Effects of Side Chain and Peptide Bond Modifications on the Targeting Properties of Stabilized Minigastrin Analogs

Different attempts have been made in the past two decades to develop radiolabeled peptide conjugates with enhanced pharmacokinetic properties in order to improve the application for tumor imaging and peptide receptor radionuclide therapy (PRRT), which targets the cholecystokinin-2 receptor (CCK2R). In this paper, the influence of different side chain and peptide bond modifications has been explored for the minigastrin analog DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 (DOTA-MGS5). Based on this lead structure, five new derivatives were synthesized for radiolabeling with trivalent radiometals. Different chemical and biological properties of the new derivatives were analyzed. Receptor interaction of the peptide derivatives and cell internalization of the radiolabeled peptides were studied in A431-CCK2R cells. The stability of the radiolabeled peptides in vivo was investigated using BALB/c mice. Tumor targeting of all 111In-labeled peptide conjugates, and of a selected compound radiolabeled with gallium-68 and lutetium-177, was evaluated in BALB/c nude mice xenografted with A431-CCK2R and A431-mock cells. All 111In-labeled conjugates, except [111In]In-DOTA-[Phe8]MGS5, showed a high resistance against enzymatic degradation. A high receptor affinity with IC50 values in the low nanomolar range was confirmed for most of the peptide derivatives. The specific cell internalization over time was 35.3–47.3% for all radiopeptides 4 h after incubation. Only [111In]In-DOTA-MGS5[NHCH3] exhibited a lower cell internalization of 6.6 ± 2.8%. An overall improved resistance against enzymatic degradation was confirmed in vivo. Of the radiopeptides studied, [111In]In-DOTA-[(N-Me)1Nal8]MGS5 showed the most promising targeting properties, with significantly increased accumulation of radioactivity in A431-CCK2R xenografts (48.1 ± 9.2% IA/g) and reduced accumulation of radioactivity in stomach (4.2 ± 0.5% IA/g). However, in comparison with DOTA-MGS5, a higher influence on the targeting properties was observed for the change of radiometal, resulting in a tumor uptake of 15.67 ± 2.21% IA/g for [68Ga]Ga-DOTA-[(N-Me)1Nal8]MGS5 and 35.13 ± 6.32% IA/g for [177Lu]Lu-DOTA-[(N-Me)1Nal8]MGS5.


Introduction
One of the most promising group of targets for both detection and treatment of different types of cancer are G-protein-coupled receptors overexpressed on the tumor cell surface [1]. The cholecystokinin-2 receptor (CCK2R) belongs to this group of targets and is overexpressed in different types of neoplasms, such as medullary thyroid carcinoma (MTC) and small cell lung cancer [2,3]. MTC is a neoplasm with low incidence but connected with limitations in sensitivity and specificity of diagnostic imaging procedures, as well as lack of effective treatment strategies [4,5]. The main treatment strategy at initial diagnosis is surgical intervention consisting of total thyroidectomy with lymphadenectomy. At an advanced

Peptide Synthesis and Radiolabeling
The investigated DOTA-conjugated MG analogs were synthesized in moderate yields of ≤20% by standard solid-phase peptide synthesis. After purification and charac-

Peptide Synthesis and Radiolabeling
The investigated DOTA-conjugated MG analogs were synthesized in moderate yields of ≤20% by standard solid-phase peptide synthesis. After purification and characterization by analytical HPLC and MALDI-TOF mass spectrometry (see Table 1), the peptide derivatives were lyophilized in aliquots of 0.5-1 mg and kept at −20 • C. The peptides were dissolved in 20-50% ethanol or phosphate buffered saline (PBS) for further experimental use. Using the standardized radiolabeling protocols described in the method section, Pharmaceuticals 2023, 16,278 4 of 17 the 111 In-labeled peptides were prepared at an apparent molar activity of 3-7 GBq/µmol and with high radiochemical purity (RCP ≥90%), allowing their use without purification in in vitro tests. Higher apparent molar activities were achieved for the biodistribution studies (indium-111:~10 GBq/µmole; gallium-68/lutetium-177:~25 GBq/µmole). To avoid the presence of radioactive impurities in the solutions used in biodistribution studies, the radiolabeling mixtures were subjected to solid phase extraction (SPE) as described before [23]. UV-traces and MALDI-TOF mass spectra of the peptide derivatives, as well as radiochromatograms after labeling with different radiometals, are shown in Figures S1-S3 in the Supplementary Materials. Table 1. Summary of the analytical data of the peptide derivatives, including previously studied DOTA-MGS5 [19].

In Vitro Characterization
The LogD evaluated for the 111 In-labeled peptides in octanol/PBS resulted in a hydrophilicity ranking of [ 111 111 In-labeled peptides are summarized in Table 1 and in Table S1 of DOTA-[(N-Me)1Nal 8 ]MGS5 labeled with different radiometals, which was also studied. A high complex stability of >90% was confirmed in PBS (n = 1).

Receptor Affinity and Cell Internalization Studies
The IC 50 values generated from the competitive binding curves obtained with [ 125 I][3iodo-Tyr 12 ,Leu 15 ]gastrin-I as radioligand and increasing concentrations of the unlabeled peptide derivatives are summarized in Table 2. For all peptides, except DOTA-MGS5[NHCH 3 ], IC 50 values in the low nanomolar (nM) range, comparable to DOTA-MGS5, were found [19]. The receptor affinity of DOTA-MGS5[NHCH 3 ] was about ten times lower. Exemplary normalized IC 50 binding curves for all peptide derivatives are shown in Figure 2. In ad-

Receptor Affinity and Cell Internalization Studies
The IC50 values generated from the competitive binding curves obtained with [ 125 I][3iodo-Tyr 12 ,Leu 15 ]gastrin-I as radioligand and increasing concentrations of the unlabeled peptide derivatives are summarized in Table 2. For all peptides, except DOTA-MGS5[NHCH3], IC50 values in the low nanomolar (nM) range, comparable to DOTA-MGS5, were found [19]. The receptor affinity of DOTA-MGS5[NHCH3] was about ten times lower. Exemplary normalized IC50 binding curves for all peptide derivatives are shown in Figure 2. In addition, the EC50 values obtained from calcium mobilization assays are shown in Table 2   The receptor-specific internalization was investigated at five different time points from 15 min to 4 h after incubation. All 111 [19]. A much lower uptake of 4.4 ± 0.9% was found for [ 111   The receptor-specific internalization was investigated at five different time points from 15 min to 4 h after incubation. All 111 [19]. A much lower uptake of 4.4 ± 0.9% was found for [ 111 Figure S4).

Metabolic Studies in BALB/c Mice
In vivo stability studies were performed only for the 111 In-labeled peptides with confirmed high stability in human serum. After intravenous injection of the radioligands in BALB/c mice, an overall improved resistance against enzymatic degradation could be confirmed for [ 111 [19]. [ 111 In]In-DOTA-[(N-Me)1Nal 8 ]MGS5 showed the highest levels of intact radiopeptide in liver and kidney with values of 94.9 ± 2.8% and 40.2 ± 4.1%, respectively, whereas a higher degree of degradation was found in the urine, resulting in much lower values of intact radiopeptide (15.9 ± 3.4%). The presence of radiometabolites with higher retention time in blood vs. urine confirms the stabilization against metabolic degradation during circulation. In Table 3, the percentage of intact radiopeptide found in all samples examined is summarized for all 111 In-labeled peptide derivatives. Exemplary radiochromatograms of the radiolabeled peptides, as well as of the analyzed samples obtained from the mice 10 min p.i., are depicted for blood and urine in Figure 3 and for liver and kidney in Figure S5 in the Supplementary Materials. Table 3. HPLC analysis of samples obtained from metabolic studies in BALB/c mice analyzing the percentage of intact radiopeptide 10 min p.i. Data of previously studied [ 111 In]In-DOTA-MGS5 are added for comparison [19].

Metabolic Studies in BALB/c Mice
In vivo stability studies were performed only for the 111 In-labeled peptides with confirmed high stability in human serum. After intravenous injection of the radioligands in BALB/c mice, an overall improved resistance against enzymatic degradation could be confirmed for [ 111 [19]. [ 111 showed the highest levels of intact radiopeptide in liver and kidney with values of 94.9 ± 2.8% and 40.2 ± 4.1%, respectively, whereas a higher degree of degradation was found in the urine, resulting in much lower values of intact radiopeptide (15.9 ± 3.4%). The presence of radiometabolites with higher retention time in blood vs. urine confirms the stabilization against metabolic degradation during circulation. In Table  3, the percentage of intact radiopeptide found in all samples examined is summarized for all 111 In-labeled peptide derivatives. Exemplary radiochromatograms of the radiolabeled peptides, as well as of the analyzed samples obtained from the mice 10 min p.i., are depicted for blood and urine in Figure 3 and for liver and kidney in Figure S5 in the Supplementary Materials.

Discussion
CCK2R targeting in patients with MTC and other malignancies characterized by CCK2R overexpression using radiolabeled peptide analogs derived from endogenous ligands has high potential for diagnostic and therapeutic application [10,25]. However, unfavorable properties such as excessive renal retention or poor enzymatic stability have led to limitations in the clinical applicability [13,25].
Various modifications to the peptide sequence have been investigated in order to stabilize the peptide derivatives against degradation in vivo and enhance the tumor targeting properties. Rapid metabolization in vivo with major cleavage sites within the C-terminal receptor specific part of the peptide sequence was reported for different CCK2R targeting peptide analogs [26]. In recent studies by our group, it was shown that by applying site-specific modifications within the C-terminal receptor-specific sequence, it is possible to prevent enzymatic degradation. This stabilization also resulted in improved tumor uptake [19,21,27]. In the peptide derivative DOTA-MGS5, two substitutions were introduced in the receptor-specific sequence "Trp-Met-Asp-Phe-NH 2 ". Oxidation-sensitive Met was replaced by (N-Me)Nle, and Phe was replace by 1Nal. The combination of these two substitutions enabled a considerable improvement of the targeting properties. DOTA-MGS5 radiolabeled with gallium-68 is currently being studied in a first clinical trial [19]. In this study, further modifications were applied to the amino acid sequence of DOTA-MGS5 and the effects of these modifications on the pharmacokinetic properties and the tumor targeting properties were evaluated. The introduction of 2Nal in replacement of 1Nal, as well as additional N-methylation of the peptide bond at Asp-Nal and of the amidated C-terminus was investigated. Furthermore, replacement of DGlu in position 1 by DLys was investigated.
The in vitro characterization confirmed that the 111 In-labeled peptide derivatives still show a favorable hydrophilicity and binding to serum proteins comparable to [ 111 In]In-DOTA-MGS5 [19]. The introduction of the bulky amino acid 1Nal/2Nal, as well as the N-methylation in different parts of the peptide backbone, resulted in somewhat lower hydrophilicity and increased serum protein binding of 28-44%. Only for [ 111 In]In-DOTA-[Phe 8 ]MGS5, which is missing the introduction of 1Nal, was a higher hydrophilicity and lower protein binding of 20% found. Increased serum protein binding in addition to a possible prolonged blood circulation, was associated with an improved stability of the radiopeptides in human serum. All compounds, except [ 111 8 ]MGS5, showed a low stability of~50% in human serum which was associated with low unfavorable tumor targeting of 1.23 ± 0.15% IA/g 4 h p.i. [21]. Due to this low stability against enzymatic degradation, no further in vivo assessments were performed for [ 111  also resulted in improved tumor accumulation. However, the introduction of the positive charge also led to considerably higher kidney uptake (~20% IA/g), reducing the tumorto-kidney ratio. A much lower tumor accumulation of 4.16 ± 1.01% IA/g at 4 h p.i. was found for [ 111 In]In-DOTA-MGS5[NHCH 3 ] with C-terminal N-methylation. Still, the tumor uptake was almost two times higher when compared to the peptide conjugate without sitespecific amino acid substitutions within the C-terminal receptor-specific region. A tumor uptake of 2.49 ± 0.92% IA/g was previously reported for 111 In-labled DOTA-MG11 (DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ) in A431-CCK2 xenografts at 4 h p.i. using an injected peptide amount of 10 pmol [28]. The results show that C-terminal N-methylation allows for improved stabilization against metabolic degradation. Despite reduced receptor affinity, receptor-specific tumor targeting can still be achieved in vivo. Previous data reported by our group confirmed complete loss of receptor affinity when replacing the C-terminal amide with a free carboxylic group [23]. [ 111 In]In-DOTA-[(N-Me)1Nal 8 ]MGS5 also showed a superior tumor-to-kidney, and especially tumor-to-stomach, ratio compared to [ 111 In]In-DOTA-MGS5. For this reason, this conjugate was further evaluated with alternative radiometals. Interestingly, a considerable influence of the radiometal on the tumor targeting profile was found for DOTA-[(N-Me)1Nal 8 ]MGS5, whereas for DOTA-MGS5 no significant influence was observed. DOTA-[(N-Me)1Nal 8 ]MGS5 radiolabeled with different radiometals generally showed a somewhat higher kidney uptake, but considerably reduced stomach uptake when compared to the radiolabeled DOTA-MGS5 counterparts. Nonspecific uptake in most of the tissues was also slightly increased. DOTA-MGS5, on the other side, showed a constant tumor uptake of~25% IA/g independently of the radiometal. For  8 ]MGS5 an improvement in tumor targeting (35.13 ± 6.32% IA/g at 4 h p.i.) was found. We believe that differences in receptor affinity, rather than pharmacokinetic properties, may explain this finding. In additional studies performed for DOTA-[(N-Me)1Nal 8 ]MGS5 labeled with different radiometals, similar serum protein binding and stability for incubation in human serum were observed. Cell uptake in A431-CCK2R cells, similarly to the varying tumor uptake, was higher for [ 111  Altogether, our results confirm that enhancing the in vivo stability of radiolabeled minigastrin analogs by site-specific substitutions within the C-terminal receptor-specific region has a beneficial effect on tumor targeting properties. When comparing our results with other radiolabeled MG analogs which are currently under clinical investigation, DOTA-MGS5 and DOTA-[(N-Me)1Nal 8 ]MGS5 exhibit a two to five-fold increase in tumor uptake. So far, only the bifunctional chelator DOTA has been used for radiolabeling with different trivalent radiometals. In our next studies, we will investigate the influence of different chelators on the biodistribution profile and targeting properties of the new peptide derivatives.
(Phenomenex Ltd., Aschaffenburg, Germany) was used. The following gradient system with flow rate set to 1 mL/min was applied: 0−3 min 10% B, 3−18 min 10−55% B, 18−20 min 80% B, 20−21 min 80−10% B, 21−25 min 10% B. The radiodetector allowed us to adjust the sensitivity for the measurement of radioactive samples. Usually, the standard loop of 5 µL was used. Samples with low radioactivity obtained from stability studies were measured with a 250 µL loop, allowing for improved sensitivity when analyzing samples with low radioactivity.

In Vitro Characterization
The distribution coefficient (LogD) in octanol/PBS was determined by mixing 0.5 mL of the radiolabeled DOTA peptides diluted in PBS (100 pmol/mL) and 0.5 mL octanol in a 1.5 mL microcentrifuge tube (n = 8). After shaking for 15 min (MS3 Basic, IKA, Staufen, Germany) at 1500 rpm and a waiting time of 10 min to ensure appropriate separation of the two phases, 100 µL aliquots of each phase were pipetted into 4.5 mL tubes. The tubes were measured using a γ-counter (2480 Wizard2 3", PerkinElmer Life Sciences and Analytical Instruments, formerly Wallac Oy, Turku, Finland) and the LogD value was determined using following equation: Protein binding was determined by incubation of the radiolabeled peptides in human serum at 37 • C (500 pmol/mL, n = 2). For the time points of 1, 4, and 24 h after incubation, samples of 25 µL were subjected to Sephadex G-50 size exclusion chromatography (GE Healthcare Illustra, Little Chalfont, UK). The column and the eluate were measured using the γ-counter and the percentage of protein binding was calculated.
In vitro stability studies were performed in human serum (n = 2). The radiolabeled peptides (1000 pmol/mL) were incubated at 37 • C and a sample of 100 µL was taken at 1, 4, and 24 h after incubation. After the precipitation of proteins with ACN at 1:1.5 (v/v), centrifugation (14,000 rpm, 2 min, centrifuge 5424, Eppendorf AG, Germany) and dilution with water at 1:1 (v/v), an aliquot of 100 µL of this solution was analyzed by HPLC. Radioligand breakdown was monitored and the percentage of intact radiopeptide was calculated using Chromeleon Dionex Software (Version 7.2.9.11323). Additionally, the radiolabeled peptides (1000 pmol/mL) were incubated in PBS (n = 1) at room temperature to confirm the complex stability as a control.

Receptor Affinity and Cell Internalization Studies
A431 human epidermoid carcinoma cells overexpressing human CCK2R through stable transfection (A431-CCK2R) and the same cell line transfected with the empty vector (A431-mock) were used for cell assays. The cells are constantly available in our laboratory and were originally provided by Dr. Luigi Aloj [33]. All cell culture media and reagents were obtained from Invitrogen Corporation (Lofer, Austria) or Sigma-Aldrich (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1% of 100x penicillin−streptomycin−glutamine was used for cell culture. The cells were passaged three times per week using a 10x 2.5% trypsin-EDTA solution in a 1:2-1:3 ratio.
Receptor affinity studies of the DOTA-conjugates for the CCK2R was competitively evaluated against [ 125 I][3-iodo-Tyr 12 ,Leu 15 ]gastrin-I. The binding assays were performed in 96-well filter plates (MultiScreenHTS-FB, Merck Group, Darmstadt, Germany) as previously described [19]. A431-CCK2R cells (200,000-400,000/well) were incubated for 1 h at RT with the peptides (concentration range of 0.01-1000 nM) and [ 125 I][3-iodo-Tyr 12 ,Leu 15 ]gastrin-I (~25,000 cpm). The incubation was arrested by filtering the medium by vacuum and rinsing twice rapidly with ice-cold binding assay buffer (10 mM TRIS/139 mM NaCl, pH 7.3). The filters were removed from the plate, transferred into 4.5 mL tubes and measured in the γ-counter. Data were normalized from 0 to 100 for graphical representation of exemplary binding curves. Origin software (Microcal Origin 6.1, Northampton, MA, USA) was used to calculate the half maximal inhibitory concentration (IC 50 ) values by nonlinear regression and expressed as mean ± sd for three independent experiments performed in triplicates. An additional cell uptake study at 2 h after incubation was performed for DOTA-[(N-Me)1Nal 8 ]MGS5 radiolabeled with gallium-68 and lutetium-177 (see Figure S4 of the Supplementary Materials).
Analog-induced receptor activation was evaluated by a calcium mobilization assay using the calcium-sensitive dye fluo-4 AM (ThermoFisher Scientific, Montigny-Le-Bretonneux, France), as previously described [22]. Briefly, A431-CCK2R cells were seeded (60,000/well) in flat clear bottom black 96-well plates (Corning, Bagneaux-sur-Loing, France) coated with poly-L-ornithine (100 µg/mL, 1 h, 37 • C) (Sigma Aldrich, Saint Quentin Fallavier, France). Cytosolic Ca 2+ variations were measured before and after application of 50 µL of graded concentrations of the investigated peptides in the vicinity of the cells, and the effects on Ca 2+ mobilization were measured. Data were background corrected and normalized for the amplitudes caused by the medium alone (0%, no self-effect) or 10 −6 M pentagastrin (100%, maximal Ca 2+ mobilization response). Prism 4.0 software was used to calculate the EC 50 values and the results were expressed as mean ± SEM for at least three independent experiments performed in triplicates.
The cell internalization of the radiolabeled peptides was measured on A431-CCK2R and A431-mock cells. The cells (1.0 × 10 6 cells per well) were seeded in six-well plates and grown for 48 h. On the day of the experiment, the medium was removed and the cells washed twice with ice-cold internalization medium (medium supplemented with 1% (v/v) fetal bovine serum). The cells were incubated with the 111 In-labeled peptides in a total volume of 1.5 mL in triplicates at 37 • C (final peptide concentration of 0.4 nM). At defined time points, the medium was removed and the cells were washed with ice-cold internalization medium, followed by treatment with 50 mM glycine buffer pH 2.8, 0.1 M NaCl (2×, 5 min) to remove the membrane-bound radioligand. The cells were separately collected with 1 M NaOH (2 × 1 mL) (internalized radioligand). The collected fractions (supernatant, acid wash, lyzed cells) were measured in the γ-counter together with a standard corresponding to the total radioactivity added to each well. For each time point, the percentage of radioactivity internalized by the cells, in relation to the total radioactivity added, was calculated. The receptor-specific uptake in A431-CCK2R cells was determined by subtraction of the non-specific uptake found in A431-mock cells. For each time point and radiopeptide studied, the mean value and standard deviation for the values obtained from three assays performed in triplicates was calculated.

Metabolic Studies in BALB/c Mice
All animal experiments were approved by the Austrian Ministry of Science (BMWF-66.011/0075-WF/V/3b/2016) and performed in accordance with the Austrian Animal Protection Act. Metabolic studies characterizing the stability of selected radioligands against enzymatic degradation were carried out in 7 to 8 week old female BALB/c mice (Charles River, Sulzfeld, Germany). The mice (two animals for each peptide) were intravenously injected into a lateral tail vein with 5-10 MBq of the 111 In-labeled peptide derivatives, corresponding to 2 nmol total peptide. At the time point of 10 min p.i. the animals were euthanatized by cervical dislocation. A sample of urine and blood was immediately collected. Liver and kidneys were dissected, mixed with 20 mM HEPES buffer pH 7.3 (1:1, w/v) and homogenized using an Ultra-Turrax T8 homogenizer (IKA-Werke, Staufen, Germany) for 1 min. All samples taken were analyzed by analytical HPLC using the low-sensitivity loop for urine and the high-sensitivity loop for blood, liver and kidney. Before injection into the HPLC system, all samples except urine were treated with ACN and diluted with water as described for stability studies in human serum. The percentage of intact radiopeptide in the analyzed samples was calculated by integration of the different peaks found in the radiochromatogram using Chromeleon Dionex Software (Version 7.2.9.11323).

Biodistribution Studies in BALB/c Nude Mice Bearing A431-CCK2R/A431-Mock Xenografts
The overall biodistribution and tumor targeting properties of selected radiolabeled peptides were evaluated in female athymic BALB/c nude mice (Charles River, Sulzfeld, Germany, age of 7 to 9 weeks). After tumor induction with 2 × 10 6 A431-CCK2R or A431-mock cells (subcutaneously injected in 200 µL DMEM into the right and left flank) and a waiting period of 10-14 days (sufficient to achieve tumors of ∼0.25 g), mice were randomly divided into groups of four and treated with a bolus injection of 150 µL of the injectable solution containing the radiolabeled peptide via a lateral tail vein to achieve an injected radioactivity of 150-200 kBq and a total injected peptide amount of~20 pmol (n = 4 for each radiopeptide). For DOTA-[(N-Me)1Nal 8 ]MGS5, additional biodistribution studies were performed with gallium-68 (~0.5 MBq, 20 pmol) and lutetium-177 (~0.5 MBq, 20 pmol). The animals were euthanatized by cervical dislocation at 1 h (gallium-68) or 4 h p.i. (indium-111/ lutetium-177). The tumors and other tissues (blood, lung, heart, muscle, spleen, intestine, liver, kidney, stomach, and pancreas) were dissected, weighed, and the radioactivity measured in the γ-counter. The rest of the body and a standard of the solution injected was also measured together with the collected samples. The percentage of injected activity per gram of tissue (% IA/g) and the tumor-to-organ activity ratio of selected tissues were calculated for each mouse and time point studied. Origin software (Microcal Origin 6.1, Northampton, MA, USA) was used for statistical analysis (independent t-test for two populations; significance level p < 0.05).

Conclusions
The significant progress achieved in recent years in the development of stabilized radiolabeled MG analogs resulted in new peptide derivatives with enhanced pharmacokinetic profile and improved targeting properties. First clinical studies have been initiated for targeted imaging and therapy of CCK2R positive malignancies. In this study, we have applied different modification strategies with a focus on the C-terminal receptor-specific sequence of the peptide. From the five new peptide derivatives studied, [ 111