a prolonged bioavailability and thus, tumor accumulation of said compound. Similar effects on albumin binding were observed in our group for SiFA-comprising PSMA-targeted compounds [29, 39]. Furthermore, overall charge of the peptide is also an important factor, as [177Lu]Lu-DOTA-rhCCK-91 displayed a higher CCK-2R affinity, yet decreased activity levels in the tumor compared to [177Lu]Lu-DOTA-rhCCK-70. We thus suggest that the positively charged SiFAlin moiety has a negative effect on tumor accumulation, which has to be further elucidated in future studies. While the lower activity levels in the tumor can be attributed to the lower CCK-2R affinity, it was surprising that activity levels in the stomach, physiologically expressing the CCK-2R, were found to be increased and similar for our novel compounds compared to [177Lu]Lu-DOTA-rhCCK-18 (70: 6.2 ± 0.9%ID/g and 91: 4.0 ± 1.2%ID/g vs. 4.3 ± 1.1%ID/g, [20]), respectively. Further experiments have to be carried out in future studies to elucidate the nature of this observation.

In summary, we could achieve our goal to design a rh-based minigastrin analog with substantially reduced kidney retention by modifying the linker section with regard to negatively charged residues. However, these pleasing results were accompanied by a negative impact on overall tumor accumulation compared to our internal benchmarks. Worth mentioning, [177Lu]Lu-DOTA-rhCCK-70 still revealed comparable, as well as higher activity levels in the tumor at 24 h p.i. than [177Lu]Lu-DOTA-MGS5 (11.0 ± 1.2%ID/g, [16]) and [177Lu]Lu-DOTA-PP-F11N (1.9 ± 0.8%ID/g, [8]), respectively; two compounds that are currently evaluated in clinical trials [5, 14]. However, kidney retention was observed to be slightly elevated (8.4 ± 0.8 vs. 1.3 ± 0.4 [16] and 3.1 ± 0.6%ID/g [8]), which resulted in decreased tumor-to-kidney ratios compared to [177Lu]Lu-DOTA-MGS5 (1.4 ± 0.1 vs. 9.4 ± 3.3 [16]) but still higher tumor-to-kidney ratios compared to [177Lu]Lu-DOTA-PP-F11N (1.4 ± 0.1 vs. 0.6 ± 0.3%ID/g). However, decreased tumor-to-stomach (2.0 ± 0.2 vs. 4.4 ± 0.7 [16] and 5.2 ± 2.2%ID/g) ratios, as well as increased lipophilicity (–1.67 ± 0.08 vs. − 4.75 ± 0.07 [16] and − 2.21 ± 0.08 [8]) was observed for [177Lu]Lu-DOTA-rhCCK-70 when compared to the reference ligands. Particularly tumor-to-stomach ratios are important, as the stomach (apart from the kidneys) is also considered a dose-limiting organ due to the physiological expression of CCK-2R.

A limitation of this study is the lack of internalization studies in vitro to get more insight into uptake pattern of the receptor. Furthermore, competition studies in vivo should be performed in order to examine receptor specificity of tumor as well as stomach uptake of our novel rhCCK derivatives. Moreover, biodistribution studies at several time points should be carried out to assess pharmacokinetics over time. Lastly, metabolic stability should be investigated in order to draw conclusions on the in vivo behavior of the new compounds. These limitations will be addressed for future compounds but were not our main interest in this study, as we primarily aimed to reduce high kidney retention observed for previously developed rhCCK derivatives (DOTA-rhCCK-18) by circumventing the synergistic effect present for compounds that contain several negative charges in proximity of a SiFA building block. Our study could demonstrate that this strategy can significantly improve kidney retention, and that DOTA-rhCCK-70 is indeed a promising lead compound, yet further optimization is necessary to pave the way for a clinical translation of rh-based minigastrin analogs for theranostic applications.