Amide-to-Triazole Switch in Somatostatin-14-Based Radioligands: Impact on Receptor Affinity and In Vivo Stability

The use of metabolically stabilized, radiolabeled somatostatin (SST) analogs ([68Ga]Ga/[177Lu]Lu-DOTA-TATE/TOC/NOC) is well established in nuclear medicine. Despite the pivotal role of these radioligands in the diagnosis and therapy of neuroendocrine tumors (NETs), their inability to interact with all five somatostatin receptors (SST1–5R) limits their clinical potential. [111In]In-AT2S is a radiolabeled DOTA-conjugate derived from the parent peptide SST-14 that exhibits high binding affinity to all SSTR subtypes, but its poor metabolic stability represents a serious disadvantage for clinical use. In order to address this issue, we have replaced strategic trans-amide bonds of [111In]In-AT2S with metabolically stable 1,4-disubstituted 1,2,3-triazole bioisosteres. From the five cyclic triazolo-peptidomimetics investigated, only [111In]In-XG1 combined a preserved nanomolar affinity for the SST1,2,3,5R subtypes in vitro and an improved stability in vivo (up to 17% of intact peptide 5 min postinjection (pi) versus 6% for [111In]In-AT2S). The involvement of neprilysin (NEP) in the metabolism of [111In]In-XG1 was confirmed by coadministration of Entresto®, a registered antihypertensive drug, in vivo releasing the selective and potent NEP-inhibitor sacubitrilat. A pilot SPECT/CT imaging study conducted in mice bearing hSST2R-positive xenografts failed to visualize the xenografts due to the pronounced kidney uptake (>200% injected activity (IA)/g at 4 h pi), likely the result of the formation of cationic metabolites. To corroborate the imaging data, the tumors and the kidneys were excised and analyzed with a γ-counter. Even if receptor-specific tumor uptake for [111In]In-XG1 could be confirmed (1.61% IA/g), further optimization is required to improve its pharmacokinetic properties for radiotracer development.


Introduction
Since the discovery of somatostatin-14 (SST-14) by Guillemin et al. in 1972, this cyclic neuropeptide has been the focus of thorough investigations revealing its paramount relevance in medicine [1].SST-14 exerts inhibitory and antisecretory actions by interacting with

Introduction
Since the discovery of somatostatin-14 (SST-14) by Guillemin et al. in 1972, this cyclic neuropeptide has been the focus of thorough investigations revealing its paramount relevance in medicine [1].SST-14 exerts inhibitory and antisecretory actions by interacting with a group of G-protein-coupled receptors (GPCRs) comprising five subtypes (somatostatin receptor subtype 1-5, SST1-5R) [2].The SST1-5R are expressed in various tissues and organs serving diverse regulatory functions, but they are also of significant importance in oncology by virtue of their high-density expression in different tumors [3,4].Thus, neuroendocrine tumors (NETs), such as growth hormone-secreting pituitary adenoma, predominantly express SST2R, but concomitant expression of different subtypes is a common finding, as for example, in breast cancer [5].Furthermore, human tumors devoid of SST2R expression may express one or more of other SSTR subtypes.For example, prostate cancer lesions predominantly express the SST1R [6,7].
The clinical importance of SST1-5R biomarkers has been explored in nuclear medicine by the development of radiometal-labeled conjugates of cyclic octapeptides (TATE/TOC/NOC) with the universal macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) [8,9].These truncated versions of SST-14 are SST2Rprefering and present higher in vivo stability as compared to the native peptide, resulting in improved pharmacokinetic properties [10].DOTA-TATE/TOC labeled with either diagnostic (gallium-68, indium-111) or therapeutic radionuclides (lutetium-177, yttrium-90) have been successfully used in the clinic for the management of NETs, due to their high affinity toward the SST2R [11].DOTA-NOC (Figure 1a) presents the widest affinity profile and is able to interact, in addition to the SST2R, with moderate (SST3R) to high affinity (SST5R) with other somatostatin receptor subtypes [9].While some of the abovementioned radioligands (LUTATHERA ® , NETSPOT ® ) have received approval by regulatory agencies for the imaging and treatment of NETs, they are still SST2R-preferring, a feature that limits their wider clinical indications [10].Several attempts have been made to develop radiolabeled somatostatin analogs with high affinity toward all five SST1-5R, referred to as pansomatostatin-like [12].SOM230 and Several attempts have been made to develop radiolabeled somatostatin analogs with high affinity toward all five SST 1-5 R, referred to as pansomatostatin-like [12].SOM230 and KE180 are two multi/pan-somatostatin receptor ligands (SST 1-3, 5 R-/SST 1-5 R-affine, respectively) used for the development of clinically tested radioligands.Interestingly, a radiolabeled version of DOTA-KE108 (KE88, Figure 1b) displayed poor retention in SST 2 Rexpressing tumors, limiting its clinical prospects [13].Furthermore, the DOTA-conjugate of SOM230 (PA1, Figure 1c) was characterized by a high liver uptake, complicating the identification of lesions in the liver [14].[ 111 In]In-AT2S (Figure 1d) is a radiolabeled pansomatostatin-like peptide based on the SST-14 scaffold following two structural modifications [15].First, the L-Trp 8 is exchanged with D-Trp 8 , a substitution reported to increase the affinity for SST 2 R and metabolic stability in vivo [16].Second, the Ala 1 residue on the N-terminus is coupled to DOTA, enabling the radiolabeling with different metallic radionuclides and serving as N-terminal capping against exopeptidases at the same time [15].Despite its pansomatostatin affinity profile, [ 111 In]In-AT2S showed disadvantageously poor metabolic stability in blood circulation.It should be noted that neutral endopeptidase (NEP) has been involved in the fast breakdown of SST-14 and its analogs cleaving the Asn 5 -Phe 6 , Phe 6 -Phe 7 , Trp 8 -Lys 9 and Thr 10 -Phe 11 bonds of the 12mer cycle, as shown in Figure 2 [17][18][19][20].
Table 1.HPLC-MS characterization of peptide and triazolo-peptidomimetic conjugates.1b) displayed poor retention in SST2R-expressing tumors, limiting its clinical prospects [13].Furthermore, the DOTA-conjugate of SOM230 (PA1, Figure 1c) was characterized by a high liver uptake, complicating the identification of lesions in the liver [14].[ 111 In]In-AT2S (Figure 1d) is a radiolabeled pansomatostatin-like peptide based on the SST-14 scaffold following two structural modifications [15].First, the L-Trp 8 is exchanged with D-Trp 8 , a substitution reported to increase the affinity for SST2R and metabolic stability in vivo [16].Second, the Ala 1 residue on the N-terminus is coupled to DOTA, enabling the radiolabeling with different metallic radionuclides and serving as N-terminal capping against exopeptidases at the same time The bonds highlighted in colors were subjected to the amide-to-triazole substitution (Table 1).The olive-green trans-amide bonds correspond to the reported positions cleaved by NEP and the turquoise position is an adjacent bond to a cleavage site.
Different approaches have been studied to address the fast degradation of SST-14 and its analogs, such as key amino acid substitutions, reduction of ring size or use of NEPinhibitors, but results have been suboptimal thus far [15,18].An alternative and yet unexplored approach in the case of SST-14 is the use of metabolically stable bioisosteres of amide bonds, which often represents an elegant strategy to improve the metabolic stability of peptides while preserving their biological function [21].Among the many mimics of amide bonds reported (e.g., sulfonamides, semicarbazides, etc.), triazole heterocycles have emerged as reliable amide bond surrogates that can be incorporated efficiently into the backbone of peptides by convenient manual or automated solid-phase peptide synthesis (SPPS), utilizing the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) [22][23][24].Metabolically, stable 1,4-disubstituted 1,2,3-triazoles (Tz) effectively mimic trans-amide nbonds whereas the 1,5-disubstituted regioisomers can serve as surrogates of cis-amide bonds [25].The amide-to-triazole substitution strategy has previously been applied to different tumor-targeting linear peptides with high affinity toward their respective targets [26,27].The obtained triazolo-peptidomimetics exhibited enhanced metabolic stabilities and maintained receptor affinities resulting in an increased tumor uptake in vivo (mice).1).The olive-green trans-amide bonds correspond to the reported positions cleaved by NEP and the turquoise position is an adjacent bond to a cleavage site.
Different approaches have been studied to address the fast degradation of SST-14 and its analogs, such as key amino acid substitutions, reduction of ring size or use of NEP-inhibitors, but results have been suboptimal thus far [15,18].An alternative and yet unexplored approach in the case of SST-14 is the use of metabolically stable bioisosteres of amide bonds, which often represents an elegant strategy to improve the metabolic stability of peptides while preserving their biological function [21].Among the many mimics of amide bonds reported (e.g., sulfonamides, semicarbazides, etc.), triazole heterocycles have emerged as reliable amide bond surrogates that can be incorporated efficiently into the backbone of peptides by convenient manual or automated solid-phase peptide synthesis (SPPS), utilizing the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) [22][23][24].Metabolically, stable 1,4-disubstituted 1,2,3-triazoles (Tz) effectively mimic trans-amide nbonds whereas the 1,5-disubstituted regioisomers can serve as surrogates of cis-amide bonds [25].The amide-to-triazole substitution strategy has previously been applied to different tumortargeting linear peptides with high affinity toward their respective targets [26,27].The obtained triazolo-peptidomimetics exhibited enhanced metabolic stabilities and maintained receptor affinities resulting in an increased tumor uptake in vivo (mice).In some cases, the receptor subtype selectivity of the peptide could be regulated or the affinity toward its target improved [28].We now report the first application of this methodology to a cyclic tumor-targeting peptide, [ 111 In]In-AT2S.Selected amide bonds in the cyclic part of SST-14, prone to cleavage by NEP (Asn 5 -Phe 6 , Phe 6 -Phe 7 , Trp 8 -Lys 9 and Thr 10 -Phe 11 ; Figure 2, Table 1), were replaced one by one by a Tz (XG1, XG2, XG3 and XG4, respectively) with an additional substitution made adjacent to a known enzymatic cleavage site (Phe 11 -Thr 12 in XG5).All analogs were functionalized with DOTA at the N-terminus for the radiolabeling with In-111, a metallic radionuclide suitable for diagnostic nuclear imaging using single-photon emission computed tomography (SPECT).
The radiolabeled triazolo-peptidomimetics were fully evaluated in vitro with special emphasis given on SST 2 R internalization and SST 1-5 R affinity profile.Additionally, the metabolic stability of [ 111 In]In-XG1 in peripheral mice blood was determined and [ 111 In]In-AT2S was used for direct comparison.Pilot studies were conducted in mice bearing hSST 2 R-positive tumors to evaluate the tumor-targeting properties of [ 111 In]In-XG1.To better understand the involvement of NEP on the metabolic stability of these analogs, we compared the metabolic stability of investigated peptides without or during NEP-inhibition.The latter was accomplished by pretreatment of mice with Entresto ® , a registered antihypertensive drug releasing the potent and selective NEP-inhibitor sacubitrilat in vivo [18,[29][30][31][32][33].
The octapeptide TATE used In uptake and saturation binding assays was synthesized using the H-L-Thr( t Bu)-2-CTC resin following conventional Fmoc/ t Bu SPPS.The HPLC-MS characterization of TATE can be found in the Supplementary Materials (Supplementary Figures S31 and S32).
The radiolabeling with [ 111 In]InCl 3 was conducted in low protein binding Eppendorf tubes.The neoBlock heater with inserts for Eppendorf tubes was used to accomplish the reaction.Alternatively, the radiolabeling reactions were conducted using a water bath.
Membrane filtration for the competition binding assays was performed on a Brandel ® Cell Harvester (Adi Hassel Ingenieur Büro, Munich, Germany) using Whatman GF/B glass fiber filters soaked 2 h in advance in an aqueous solution of polyethylenimine (PEI, 1%).
Samples were measured for 1 min with a 50-500 keV energy window with the γcounter 2480 Wizard2 (PerkinElmer, Waltham, MA, USA) or the Canberra Packard Cobra TM Quantum U5003/1, Auto-Gamma ® counting system.Measurements were also conducted with an automated well-type γ-counter (an automated multisample well-type instrument with a NaI(Tl) 3" crystal, Canberra Packard Cobra TM Quantum U5003/1, Auto-Gamma ® counting system; Little Rock, AR/USA).
Cell culture reagents were purchased from Thermo Fisher Scientific (Vienna, Austria) or Sigma-Aldrich (Vienna, Austria).The AR42J cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), while the CHO-K1 cell line transfected with the hSSt 1 R (CHO-hSSt 1 R) was obtained from the European Collection of Authenticated Cell Culture (ECACC, Braunschweig, Germany).HEK293 cells transfected to stably express each of hSST 2,3,5 R were kindly provided by Prof. S. Schultz (Jena, Germany).

Synthesis
The synthesis of the α-amino alkyne building block required for the insertion of Tz into the backbone of peptides was synthesized as previously reported.For a detailed description, see the Supplementary Materials.Manual SPPS was performed as previously reported [26].In brief, the Cys(Trt)-preloaded-2-CTC resin was swollen and the amino acids were mounted by the general procedure 1 followed by Fmoc deprotection specified in general procedure 2. The incorporation of the triazole heterocycle into the peptide backbone was accomplished by a diazotransfer reaction (general procedure 3) followed by the CuAAC reaction (general procedure 4) with the corresponding α-amino alkynes (for complete synthesis and characterization, see Supplementary Materials, pp.[3][4][5][6][7][8][9][10][11][12][13][14][15][16].The peptide was elongated until completion of the DOTA-containing sequence and the cleavage, cyclization and full deprotection were performed in accordance with general procedure 5. Finally, the peptide was purified by preparative-RP-HPLC as described in general procedure 5 and characterized by mass spectrometry.

General Procedure 3. On Resin Azido Functionalization of Peptide N-Terminus
A solution of ISA•HCl (0.15 mmol, 5 equiv.),and DIPEA (0.18 mmol, 6 equiv.) in DMF (3 mL) was added to the resin.The mixture was shaken for 2 h at RT.The solvent was removed by filtration and the resin washed with DCM (3 × 3 mL) and DMF (3 × 3 mL).The presence of the azido functional group was determined by the Punna-Finn colorimetric test [37].

General Procedure 5. Peptide Cleavage, Cyclization, Complete Deprotection and Purification
To cleave the peptide from the resin and selectively remove the side-chain protecting groups of the cysteine (Cys) residues, 3 mL of a cleavage cocktail (DCM/TFE/AcOH 7:2:1) were added to the syringe containing the resin with the full sequence.After shaking for 2 h at RT, the solution was added over a 0.8 M iodine (0.3 mmol, 10 equiv.)solution in a DCM/TFE/AcOH (7:2:1) mixture.The cyclization proceeded for 20-30 min at RT and the formation of the disulfide bridge was checked by HPLC-MS.The reaction was quenched with a 1 M ascorbic acid aqueous solution (2 mL) and extracted with DCM (3 × 3 mL).The organic phases were combined and washed with an aqueous 5% NaCl/citric acid (1:1) solution (3 × 3 mL).The organic phase containing the product was concentrated under reduced pressure and 4 mL of a cocktail containing TFA/TIPS/H 2 O (9.5:0.25:0.25)was added for the removal of the remaining side-chain protecting groups.The reaction was let to stir for 4 h at RT and the resulting solution was washed with heptane (3 × 2 mL) and concentrated with a stream of argon.The peptide was precipitated with MTBE (10 mL), vortexed, sonicated and centrifuged (4000 rpm, 5 min).The crude peptides (typically 10 mg) were dissolved in 1 mL of a H 2 O/MeCN (80:20) mixture containing 0.1% TFA and purified by preparative-HPLC eluted at a flow rate of 17 mL/min with the linear gradient from 80% A/20% B to 40% A/60% B in 30 min (A: 0.1% aqueous TFA and B: MeCN).The purification afforded the peptides in moderate yields (10-20%) and excellent purities (>95%).
[M+2H] For the determination of the IC 50 values, XG1 was complexed with nonradioactive indium(III).The peptide-conjugate (0.5 mg, 0.25 µmol) was dissolved in 250 µL of H 2 O and a nat InCl 3 (50 µL, 2.5 µmol, 0.05 M) solution in HCl 0.05 M mixed with 60 µL of a 0.3 M aq.NaOAC buffer solution was added.The final pH (4.5-5) of the solution was determined using pH stripes.The reaction was heated for 20 min at 95 • C in a heating block and the completion of the reaction was monitored by HPLC-MS.The metal-tagged conjugate was purified with a SEP-PAK ® C-18 cartridge (conditioned with 1 mL of EtOH followed by 3 mL of H 2 O).Following the conditioning, the reaction mixture was loaded and the unbound nat InCl 3 was washed out with 3 mL of H 2 O. nat In-XG1 was eluted with a H 2 O/MeCN (50/50) mixture (2 mL).The samples were lyophilized yielding a white solid (80%) of high purity (>95%, Figures S29 and S30).

Radiolabeling with [ 111 In]InCl 3
Aliquots of the purified peptides were prepared at a concentration of 1 µM by dissolving 1 mg of peptide in Millipore Water/MeCN (9/1).The concentration of the peptides was determined at 280 nm (ε = 5625 L•mol −1 •cm −1 ) with the Nanodrop Micro-UV/Vis system.The radiolabeling solution was prepared by mixing 50 µL of [ 111 In]InCl 3 (20-30 MBq) in 0.050 M HCl, 10 µL of aq.NaOAc buffer (0.3 M) and 5 µL of peptide solution (1 µM) with final pH values of 4.2-4.5 (determined with pH stripes, Macherey Nagel © , Düren, Germany).The radiolabeling of the DOTA-peptide conjugates was carried out by incubation at 95 • C for 10-15 min.Samples for quality control were prepared by mixing 1 µL of the labeling solution with 1 µL of a DTPA solution and 40 µL of Millipore Water (+0.1% TFA).The quality control was achieved by γ-HPLC at a flow rate of 3 mL/min on a Jupiter Proteo column (90 Å, 4 µm, 4.6 mm × 250 mm) with the linear gradient from 90% A/10% B to 50% A/50% B in 14 min (A: 0.1% aqueous TFA and B: MeCN).The radiopeptides were obtained in >95% radiochemical yields and radiochemical purity of >95% and used for in vitro and in vivo experiments without further purification (see Supplementary Materials for the γ-HPLC chromatograms, Figures S33-S38).

Cell Binding and Internalization Assays and Blocking Experiments with AR42J Cells
AR42J cells were seeded in six-well plates (1 × 10 6 cells/well) 2 days prior to the internalization assay resulting in 70-80% confluency.On the day of the experiment, the cells were counted with the LUNA TM Automated cell counter (approximately 2 × 10 6 cells/well).One hour before the experiment, the medium was removed, the cells were washed with PBS (1 × 1 mL) and fresh internalization medium (DMEM with Glutamax-1 and supplemented with 1% FBS (v/v), 1.3 mL/well) was added.The radiolabeled peptide was added to each well (0.250 pmol, 5 KBq, in 100 µL of PBS).For the determination of the nonspecific binding, blocking experiments (Figures S39 and S40) were conducted by adding a 1000-fold excess of TATE (250 pmol in 100 µL PBS) together with the radioligand.Next, the cells were incubated in culture conditions (37 • C, 5% CO 2 ) for 10, 30, 60 and 120 min; the medium was removed followed by two washes with PBS (1 mL).The collected supernatants correspond to the nonbound fraction of the radiotracers.For the determination of the membrane-bound fraction, the six-well plates were placed on ice and washed twice for 5 min with 1 mL of ice-cold acidic glycine solution (100 mM NaCl, 50 mM glycine, pH 2.8).The cells were then lysed upon incubation with a 1 M NaOH aqueous solution (1 mL) for 10 min and the wells were washed with the NaOH solution (2 × 1 mL) representing the internalized fraction.Standards were prepared in triplicates by adding 0.250 pmol (5 KBq) of the radiopeptide in PBS (100 µL) to 1.4 mL of PBS.All collected fractions were measured in a γ-counter.Experiments were conducted in triplicates (n = 3) and data was analyzed using GraphPad Prism 8.0 TM 2.4.3.Receptor Saturation Binding Assay with SST 2 R-Expressing in AR42J Cells AR42J cells were seeded in six-well plates (1 × 10 6 cells/well) 2 days before the assay.One hour prior to the experiment, the medium was removed, the cells were washed with PBS (1 mL) and fresh assay medium (DMEM with Glutamax-1 and supplemented with 1% FBS (v/v)) was added (1.3 mL/well).The study of the [ 111 In]In-AT2S reference was conducted by adding the radioligand in 100 µL of PBS at different concentrations (1, 10, 25, 50, 75, 100 nM).For [ 111 In]In-XG1 and [ 111 In]In-XG2 a different concentration range was chosen (10, 30, 60, 90, 120, 150 nM).The determination of the unspecific binding was conducted by incubating each test radioligand in the presence of excess TATE, which was added in 100 µL of PBS to a final concentration of 5 µM.The six-well plates were incubated under standard culturing conditions for 2 h; the medium was removed, the cells were washed with PBS (2 × 1 mL) and lysed as described above.The cell lysates were collected and measured in the γ-counter.The dissociation constant (K D ) was calculated by nonlinear regression using the GraphPad Prism 8.0 TM program (n = 2-3 in triplicates).C, the reaction was stopped by dilution with ice-cold washing buffer (10 mM HEPES at pH 7.4, 150 mM NaCl, 3 mL).The samples were filtered through 1%PEI presoaked glass fiber filters using a Brandel ® cell harvester.After thorough washing with ice-cold washing buffer (2 × 3 mL), the filters were collected and measured in the γ-counter.The IC 50 (50% inhibitory concentration) values were calculated with GraphPad Prism 8.0 TM by normalized nonlinear regression (n = 3-4 in triplicate, for membrane validation and representative competition experiments, see Figures S41-S51).

In Vitro Blood Plasma Stability Studies
Plasma stability studies were conducted in vitro by incubating the radioligands (in 100 µL PBS) in 1400 µL of human blood plasma (H4522-20ML, Sigma-Aldrich, Vienna, Austria) to a final concentration of 250 nM (approximately 5 MBq).After an incubation period of 6 h at 37 • C, aliquots of 100 µL were mixed with 100 µL of MeCN for protein precipitation.The mixtures were vortexed and centrifuged for 10 min (15,000× g) at 5 • C and the supernatants were collected.The supernatants (50 µL) were mixed with 0.1% aqueous TFA (50 µL) for posterior γ-HPLC analyses on a Chromolith ® RP-18 performance column (2 µm, 4.6 mm × 100 mm; Merck Millipore, Vienna, Austria) eluted at a flow rate of 3 mL/min with the linear gradient from 90% A/10% B to 50% A/50% B in 14 min (A: 0.1% aqueous TFA and B: MeCN) (n = 2).

In Vivo Studies
All mice experiments were conducted in licensed facilities (EL 25 BIO exp021) and complied with European and national regulations.The study protocols were approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (#1609, 24 April 2019 for the stability studies and #1610, 24 April 2019 for the biodistribution studies).

Metabolic Stability Studies
Healthy male Swiss albino mice (>8 weeks of age, body weight: 30 ± 5 g) provided by the NCSR "Demokritos" Animal House (Athens, Greece) were used in the in vivo stability studies.Animals (3 per compound administered with a 100 µL bolus (saline/EtOH 9/1 v/v) containing the test radioligand (11-22 MBq, 3 nmol) via the tail vein either without pretreatment (controls) or 20-30 min following per os administration of Entresto ® (12 mg/200 µL per animal, Entresto ® groups).Individual Entresto ® doses were prepared from Entresto ® pills purchased from a local pharmacy (200 mg corresponding to 24 mg/26 mg sacubitril/valsartan per pill), containing the prodrug sacubitril (Novartis, Basel, Switzerland) [39].The pills were ground to a fine powder in a mortar, divided and suspended in tap water to the desired individual doses, as previously reported [29].Mice were sacrificed 5 min pi and blood was withdrawn from the heart in a precooled syringe.The blood was immediately transferred in a low protein binding Eppendorf tube on ice containing 40 µL of an aqueous solution of ethylenediaminetetraacetic acid (EDTA, 50 mM Na 2 EDTA).Samples were centrifuged for 10 min at 4 • C (2000× g), the supernatant was separated and an equal volume of MeCN was added for protein precipitation.After a centrifugation period of 10 min at 4 • C (15,000× g), the supernatant was collected and concentrated under a gentle stream of N 2 at 40 • C to an approximate volume of 50-100 µL.Physiological saline was added (400 µL) and the solution was filtered through a Millex GV filter.The samples were analyzed by γ-HPLC to detect radiometabolites formed in vivo.For analyses on a Symmetry Shield RP18 cartridge column (5 µm, 3.9 mm × 20 mm; Waters, Eschborn, Germany) eluted at a flow rate of 1 mL/min with the linear gradient from 100% A/0% B to 60% A/40% B in 40 min (A: 0.1% aqueous TFA and B: MeCN).The r t (retention time) of intact radioligand in this system was verified by co-injecting the serum sample with an aliquot of the labeling solution (HPLC chromatograms can be found in Supplementary Materials, Figure S54).

SPECT/CT Imaging and Ex Vivo Determination of Tumor/Kidney Uptake
Three male severe combined immunodeficiency (SCID) mice (23.1 ± 1.6 g body weight, 6 weeks of age on arrival day; NCSR "Demokritos" Animal House, Athens, Greece) were used for SPECT/CT imaging.Animals were subcutaneously inoculated with a sterile suspension of freshly harvested HEK293-hSST 2 R (1.2 × 10 7 cells) in the right flank and with wtHEK293 cells (0.6 × 10 7 cells) in the left flank (negative control).After 4 weeks, the mice developed well-palpable tumors at the implantation sites.During this period, mice were housed in suitable facilities under aseptic conditions with 12 h day/night cycles and were provided with sterilized chow food and drinking water ad libitum.At the date of the experiment, one untreated animal received a bolus of [ 111 In]In-XG1 (100 µL, 3 nmol in vehicle: normal saline/EtOH 9/1 v/v, control, 11-22 MBq) through the tail vein, while one mouse received per os Entresto ® (100 µL, 12 mg; Entresto ® ) 30 min in advance.Mice were euthanized at 4 h pi and SPECT/CT images were obtained on a x-CUBE & γ-CUBE (Molecubes, BIOMTECH, Athens, Greece) instrument.For SPECT, 40 min acquisition at 4 pi, MLEM (maximum-likelihood expectation maximization) reconstruction with 250 µm voxel size and 100 iterations protocol was adopted, while for CT acquisition, a high-resolution protocol (50 kVp), ISRA reconstruction with 100 um voxel size was applied, as previously described.After completion of the imaging experiment, mice were dissected and the implanted tumors as well as the kidneys were excised, weighed and measured for radioactivity in the γ-counter together with proper standards of the injected dose.Results were calculated as percentage of injected activity per gram tissue (%IA/g, see the Figure S56 detailed results).

Synthesis and (Radio)Metal Labeling of Triazolo-Peptidomimetics
The trans-amide bonds subjected to the amide-to-triazole substitution were selected based on the enzymatic cleavage sites reported for SST-14.Thus, the zinc-dependent endopeptidase NEP has been identified as the key degrading protease of native SST-14, cleaving several trans-amide bonds within the 12mer cycle of the 14peptide backbone (Figure 2) [19,40,41].These bonds were substituted by a Tz and in addition the Phe 11 -Thr 12 bond adjacent to a NEP cleavage site was also replaced (XG5).The inclusion of a Tz in a position adjacent to a cleavage site has been reported to effectively enhance the metabolic stability of NEP-substrates [29].
The synthesis of five cyclic triazolo-peptidomimetic AT2S analogs was accomplished by adapting protocols previously described (Scheme 1, Table 1) [26].In brief, following the orthogonal Fmoc/ t Bu SPPS, the 1,4-disubstituted 1,2,3-Tz was inserted in the peptide backbone in two steps.First, the Fmoc protecting group was removed from the N-terminal amine and the amide-to-azide conversion was accomplished using the imidazole-1-sulfonyl azide hydrochloride (ISA•HCl) as diazotransfer reagent [42].The subsequent copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) click reaction was conducted using a suitable α-amino alkyne derivative of the next amino acid in the sequence employing tetrakis(acetonitrile)copper(I) hexafluorophosphate ([Cu(CH 3 CN) 4 ]PF 6 ) as catalyst [43].The amino acid sequence was then further elongated and completed by appending the DOTA chelator to the N-terminal amine of the peptide.The peptide was cleaved from the resin and the trityl protecting groups of the two cysteine residues were selectively removed with a cocktail containing acetic acid and trifluoroethanol (TFE).The disulfide-bond driven cyclization was accomplished using an excess of iodine and the remaining protecting groups were subsequently removed by treatment with a cocktail containing trifluoroacetic acid (TFA) and scavengers.Purification by HPLC yielded the triazolo-peptidomimetics in excellent purities (>95%, see supporting information (Supplementary Materials) for complete synthetic scheme and HPLC as well as for mass spectrometry (MS) data, Figures S17-S30) demonstrating for the first time that the amide-to-triazole switch methodology is applicable to the synthesis of cyclic triazolo-peptidomimetics (Figure 1, Table 1).The obtained five DOTA-conjugates were radiolabeled with [ 111 In]InCl 3 in radiochemical purities and yields (RCP, RCY) of >95% (Figures S33-S38) at molar activities of 3.7-7.3MBq/nmol (decay corrected) and were used in vitro and in vivo without further purification.The synthesis of non-radioactive nat In-XG1 was accomplished in a NaOAc buffer by mixing nat InCl 3 with an aqueous solution of XG1 (final pH = 4.5-5).Following purification via a Sep-PAK ® C-18 cartridge, nat In-XG1 was isolated with high purity (>95%) and yield (80%). chemical purities and yields (RCP, RCY) of > 95% (Figures S33-S38) at molar activities of 3.7-7.3MBq/ nmol (decay corrected) and were used in vitro and in vivo without further purification.The synthesis of non-radioactive nat In-XG1 was accomplished in a NaOAc buffer by mixing nat InCl3 with an aqueous solution of XG1 (final pH = 4.5-5).Following purification via a Sep-PAK ® C-18 cartridge, nat In-XG1 was isolated with high purity (>95%) and yield (80%).Scheme 1. Synthesis of the 1,4-Tz-containing peptidomimetic XG1 with the side-chains of the cysteine residues used in the cyclization in magenta and the Tz in olive-green.CTC refers to the 2chlorotrityl resin.

In Vitro Evaluation
The 111 In-radioligands were tested in vitro for their cell binding and internalization properties first for the SST2R subtype, due to its predominant expression across a wide range of tumors [10].Thus, only compounds with a retained high affinity toward the Scheme 1. Synthesis of the 1,4-Tz-containing peptidomimetic XG1 with the side-chains of the cysteine residues used in the cyclization in magenta and the Tz in olive-green.CTC refers to the 2-chlorotrityl resin.

In Vitro Evaluation
The 111 In-radioligands were tested in vitro for their cell binding and internalization properties first for the SST 2 R subtype, due to its predominant expression across a wide range of tumors [10].Thus, only compounds with a retained high affinity toward the SST 2 R were considered for further investigations.For this purpose, the rat SST 2 R-expressing AR42J cell line was used.To verify SST 2 R-specificity of the radiolabeled peptides, a 1000-fold excess of the SST 2 R-prefering TATE was used for receptor blocking experiments (Figures S31 and S32).In accordance with reported data, a rapid and SST 2 R specific cell association and internalization was observed for [ 111 In]In-AT2S (Figure 3a) with 22% of the applied activity (A.A) was found associated to the cells after 2 h, from which 80% (17% A.A) corresponds to the internalized fraction [15].A similar trend was observed for [ 111 In]In-XG1, which displayed an equally fast but superior cell binding and internalization capacity (24% of A.A was internalized, Figure 3).During receptor blockade in the presence of an excess TATE, minimal binding and internalization (1% A.A) of the radioligands were observed, thus confirming their SST 2 R-specificity.In case of [ 111 In]In-XG2, the cell associated radioactivity was found to be considerably less (6% A.A).Given the low SST 2 R-specific cell associated activity observed for [ 111 In]In-XG3, XG4 and XG5 (< 1% A.A), they were excluded from further studies.We hypothesize that the proximity of the introduced Tz-modifications to the pharmacophore of the peptide (Phe 7 -D-Trp 8 -Lys 9 -Thr 10 ) resulted in structural changes, detrimental for interaction with the SST 2 R [44].
were conducted by incubation of [ 111 In]In-XG1 and [ 111 In]In-XG2 at increasing concentrations in AR42J cells (Figure 3) and the KD of each compound was determined (Figure 3).Interestingly, despite the superior internalization of [ 111 In]In-XG1, the [ 111 In]In-AT2S reference displayed higher affinity toward SST2R (KD = 12.04 ± 3.88 nM, Figure 3a) compared to the triazolo-peptidomimetic (KD = 25.11± 4.32 nM, Figure 3b).This partial loss of affinity for SST2R was more pronounced for [ 111 In]In-XG2, the KD value of which was found to be much higher (>50 nM), compromising its prospects for clinical applicability.Consequently, only [ 111 In]In-XG1 was selected for further in vitro experiments to determine the SST1-3,5R affinity profile.Given the retained ability of [ 111 In]In-XG1 and [ 111 In]In-XG2 to internalize via SST 2 R, we set out to determine their affinity toward this receptor.Thus, SST 2 R-saturation studies were conducted by incubation of [ 111 In]In-XG1 and [ 111 In]In-XG2 at increasing concentrations in AR42J cells (Figure 3) and the KD of each compound was determined (Figure 3).Interestingly, despite the superior internalization of [ 111 In]In-XG1, the [ 111 In]In-AT2S reference displayed higher affinity toward SST 2 R (KD = 12.04 ± 3.88 nM, Figure 3a) compared to the triazolo-peptidomimetic (KD = 25.11± 4.32 nM, Figure 3b).This partial loss of affinity for SST 2 R was more pronounced for [ 111 In]In-XG2, the KD value of which was found to be much higher (>50 nM), compromising its prospects for clinical applicability.Consequently, only [ 111 In]In-XG1 was selected for further in vitro experiments to determine the SST 1-3,5 R affinity profile.
The pansomatostatin profile of [ 111 In]In-XG1 was investigated by competition binding assays of XG1 and the nat In-XG1 surrogate against the pansomatostatin [ 125 I][I-Tyr 25 ]LTT-SST-28 radioligand.The IC 50 values were determined using membrane homogenates from cells transfected to stably express one of the hSST 1-3,5 R (Figure 4).hSST 4 R was not included in this study due to its minor relevance in oncology [3,12].nat In-XG1 exhibited a similar pansomatostatin profile when compared to AT2S, with slightly lower affinities for hSST 1 R and hSST 2 R (Table 2).The observed small reduction in the hSST 2 R-affinity is in line with the respective K D values acquired during saturation binding assays in SST 2 R-positive Pharmaceutics 2024, 16, 392 14 of 20 AR42J cells.In addition, we investigated the affinity profile of the nonmetal-tagged XG1 in order to assess the influence of the metal on the binding affinity toward hSSTRs.XG1 and nat In-XG1 have a similar affinity profile but they differ in the affinity toward hSST 1 R, where nat In-XG1 displayed clearly superior affinity compared to nonmetal-tagged XG1 (Table 2).It is reported that GPCRs with a high degree of glycosylation can influence the binding affinities of chelator/metal-chelate-modified ligands due to distinct electrostatic interactions (this phenomenon has mainly been described for SST 2 R) [45].It has been reported that sialic acid is present in somatostatin receptors, and this carbohydrate could be responsible for the aforementioned electrostatic repulsive interactions.
SST-28 radioligand.The IC50 values were determined using membrane homogenates from cells transfected to stably express one of the hSST1-3,5R (Figure 4).hSST4R was not included in this study due to its minor relevance in oncology [3,12].nat In-XG1 exhibited a similar pansomatostatin profile when compared to AT2S, with slightly lower affinities for hSST1R and hSST2R (Table 2).The observed small reduction in the hSST2R-affinity is in line with the respective KD values acquired during saturation binding assays in SST2R-positive AR42J cells.In addition, we investigated the affinity profile of the nonmetal-tagged XG1 in order to assess the influence of the metal on the binding affinity toward hSSTRs.XG1 and nat In-XG1 have a similar affinity profile but they differ in the affinity toward hSST1R, where nat In-XG1 displayed clearly superior affinity compared to nonmetal-tagged XG1 (Table 2).It is reported that GPCRs with a high degree of glycosylation can influence the binding affinities of chelator/metal-chelate-modified ligands due to distinct electrostatic interactions (this phenomenon has mainly been described for SST2R) [45].It has been reported that sialic acid is present in somatostatin receptors, and this carbohydrate could be responsible for the aforementioned electrostatic repulsive interactions.In vitro stability studies were conducted for [ 111 In]In-AT2S and the [ 111 In]In-XG1 in human plasma.Both were found to be highly stable over 6 h of incubation at 37 °C (>90% of intact radiopeptides).This result is in disagreement with the poor in vivo stability reported for [ 111 In]In-AT2S (only 6% of intact peptide after 5 min in blood circulation in mice) [15].It is reported that the zinc-dependent endopeptidase NEP, an ectoenzyme anchored on epithelial cells on vessel walls and major tissues/organs of the body but not in the solute, plays a pivotal role in the degradation of SST-14 and its analogs.Hence, its action is not shown in  In vitro stability studies were conducted for [ 111 In]In-AT2S and the [ 111 In]In-XG1 in human plasma.Both were found to be highly stable over 6 h of incubation at 37 • C (>90% of intact radiopeptides).This result is in disagreement with the poor in vivo stability reported for [ 111 In]In-AT2S (only 6% of intact peptide after 5 min in blood circulation in mice) [15].It is reported that the zinc-dependent endopeptidase NEP, an ectoenzyme anchored on epithelial cells on vessel walls and major tissues/organs of the body but not in the solute, plays a pivotal role in the degradation of SST-14 and its analogs.Hence, its action is not shown in the in vitro plasma studies [19].In order to have a reliable measure of the metabolism of the peptides, in vivo stability studies of [ 111 In]In-XG1 were conducted in mice.

In Vivo Evaluation
The metabolic stability of [ 111 In]In-XG1 in peripheral mice blood was compared to the rapidly metabolized [ 111 In]In-AT2S in order to determine potential improvements in stability upon introduction of Asn 5 -Ψ[Tz]-Phe 6 motif.Compared to the reference [ 111 In]In-AT2S (6% intact peptide 5 min pi, Figure 5b), a significant improvement was achieved by [ 111 In]In-XG1 (up to 17% of intact peptide, p < 0.0001, Figure 5c).It is interesting to note that the radiometabolite pattern notably differs between [ 111 In]In-AT2S and [ 111 In]In-XG1.Degradation of [ 111 In]In-AT2S results in the formation of hydrophilic, likely low-molecularweight metabolites r t = approx.0.5 min.Figure 5b) barely retained by the HPLC column.On the other hand, the major radiometabolite of [ 111 In]In-XG1 (r t = approx.23 min) eluted close to the intact parent compound ([ 111 In]In-XG1, r t = approx.28 min, Figure 5c) indicating the presence of a higher molecular weight metabolites.We hypothesize that the introduction of the Tz at the position Asn 5 -Phe 6 of the peptide impedes with the complete cleavage of the pharmacophore of the peptide (Phe 7 -D-Trp 8 -Lys 9 -Thr 10 ), thereby preventing further cleavage of amide bonds.In the case of [ 111 In]In-AT2S, this process was not hampered resulting in the formation of hydrophilic metabolites (Figure 5b).
Pharmaceutics 2024, 16, x FOR PEER REVIEW 16 of 20 5c).According to our hypothesis as outlined above, cleavage of the bond between Thr 10 -Phe 11 amino acid residues would result in a radiometabolite that preserves two Lys residues (Lys 4 and Lys 9 , Figure 5a).It is known that radiopeptides (or metabolites thereof) containing primary amines, such as in Lys residues, tend to accumulate in the kidneys.Interaction of the protonated amines under physiological conditions with the negatively charged glomerulus basement membrane is most probably key to this process [47].A similar trend was observed for [ 111 In-DOTA]LTT-SST-28, a pansomatostatin-like radiotracer with three Lys residues [12].In the case of [ 111 In]In-AT2S on the other hand, the absence of the Tz in the position Asn 5 -Phe 6 would likely result in further degradation resulting in low molecular weight, hydrophilic metabolites lacking multiple positive charges and thus efficient clearance via the urine.The mouse pretreated with Entresto ® showed an increased uptake of [ 111 In]In-XG1 in the HEK293-hSST2R tumor (2.57% IA/g versus 1.61% IA/g for the untreated mice), confirming a positive outcome of this approach [18].Yet, the use of Entresto ® did not alleviate the high renal uptake of [ 111 In]In-XG1, in support of our hypothesis that the observed pronounced kidney uptake is a result of the intrinsic structural properties of the radiotracer.Different strategies to reduce the kidney uptake of radiolabeled peptides have been reported, some of which have found routine applications in the clinic.Examples include the infusion of solutions of basic amino acids (Lys, Arg) prior and/or during application of radiolabeled peptide or the use of plasma expanders [48][49][50].Furthermore, the replacement of Lys by Arg was shown to effectively lead to reduced kidney uptake of radiolabeled peptides or metabolites thereof [50,51].Finally, the use of cleavable linkers specific for enzymes present at the renal brush border membrane of kidneys have shown promising results [52,53].Such approaches will be investigated in the future in order to improve the pharmacokinetic properties of our lead compound [ 111 In]In-XG1.In order to assess the involvement of NEP in the metabolic fate of [ 111 In]In-XG1, we compared the metabolic patterns of the radioligand injected alone (controls) or after pretreatment of mice with Entresto ® inducing in situ NEP-inhibition (Figure S53) [46].As shown in Figure 5, [ 111 In]In-XG1 appears to be considerably more stable in the Entresto ® group (75% of intact [ 111 In]In-XG1 at 5 min pi).It is interesting to note that the same major radiometabolite is formed (r t = approx.23 min), albeit to a much lesser extent than in controls.These findings indicate that indeed NEP is the main peptidase responsible for the degradation of [ 111 In]In-XG1 in blood.Based on these studies, we hypothesize that the Tz does not only impede with the cleavage of the Asn 5 -Phe 6 bond, but also serves to prevent the hydrolysis of the amide between the Phe 6 -Phe 7 bond.However, we infer that the amide bond between the Thr 10 -Phe 11 residues remains vulnerable to the cleavage by NEP, which in turn could result in the formation of the radiometabolite observed at r t 23 min (Figure 5a, turquoise structure).Since the introduction of a Tz in this position (Thr 10 -Phe 11 ) was shown to be detrimental for SST 2 R-affinity, employment of alternative amide bond surrogates, in combination with the already incorporated Tz at Asn 5 -Ψ[Tz]Phe 6 , represents an interesting and promising alternative to enhance the stability in vivo while preserving the pansomatostatin profile of [ 111 In]In-XG1.This work is currently ongoing and will be reported in due time.
Next, a pilot SPECT/CT imaging study was conducted in two male SCID mice bearing HEK293-hSST 2 R (left flank) and control hSST 2 R-negative wtHEK293 (right flank) tumors.One animal served as untreated control, whereas the other was pretreated with Entresto ® as described above.At 4 h pi of [ 111 In]In-XG1, animals were euthanized and static images were obtained (Figure 6).To quantify results, the tumors and the kidneys were resected, and the % IA/g values were measured on a γ-counter (Figure S56).In contrast to [ 111 In]In-AT2S, [ 111 In]In-XG1 exhibited a pronounced kidney uptake in untreated mice (>200% IA/g versus 23.20 ± 7.04% IA/g for [ 111 In]In-AT2S [15]), which impeded with the visualization of the HEK293-hSST 2 R xenografts by SPECT/CT (Figure 6).The untreated mouse showed higher accumulation of [ 111 In]In-XG1 in the HEK293-hSST 2 R tumor (1.61% IA/g) as compared to the SST 2 R-negative wtHEK293 control tumor (0.56% IA/g), demonstrating SST 2 R-specific uptake.However and despite the enhanced in vivo stability, the tumor uptake of In-XG1 could be attributed to its distinctive metabolism (Figure 5c).According to our hypothesis as outlined above, cleavage of the bond between Thr 10 -Phe 11 amino acid residues would result in a radiometabolite that preserves two Lys residues (Lys 4 and Lys 9 , Figure 5a).It is known that radiopeptides (or metabolites thereof) containing primary amines, such as in Lys residues, tend to accumulate in the kidneys.Interaction of the protonated amines under physiological conditions with the negatively charged glomerulus basement membrane is most probably key to this process [47].A similar trend was observed for [ 111 In-DOTA]LTT-SST-28, a pansomatostatin-like radiotracer with three Lys residues [12].In the case of [ 111 In]In-AT2S on the other hand, the absence of the Tz in the position Asn 5 -Phe 6 would likely result in further degradation resulting in low molecular weight, hydrophilic metabolites lacking multiple positive charges and thus efficient clearance via the urine.

Conclusions
We have applied the amide-to-triazole substitution strategy to the synthesis of five novel cyclic somatostatin-based triazolo-peptidomimetics.Only [ 111 In]In-XG1 preserved the distinctive in vitro high binding affinity toward SST1,2,3,5R as in the case of reference compound [ 111 In]In-AT2S, as well as demonstrating enhanced in vivo stability.When tested in mice bearing hSST2R-expressing tumors, the triazolo-peptidomimetic [ 111 In]In-XG1 displayed receptor-specific tumor uptake.Yet, the introduction of the Tz in the pep- The mouse pretreated with Entresto ® showed an increased uptake of [ 111 In]In-XG1 in the HEK293-hSST 2 R tumor (2.57% IA/g versus 1.61% IA/g for the untreated mice), confirming a positive outcome of this approach [18].Yet, the use of Entresto ® did not alleviate the high renal uptake of [ 111 In]In-XG1, in support of our hypothesis that the observed pronounced kidney uptake is a result of the intrinsic structural properties of the radiotracer.Different strategies to reduce the kidney uptake of radiolabeled peptides have been reported, some of which have found routine applications in the clinic.Examples include the infusion of solutions of basic amino acids (Lys, Arg) prior and/or during application of radiolabeled peptide or the use of plasma expanders [48][49][50].Furthermore, the replacement of Lys by Arg shown to effectively lead to reduced kidney uptake of radiolabeled peptides or metabolites thereof [50,51].Finally, the use of cleavable linkers specific for enzymes present at the renal brush border membrane of kidneys have shown promising results [52,53].Such approaches will be investigated in the future in order to improve the pharmacokinetic properties of our lead compound [ 111 In]In-XG1.

Conclusions
We have applied the amide-to-triazole substitution strategy to the synthesis of five novel cyclic somatostatin-based triazolo-peptidomimetics.Only [ 111 In]In-XG1 preserved the distinctive in vitro high binding affinity toward SST 1,2,3,5 R as in the case of reference compound [ 111 In]In-AT2S, as well as demonstrating enhanced in vivo stability.When tested in mice bearing hSST 2 R-expressing tumors, the triazolo-peptidomimetic [ 111 In]In-XG1 displayed receptor-specific tumor uptake.Yet, the introduction of the Tz in the peptide backbone had a marked effect on the uptake of radioactivity in the kidneys, which was found considerably higher for [ 111 In]In-XG1 than for [ 111 In]In-AT2S.We hypothesize that this is a result of the distinctive metabolism of [ 111 In]In-XG1, which may lead to the formation of cationic radiometabolites that are trapped in the proximal tubule of the kidneys.The in vivo metabolic studies conducted with Entresto ® , showed the efficacy of this antihypertensive drug at preserving the integrity of [ 111 In]In-XG1 in blood through the inhibition of NEP.However, the pronounced kidney uptake was not influenced by the use of Entresto ® in tumor uptake studies.We therefore conclude that the use of [ 111 In]In-XG1 could benefit from alternative strategies aiming at the reduction of renal uptake, such as the use of plasma expanders (e.g., Gelofusine ® ) or/and basic amino acid solutions.In addition, with the aim to further increase the metabolic stability of [ 111 In]In-XG1, complementary structural modifications in combination with the triazole motif (Asn 5 -Ψ[Tz]-Phe 6 ) are currently being explored.

Figure 2 .
Figure 2. Structures of: (a) the reference compound [ 111 In]In-AT2S; (b) the triazolo-peptidomimetic [ 111 In]In-XG1 (b).The bonds highlighted in colors were subjected to the amide-to-triazole substitution (Table1).The olive-green trans-amide bonds correspond to the reported positions cleaved by NEP and the turquoise position is an adjacent bond to a cleavage site.

Figure 2 .
Figure 2. Structures of: (a) the reference compound [ 111 In]In-AT2S; (b) the triazolo-peptidomimetic [ 111 In]In-XG1 (b).The bonds highlighted in colors were subjected to the amide-to-triazole substitution (Table1).The olive-green trans-amide bonds correspond to the reported positions cleaved by NEP and the turquoise position is an adjacent bond to a cleavage site.

Figure 3 .
Figure 3.In vitro characterization of (a,b) [ 111 In]In-AT2S and (c,d) [ 111 In]In-XG1 in SST2R-expressing AR42J cells where cell binding and internalization kinetics were studied (a,c, n = 3 in triplicates) and (b,d) the KD were determined in saturation binding assays (n = 2-3 in triplicates) using GraphPad

Figure 3 .
Figure 3.In vitro characterization of (a,b) [ 111 In]In-AT2S and (c,d) [ 111 In]In-XG1 in SST 2 R-expressing AR42J cells where cell binding and internalization kinetics were studied (a,c, n = 3 in triplicates) and (b,d) the K D were determined in saturation binding assays (n = 2-3 in triplicates) using GraphPad Prism 8.0.Data points show mean values ± standard deviation (SD).NSB stands for nonspecific binding.

Table 2 .
Affinity profiles of AT2S, XG1 and nat In-XG1 for hSST1-3,5R; mean IC50 ± sd (in nanomolar), n to the unmodified reference and nat In-XG1 to the triazolo-peptidomimetic XG1 tagged with natural indium; 2 membranes individually expressing each receptor subtype were used in competition binding assays.
[ 111 In]In-XG1 was not superior to the reported data of [ 111 In]In-AT2S (1.49 ± 0.2% IA/g) [15].A direct comparison of the in vivo data should be drawn with caution because of different amounts of radiotracers applied (for this study: 11-22 MBq/3 nmol [ 111 In]In-XG1 versus reported dose for [ 111 In]In-AT2S of 34-74 KBq/10 pmol).The higher kidney uptake of [ 111 In]

Figure 6 .
Figure 6.Static whole-body SPECT/CT images of SCID mice bearing HEK293-hSST2R tumors in their left flank (green arrows) and hSST2R-negative wtHEK293 control tumors in their right flank (orange arrow) at 4 h pi of [ 111 In]In-XG1: (a) without pretreatment with Entresto ® or (b) 30 min following oral administration of Entresto ® .The kidneys are indicated with blue arrows and the color bars represent the difference in accumulated activity (purple being the lowest and white the highest level of accumulation).

Figure 6 .
Figure 6.Static whole-body SPECT/CT images of SCID mice bearing HEK293-hSST 2 R tumors in their left flank (green arrows) and hSST 2 R-negative wtHEK293 control tumors in their right flank (orange arrow) at 4 h pi of [ 111 In]In-XG1: (a) without pretreatment with Entresto ® or (b) 30 min following oral administration of Entresto ® .The kidneys are indicated with blue arrows and the color bars represent the difference in accumulated activity (purple being the lowest and white the highest level of accumulation).