Preclinical Characterization of a Stabilized Gastrin-Releasing Peptide Receptor Antagonist for Targeted Cancer Theranostics

Radiolabeled gastrin-releasing peptide receptor (GRPR) antagonists have shown great promise for the theranostics of prostate cancer; however, their suboptimal metabolic stability leaves room for improvements. It was recently shown that the replacement of Gly11 with Sar11 in the peptidic [D-Phe6,Leu13-NHEt,des-Met14]BBN(6–14) chain stabilized the [99mTc]Tc-DB15 radiotracer against neprilysin (NEP). We herein present DOTAGA-PEG2-(Sar11)RM26 (AU-RM26-M1), after Gly11 to Sar11-replacement. The impact of this replacement on the metabolic stability and overall biological performance of [111In]In-AU-RM26-M1 was studied using a head-to-head comparison with the unmodified reference [111In]In-DOTAGA-PEG2-RM26. In vitro, the cell uptake of [111In]In-AU-RM26-M1 could be significantly reduced in the presence of a high-excess GRPR-blocker that demonstrated its specificity. The cell uptake of both radiolabeled GRPR antagonists increased with time and was superior for [111In]In-AU-RM26-M1. The dissociation constant reflected strong affinities for GRPR (500 pM for [111In]In-AU-RM26-M1). [111In]In-AU-RM26-M1 showed significantly higher stability in peripheral mice blood at 5 min pi (88 ± 8% intact) than unmodified [111In]In-DOTAGA-PEG2-RM26 (69 ± 2% intact; p < 0.0001). The administration of a NEP inhibitor had no significant impact on the Sar11-compound (91 ± 2% intact; p > 0.05). In vivo, [111In]In-AU-RM26-M1 showed high and GRPR-mediated uptake in the PC-3 tumors (7.0 ± 0.7%IA/g vs. 0.9 ± 0.6%IA/g in blocked mice) and pancreas (2.2 ± 0.6%IA/g vs. 0.3 ± 0.2%IA/g in blocked mice) at 1 h pi, with rapid clearance from healthy tissues. The tumor uptake of [111In]In-AU-RM26-M1 was higher than for [111In]In-DOTAGA-PEG2-RM26 (at 4 h pi, 5.7 ± 1.8%IA/g vs. 3 ± 1%IA/g), concordant with its higher stability. The implanted PC-3 tumors were visualized with high contrast in mice using [111In]In-AU-RM26-M1 SPECT/CT. The Gly11 to Sar11-substitution stabilized [111In]In-DOTAGA-PEG2-(Sar11)RM26 against NEP without negatively affecting other important biological features. These results support the further evaluation of AU-RM26-M1 for prostate cancer theranostics after labeling with clinically relevant radionuclides.


Introduction
Prostate cancer is one of the most commonly diagnosed cancers in men and the cause of many cancer-related deaths [1]. A lot of effort has been focused on targeting prostate cancer cell markers, such as the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR), to improve diagnosis and therapy. Owing to its overexpression in the vast majority of prostate cancer lesions [2], PSMA has been extensively studied and a number of FDA-approved PSMA-radioligands are currently commercially available for the D Phe 6 ,Sar 11 ,LeuNHEt 13 ]BBN (6)(7)(8)(9)(10)(11)(12)(13); N 4 : 6-carboxy-1,4,8,11-tetraazaundecane, AMA: paminomethylaniline, DGA: diglycolic acid) led to full radioligand resistance to NEP in vivo linked with a high tumor uptake in mice models and in breast cancer patients [32,33]. The impact of this modification on metabolic stability and several other biological features was NEP in vivo linked with a high tumor uptake in mice models and in breast cancer patients [32,33]. The impact of this modification on metabolic stability and several other biological features was assessed in a head-to-head comparison of [ 111 In]In-AU-RM26-M1 with the unmodified [ 111 In]In-DOTAGA-PEG2-RM26 reference.

Materials and Methods
PC-3 cells were purchased from ATCC (Manassas, VA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with L-Glutamine was purchased from Biowest (Nuaillé, France), fetal bovine serum was purchased from Sigma-Aldrich (St. Louis, MO, USA), and penicillin-streptomycin (10,000 U/mL penicillin and 10,000 µg/mL streptomycin) and trypsin-EDTA were purchased from Biochrom AG (Berlin, Germany). For stability studies, Entresto ® pills (200 mg corresponding to 24 mg/26 mg sacubitril/valsartan per pill) containing the prodrug sacubitril (in vivo releasing the potent NEP inhibitor sacubitrilat) [34] were purchased from a local pharmacy. Pills were ground to a fine powder in a mortar, divided, and suspended in tab water to individual 12 mg/200 mL doses of pill per animal administered per os (Entresto ® group) [35].
Activity measurements were performed on a 2480 Wizard 2® automatic gamma counter from PerkinElmer (Waltham, MA, USA). Instant thin-layer chromatography (ITLC) strips (Agilent Technologies, Santa Clara, CA, USA) were used to estimate the radiochemical yield using a Cyclone ® Plus Storage Phosphor System from PerkinElmer (Waltham, MA, USA). Real-time affinity measurements were performed using LigandTracer ® from Ridgeview Instruments AB (Uppsala, Sweden). Data analysis was performed by applying the unpaired two-tailed t-test of GraphPad Prism 8 (GraphPad, San Diego, CA, USA).
The in vivo targeting specificity, biodistribution over time, and SPECT/CT studies performed were approved by the Ethics Committee for Animal Research in Uppsala (Sweden) and followed the national legislation on the protection of laboratory animals (Ethics permit: 5.8.18-00473/2021, 26 February 2021). The in vivo stability study was conducted in a licensed facility (EL 25 BIO exp021) and the protocol was approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (#1609, 24 April 2019).

Cell Culture
PC-3 cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin and 1% L-Glutamine. The cells were incubated at 37 °C and in 5% CO2 in a humidified atmosphere.

Materials and Methods
PC-3 cells were purchased from ATCC (Manassas, VA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with L-Glutamine was purchased from Biowest (Nuaillé, France), fetal bovine serum was purchased from Sigma-Aldrich (St. Louis, MO, USA), and penicillin-streptomycin (10,000 U/mL penicillin and 10,000 µg/mL streptomycin) and trypsin-EDTA were purchased from Biochrom AG (Berlin, Germany). For stability studies, Entresto ® pills (200 mg corresponding to 24 mg/26 mg sacubitril/valsartan per pill) containing the prodrug sacubitril (in vivo releasing the potent NEP inhibitor sacubitrilat) [34] were purchased from a local pharmacy. Pills were ground to a fine powder in a mortar, divided, and suspended in tab water to individual 12 mg/200 mL doses of pill per animal administered per os (Entresto ® group) [35].
Activity measurements were performed on a 2480 Wizard 2® automatic gamma counter from PerkinElmer (Waltham, MA, USA). Instant thin-layer chromatography (ITLC) strips (Agilent Technologies, Santa Clara, CA, USA) were used to estimate the radiochemical yield using a Cyclone ® Plus Storage Phosphor System from PerkinElmer (Waltham, MA, USA). Real-time affinity measurements were performed using LigandTracer ® from Ridgeview Instruments AB (Uppsala, Sweden). Data analysis was performed by applying the unpaired two-tailed t-test of GraphPad Prism 8 (GraphPad, San Diego, CA, USA).
The in vivo targeting specificity, biodistribution over time, and SPECT/CT studies performed were approved by the Ethics Committee for Animal Research in Uppsala (Sweden) and followed the national legislation on the protection of laboratory animals (Ethics permit: 5.8.18-00473/2021, 2021-02-26). The in vivo stability study was conducted in a licensed facility (EL 25 BIO exp021) and the protocol was approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (#1609, 24-04-2019).

Cell Culture
PC-3 cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin and 1% L-Glutamine. The cells were incubated at 37 • C and in 5% CO 2 in a humidified atmosphere.

Radiometal Complex Stability
The radiochemical stability to transchelation was assessed by incubation of the radiolabeled peptide either with 1000× molar excess of EDTA or by incubation in human serum for 1 h at 37 • C. Incubation of radiolabeled peptide in PBS was used as control. The leakage of radiometal from the radiometal-chelate was determined with ITLC.

In Vivo Metabolic Stability
The in vivo stability was determined in healthy male Swiss albino mice (12 mice in total, body weights 30 ± 5 g). Briefly, each radiolabeled peptide was injected as a bolus (100 µL, 11-22 MBq, 3 nmol of total peptide in saline/EtOH 9/1 v/v) in the tail vein of mice either untreated (controls) or 20 min after having received per os a slurry of an Entresto ® pill (individual 12 mg/200 mL doses of pill per animal; Entresto ® -group; Novartis, Basel, Switzerland), as previously described [34,35]. Blood was collected 5 min post-injection (pi) in a chilled syringe directly from the heart (0.5-1 mL) and samples were processed prior to being analyzed by HPLC for the detection of forming radiometabolites, as previously described [25,35]. For analyses, an XBridge Shield RP18 (5 µm, 4.6 mm × 20 mm) column (Waters, Vienna, Austria) was eluted at a flow rate of 1 mL/min with 0.1% TFA in H 2 O (A) and MeCN (B) with the following linear gradient system: 100%A/0%B at 0 min, with B linearly increasing by 1%/min to 60%A/40%B (system 2). The t R of the intact radiopeptide was determined by coinjection with the respective [ 111 In]In-DOTAGA-PEG 2 -RM26 (t R = 30 min)/[ 111 In]In-AU-RM26-M1 (t R = 29 min) reference in the HPLC.

Cell Binding and Internalization
The binding of radiolabeled peptides to GRPR was tested in PC-3 cells (GRPR positive). Peptides were added to cells (10 6 cells/well) in concentration of 2 nM, with or without pre-blocking of receptors with 1 µM of NOTA-PEG 2 -RM26 for 10 min at room temperature. After incubation for 1 h at 37 • C, the cells were detached, collected, and measured for their activity content.
The internalization assay was performed at 1, 2, 6, and 24 h of incubation with 2 nM solution of radiolabeled peptides as previously described [36]. All in vitro experiments were performed in triplicates.

SPECT/CT Imaging
The PC-3 tumor-bearing Balb/c nu/nu mice were injected with 1 MBq (40 pmol) of [ 111 In]In-AU-RM26-M1 and imaged at 1, 4, and 24 h pi on the nanoScan ® SPECT/CT system from Mediso Medical Imaging Systems (Budapest, Hungary). SPECT raw data were reconstructed using Tera-Tomo™ 3D SPECT reconstruction technology (Mediso Medical Imaging Systems Ltd., Budapest, Hungary). CT data were reconstructed using Filter Back Projection in Nucline 2.03 Software (Mediso Medical Imaging Systems Ltd., Budapest, Hungary). SPECT and CT files were fused using the Nucline 2.03 Software and are presented as maximum intensity projections in the RGB color scale.

Radiolabeling and Radiochemical Stability
The GRPR antagonists were radiolabeled with In-111 in high radiochemical yields (>98%), as determined with ITLC and RP-HPLC ( Figure 2). The presence of small additional peaks could be explained by the use of an enantiomeric mixture of R-and S-DOTAGA for the synthesis of the studied peptides [37]. The molar activity of the labeled peptides was 13.3 MBq/nmol. The radiometal complexes were stable during competition with EDTA and when incubated with human sera (Table 1).

SPECT/CT Imaging
The PC-3 tumor-bearing Balb/c nu/nu mice were injected with 1 MBq (40 pmol) of [ 111 In]In-AU-RM26-M1 and imaged at 1, 4, and 24 h pi on the nanoScan ® SPECT/CT system from Mediso Medical Imaging Systems (Budapest, Hungary). SPECT raw data were reconstructed using Tera-Tomo™ 3D SPECT reconstruction technology (Mediso Medical Imaging Systems Ltd., Budapest, Hungary). CT data were reconstructed using Filter Back Projection in Nucline 2.03 Software (Mediso Medical Imaging Systems Ltd., Budapest, Hungary). SPECT and CT files were fused using the Nucline 2.03 Software and are presented as maximum intensity projections in the RGB color scale.

Radiolabeling and Radiochemical Stability
The GRPR antagonists were radiolabeled with In-111 in high radiochemical yields (>98%), as determined with ITLC and RP-HPLC ( Figure 2). The presence of small additional peaks could be explained by the use of an enantiomeric mixture of R-and S-DOTAGA for the synthesis of the studied peptides [37]. The molar activity of the labeled peptides was 13.3 MBq/nmol. The radiometal complexes were stable during competition with EDTA and when incubated with human sera (Table 1).

Cell Binding and Internalization
Both radiolabeled GRPR antagonists showed a significantly higher uptake in PC-3 cells when incubated alone than in the presence of excess GRPR-blocker ( Figure 3). The internalization of both radiolabeled GRPR antagonists was slow as shown in Figure 4.

Cell Binding and Internalization
Both radiolabeled GRPR antagonists showed a significantly higher uptake in PC-3 cells when incubated alone than in the presence of excess GRPR-blocker ( Figure 3). The internalization of both radiolabeled GRPR antagonists was slow as shown in Figure 4.

Affinity Measurements
The measured rates of association (k on ) and dissociation (k off ) of each radiolabeled peptide to GRPR-positive PC-3 cells were analyzed using one-to-one and one-to-two fitting models, and the last one demonstrated the best fitting for both radiolabeled peptides ( Figure A1). The affinity measurements of the radiolabeled GRPR antagonists showed dissociation constant (K D ) values in the low nanomolar range, as shown in Table 2.

Affinity Measurements
The measured rates of association (kon) and dissociation (koff) of each radiolabeled peptide to GRPR-positive PC-3 cells were analyzed using one-to-one and one-to-two fitting models, and the last one demonstrated the best fitting for both radiolabeled peptides ( Figure A1). The affinity measurements of the radiolabeled GRPR antagonists showed dissociation constant (KD) values in the low nanomolar range, as shown in Table 2.

In Vivo Studies
The metabolic stability study revealed a 69 ± 2% of [ 111 In]In-DOTAGA-PEG2-RM26 remaining intact in peripheral mice blood at 5 min pi for controls ( Figure 5A). The respective value in the Entresto ® -treated group was found to be significantly higher (98.3 ± 0.7% intact; p < 0.0001). These data were in good agreement with the published data for [ 111 In]In-DOTAGA-PEG2-RM26 [31]. In the case of Sar 11 -substituted [ 111 In]In-AU-RM26-M1, the percentage of intact radioligands in the control group was significantly higher than the reference, [ 111 In]In-DOTAGA-PEG2-RM26 (88 ± 8% intact at 5 min pi; p < 0.0001, n = 3). This value slightly, but not significantly, increased in the Entresto ® -treated group (91 ± 2% intact at 5 min pi; p > 0.05) ( Figure 5B). The in vivo GRPR-targeting specificity study for [ 111 In]In-AU-RM26-M1 in mice bearing PC-3 tumors (GRPR-positive) showed a significant reduction in tumor activity uptake in the GRPR-blocking group at 1 h pi (0.9 ± 0.6%IA/g vs. 7.0 ± 0.7%IA/g for controls; p < 0.001, n = 4). The radioligand uptake in the DU-145 tumors (GRPR-negative) was also significantly lower than in the positive control group at the same time point (0.9 ± 0.2%IA/g vs. 7.0 ± 0.7%IA/g; p < 0.001, n = 4). Furthermore, the uptake in the pancreas (GRPR-positive organ) for the positive control group was 2.2 ± 0.6%IA/g, for the negative control group was 2.2 ± 0.7%IA/g, and for the blocked group was 0.3 ± 0.2%IA/g, with statistically significant differences between each control group and the blocked group, as presented in Figure 6. The in vivo GRPR-targeting specificity study for [ 111 In]In-AU-RM26-M1 in mice bearing PC-3 tumors (GRPR-positive) showed a significant reduction in tumor activity uptake in the GRPR-blocking group at 1 h pi (0.9 ± 0.6%IA/g vs. 7.0 ± 0.7%IA/g for controls; p < 0.001, n = 4). The radioligand uptake in the DU-145 tumors (GRPR-negative) was also significantly lower than in the positive control group at the same time point (0.9 ± 0.2%IA/g vs. 7.0 ± 0.7%IA/g; p < 0.001, n = 4). Furthermore, the uptake in the pancreas (GRPR-positive organ) for the positive control group was 2.2 ± 0.6%IA/g, for the negative control group was 2.2 ± 0.7%IA/g, and for the blocked group was 0.3 ± 0.2%IA/g, with statistically significant differences between each control group and the blocked group, as presented in Figure 6.  Table A1) in tumor-bearing mice at 1, 4, and 24 h pi reveal a similar profile. While the majority of examined healthy organs had low activity uptake already at 1 h pi, tumors, kidneys, pancreas and-to a lesser extent-intestinal wall, displayed the highest background levels. Tumor and kidney uptake was higher for the Sar 11 -modified analog [ 111 In]In-AU-RM26-M1 compared with the Gly 11 -reference [ 111 In]In-DOTAGA-PEG2-RM26. Thus, the uptake was significantly higher at 1 and 4 h pi for kidneys and at 4 h pi for tumors. The area under the tumor uptake curve (AUC, Figure  8) was about twice as high for the metabolically stabilized [ 111 In]In-AU-RM26-M1 as for the reference (98 ± 21 vs. 56 ± 11, p < 0.0125).
The tumor-to-organ ratios for [ 111 In]In-DOTAGA-PEG2-RM26 and [ 111 In]In-AU-RM26-M1 at the three investigated time points were comparable with minimal differences, as summarized in Table A2.   Table A1) in tumor-bearing mice at 1, 4, and 24 h pi reveal a similar profile. While the majority of examined healthy organs had low activity uptake already at 1 h pi, tumors, kidneys, pancreas and-to a lesser extent-intestinal wall, displayed the highest background levels. Tumor and kidney uptake was higher for the Sar 11 -modified analog [ 111 In]In-AU-RM26-M1 compared with the Gly 11 -reference [ 111 In]In-DOTAGA-PEG2-RM26. Thus, the uptake was significantly higher at 1 and 4 h pi for kidneys and at 4 h pi for tumors. The area under the tumor uptake curve (AUC, Figure  8) was about twice as high for the metabolically stabilized [ 111 In]In-AU-RM26-M1 as for the reference (98 ± 21 vs. 56 ± 11, p < 0.0125).
The tumor-to-organ ratios for [ 111 In]In-DOTAGA-PEG2-RM26 and [ 111 In]In-AU-RM26-M1 at the three investigated time points were comparable with minimal differences, as summarized in Table A2.

Discussion
The high incidence and mortality of prostate cancer necessitate the development of new, improved theranostic options. Due to the limitations of PSMA-targeting, other prostate cancer cell markers, including GRPR, need to be further explored. Numerous GRPR radioantagonists have been developed and evaluated thus far, showing great potential in prostate cancer management [28]. The metabolic stability of circulating peptide radioligands was shown to be an essential factor for GRPR-targeting of pathological lesions, improving imaging quality and radiotherapy output [25,27,38]. High in vivo stability of radiopeptide has resulted in a higher tumor uptake and, in most cases, improved tumor-to-background ratios. The major protease involved in the rapid in vivo The tumor-to-organ ratios for [ 111 In]In-DOTAGA-PEG 2 -RM26 and [ 111 In]In-AU-RM26-M1 at the three investigated time points were comparable with minimal differences, as summarized in Table A2.
SPECT/CT imaging of [ 111 In]In-AU-RM26-M1 in PC-3 tumor-bearing mice showed clear visualization of tumor uptake at 1, 4, and 24 h pi (Figure 9). No other organs displayed any detectable uptake except for the kidneys. The SPECT images were in agreement with ex vivo biodistribution data.

Discussion
The high incidence and mortality of prostate cancer necessitate the development of new, improved theranostic options. Due to the limitations of PSMA-targeting, other prostate cancer cell markers, including GRPR, need to be further explored. Numerous GRPR radioantagonists have been developed and evaluated thus far, showing great potential in prostate cancer management [28]. The metabolic stability of circulating peptide radioligands was shown to be an essential factor for GRPR-targeting of pathological lesions, improving imaging quality and radiotherapy output [25,27,38]. High in vivo stability of radiopeptide has resulted in a higher tumor uptake and, in most cases, improved tumor-to-background ratios. The major protease involved in the rapid in vivo

Discussion
The high incidence and mortality of prostate cancer necessitate the development of new, improved theranostic options. Due to the limitations of PSMA-targeting, other prostate cancer cell markers, including GRPR, need to be further explored. Numerous GRPR radioantagonists have been developed and evaluated thus far, showing great potential in prostate cancer management [28]. The metabolic stability of circulating peptide radioligands was shown to be an essential factor for GRPR-targeting of pathological lesions, improving imaging quality and radiotherapy output [25,27,38]. High in vivo stability of radiopeptide has resulted in a higher tumor uptake and, in most cases, improved tumor-to-background ratios. The major protease involved in the rapid in vivo degradation of bombesin-like peptides (both GRPR agonists and antagonists) in blood circulation is the ectoenzyme NEP, the action of which remained long elusive [24][25][26]39].
Like other peptides, peptide-based GRPR antagonists are prone to the action of proteases that cleave specific peptide bonds in the chain. For peptide radioligands in circulation, this action translates into a lower percentage of intact peptides reaching the target following intravenous administration [24]. Many attempts have been made over the years to improve the metabolic stability of GRPR antagonists with the substitution of selected amino acids, such as Gly 11 and Gln 7 by unnatural residues [28]. In this study, we introduce AU-RM26-M1 generated with the Gly 11 /Sar 11 -substitution in DOTAGA-PEG 2 -RM26 (Figure 1), previously reported for good therapeutic efficacy in a murine model [31]. The same substitution previously led to the SPECT radiotracer [ 99m Tc]Tc-DB15 showing an attractive profile in GRPR-positive lesions in animal models and in breast cancer patients [32,33]. It should be noted that other Gly 11 -replacements have not been equally successful. For example, [ 111 In]In-SB4, where Gly 11 is replaced by DAla 11 , despite its high in vivo metabolic stability demonstrated lower GRPR-affinity and cell uptake, eventually translating into an inferior tumor uptake compared with the unmodified parent [ 111 In]In-SB3 [38].
The low and slowly increasing internalized fraction of [ 111 In]In-AU-RM26-M1 in PC-3 cells is concordant with a GRPR antagonist profile ( Figure 4). This finding indicates that the antagonistic features of [ 111 In]In-AU-RM26-M1 were not affected by the adopted Gly 11 /Sar 11 -substitution, as after binding to GRPR the bulk of cell-associated activity remained attached to the cell membrane. However, it is interesting to note that [ 111 In]In-AU-RM26-M1 internalized faster compared to the unmodified [ 111 In]In-DOTAGA-PEG 2 -RM26, most probably via a non-ligand-induced GRPR cellular recycling process. This feature of [ 111 In]In-AU-RM26-M1 may turn out to be advantageous for the therapeutic application of AU-RM26-M1 labeled with beta emitters, such as Lu-177, by prolonging tumor residence times. Indeed, while the initial activity uptake of [ 111 In]In-AU-RM26-M1 in the GRPR-positive PC-3 tumors was only slightly higher than the parent radioligand, tumor retention was clearly superior at 4 h pi, as shown by the two-fold higher tumor values ( Figure 7 and Table A1).
The real-time receptor affinity measurements revealed a comparably high affinity for both radioligands to GRPR, with equilibrium dissociation constants found in the low nanomolar range (Table 2). Interestingly, the interaction of both peptides with GRPR measured in real time on living PC-3 cells fits better to the one-to-two model than to the one-to-one model ( Figure A1). A single value of 0.44 ± 0.05 nM (one-to-one fitting) was previously reported for [ 111 In]In-DOTAGA-PEG 2 -RM26 [30]. However, when the GRPR affinity of the closely related antagonist [ 111 In]In-NOTA-PEG 6 -RM26 was studied in more detail in similar experimental settings, two different interactions were found [40]. The authors hypothesized that either the GRPR is present in two conformations on PC-3 cells, or a conformation change occurs after the formation of the receptor-ligand complex. In the second case, k a1 and k a2 should have close values. We can speculate that in this study, we observed the presence of two conformations rather than a conformation change ( Table 2). The coexistence of two types of binding interactions could decrease the radioligand uptake in target cells if these interaction types are considerably different in strength. However, the two dissociation constants measured for [ 111 In]In-AU-RM26-M1 were not markedly different (0.53 ± 0.46 and 1.6 ± 0.3 nM).
Our initial hypothesis that the Gly 11 /Sar 11 -substitution may enhance the resistance of BBN-like radioligands to the rapid proteolytic action of NEP was confirmed using the comparative in vivo stability assay performed for [ 111 In]In-DOTAGA-PEG 2 -RM26 and its Sar 11 -version, [ 111 In]In-AU-RM26-M1 ( Figure 5). Thus, about 30% of [ 111 In]In-DOTAGA-PEG 2 -RM26 was degraded in peripheral mice blood already at 5 min pi, while in situ inhibition of NEP by pre-administration of Entresto ® per os led to stabilization of the radioligand in this time interval. It should be noted that the commercially available drug Entresto ® contains the prodrug sacubitril, in vivo releasing the selective and potent NEP inhibitor sacubitrilat [34,35]. This finding pin-points NEP as the major degrading protease of [ 111 In]In-DOTAGA-PEG 2 -RM26, in agreement with previous findings with other BBN-like radioligands [24,25,27,38]. Remarkably, no significant difference was found in the stability of [ 111 In]In-AU-RM26-M1 between untreated mice and those treated with sacubitril (Entresto ® ), suggesting that Gly 11 /Sar 11 -subsitution rendered [ 111 In]In-AU-RM26-M1 resistant to NEP, in line with previous reports [33]. Overall, the in vivo stability of [ 111 In]In-AU-RM26-M1 was significantly better than the parent [ 111 In]In-DOTAGA-PEG 2 -RM26. This is an advantageous quality of [ 111 In]In-AU-RM26-M1 when clinical translation is considered because it facilitates its clinical use without the need for co-medication.
The new Sar 11 -modified [ 111 In]In-AU-RM26-M1 successfully targeted the experimental PC-3 tumors and the GRPR-rich pancreas in mice via a GRPR-specific process, as verified by the significant decrease in tumor and pancreas uptake in the mice that received a high excess of a GRPR-specific peptide for an in vivo GRPR blockade. The specificity of the PC-3 tumor uptake (GRPR-positive) was further confirmed by the significantly lower uptake of the radioligand in the DU-145 tumors (GRPR-negative) that was at the same level as the GRPR-blocked group ( Figure 6). In contrast, the pancreatic uptake was indistinguishable between the groups of PC-3 and DU-145 tumor-bearing mice. As expected, in both groups pancreatic uptake was found significantly higher than in the GRPR-blocked group of PC-3 tumor-bearing mice. The SPECT/CT images of tumor-bearing mice after injection of [ 111 In]In-AU-RM26-M1 were in agreement with the biodistribution profile, with clear visualization of the GRPR-positive tumors ( Figure 9).
The comparison between the biodistribution of [ 111 In]In-AU-RM26-M1 and [ 111 In]In-DOTAGA-PEG2-RM26 over time indicated a tendency of the Sar 11 -radioligand for improved GRPR-mediated tumor uptake. Accordingly, a slower wash out of radiolabeled peptide (Figure 8) is expected to translate to an increased absorbed dose for tumors and be beneficial for radiotherapy.
Overall, tumor-to-organ ratios were higher for [ 111 In]In-AU-RM26-M1 than for [ 111 In]In-DOTAGA-PEG 2 -RM26 due to better activity retention in tumors (Table A2). Interestingly, however, no statistically significant differences were found in the tumor uptake between the two radioligands at 1 and 24 h pi. On the other hand, [ 111 In]In-AU-RM26-M1 displayed a higher kidney uptake than [ 111 In]In-DOTAGA-PEG 2 -RM26 with minor uptake differences in the rest of the organs. Overall, the biodistribution profile of [ 111 In]In-AU-RM26-M1 has been improved as far as tumor uptake and tumor-to-organ ratios are concerned, with the exception of kidney uptake. Indeed, the biodistribution profile of [ 111 In]In-AU-RM26-M1 (as a prototype to the agent for targeted radiotherapy) has not shown marked improvements, taking into account the tumor-to-kidney ratios in our mice model, despite the enhanced metabolic stability achieved. Certainly, extrapolation of biodistribution results from In-111 to Lu-177, as well as from mice to humans should be made with caution. In general, renal excretion of radiolabeled peptides has been shown to involve reabsorption in the proximal tubules, as a part of a mechanism aimed to prevent the excessive loss of proteins. Several systems and processes responsible for reabsorption have been identified thus far, such as the megalin/cubilin endocytic receptor system, pinocytosis, organic anion, organic cation, and oligopeptide transporter families [41]. Coinfusion of positively charged amino acids (lysine/arginine) and negatively charged succinylated gelatin (Gelofusine) are routinely applied to decrease the reabsorption of radiopeptides used clinically for targeted radiotherapy [41]. The optimization of injected peptide amount is another way to increase the ratio of absorbed dose in tumors to that in kidneys. For example, kidney uptake decreased two-fold when the peptide dose for GRPR-targeting peptide [ 177 Lu]Lu-NeoB increased from 400 to 1200 pmol [42].

Conclusions
In this study, we reported the effects of Gly 11 /Sar 11 -substitution on the performance of the GRPR-antagonist-based radioligand [ 111 In]In-AU-RM26-M1 after a head-to-head comparison with the [ 111 In]In-DOTAGA-PEG 2 -RM26 parent. This modification resulted in significant improvement in metabolic stability of circulating [ 111 In]In-AU-RM26-M1 and translated into better uptake and retention in a GRPR-positive tumor mice model. However, the overall biodistribution profile of [ 111 In]In-AU-RM26-M1 was not notably better, especially as far as kidney uptake in mice is concerned. These results call for further modifications with the aim of decreasing the renal uptake and retention while maintaining the high metabolic stability achieved for [ 111 In]In-AU-RM26-M1, especially when radionuclide therapy with beta or alpha emitters is considered.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data generated during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.