SPECT Imaging of SST2-Expressing Tumors with 99mTc-Based Somatostatin Receptor Antagonists: The Role of Tetraamine, HYNIC, and Spacers

[99mTc]Tc-HYNIC-TOC is the most widely used 99mTc-labeled somatostatin receptor (SST) agonist for the SPECT imaging of SST-expressing tumors, such as neuroendocrine tumors. Recently, radiolabeled SST antagonists have shown improved diagnostic efficacy over agonists. 99mTc-labeled SST antagonists are lacking in clinical practice. Surprisingly, when [99mTc]Tc-HYNIC was conjugated to the SST2 antagonist SS01, SST2 imaging was not feasible. This was not the case when [99mTc]Tc-N4 was conjugated to SS01. Here, we assessed the introduction of different spacers (X: β-Ala, Ahx, Aun and PEG4) among HYNIC and SS01 with the aim of restoring the affinity of HYNIC conjugates. In addition, we used the alternative antagonist JR11 for determining the suitability of HYNIC with 99mTc-labeled SST2 antagonists. We performed a head-to-head comparison of the N4 conjugates of SS01 and JR11. [99mTc]Tc-HYNIC-TOC was used as a reference, and HEK-SST2 cells were used for in vitro and in vivo evaluation. EDDA was used as a co-ligand for all [99mTc]Tc-HYNIC conjugates. The introduction of Ahx restored, to a great extent, the SST2-mediated cellular uptake of the [99mTc]Tc-HYNIC-X conjugates (X: spacer), albeit lower than the corresponding [99mTc]Tc-N4-conjugates. SPECT/CT images showed that all 99mTc-labeled conjugates accumulated in the tumor and kidneys with [99mTc]Tc-HYNIC-PEG4-SS01, [99mTc]Tc-N4-SS01 and [99mTc]Tc-N4-JR11 having notably higher kidney uptake. Biodistribution studies showed similar or better tumor-to-non-tumor ratios for the [99mTc]Tc-HYNIC-Ahx conjugates, compared to the [99mTc]Tc-N4 counterparts. The [99mTc]Tc-HYNIC-Ahx conjugates of SS01 and JR11 were comparable to [99mTc]Tc-HYNIC-TOC as imaging agents. HYNIC is a suitable chelator for the development of 99mTc-labeled SST2 antagonists when a spacer of appropriate length, such as Ahx, is used.

Despite the increasing application of PET-based radiopharmaceuticals, more than 70% of nuclear medicine procedures still use 99m Tc. The ease and cost-effective availability of 99m Tc, in combination with the new-generation SPECT scanners with improved spatial resolution and sensitivity, are expected to revive the interest in 99m Tc-radiopharmaceuticals. 99m Tc-radiopharmaceuticals for imaging NETs are practically based on [Tyr 3 ]-octreotide (TOC) conjugated to the monodentate ligand hydrazinonicotinamide (HYNIC) and labeled with 99m Tc(V) using ethylenediamine N,N diacetic acid (EDDA) as a co-ligand [6][7][8].
The indications that SST antagonists may have higher sensitivity in imaging NETs than agonists, and the increased interest in 99m Tc-radiopharmaceuticals, encouraged us and other groups to develop 99m Tc-based SST antagonists for SPECT imaging [11][12][13][14][15][16]. The HYNIC/EDDA and N4 ligands were used, comparatively, for 99m Tc-labeling of the SST2 antagonist SS01 (Cpa-c(DCys-Tyr-DTrp-Lys-Thr-Cys)DTyr-NH 2 [15]. Interestingly, while [ 99m Tc]Tc-N4-SS01 demonstrated high affinity for SST2, [ 99m Tc]Tc-HYNIC/EDDA-SS01 lost affinity completely [15]. This indicated that HYNIC, one of the most commonly used ligands for 99m Tc, is unsuitable in combination with SST antagonists, although it demonstrates excellent performance with SST agonists. It remains unclear, however, if this is the case only for SS01 or also for other SST antagonists. In addition, considering that the [ 99m Tc]Tc-N4 conjugates showed very high accumulation in the kidneys [15,16], probably due to the positively charged [ 99m Tc]Tc(O) 2 (N 4 )] +1 , it is worth investigating if HYNIC can still be used in combination with SST2 antagonists.

Synthesis, Radiolabeling and Partition Coefficients (log D)
The chemical structures of the radiolabeled peptide conjugates investigated in this study are shown in Figure 1, and their analytical data are summarized in Table 1. All conjugates were synthesized with a maximum yield of 30-40% by following an Fmoc-based solid phase peptide synthesis. SS01 was coupled either directly to HYNIC or via different spacers, namely, β-Ala, Ahx, Aun and PEG 4 ( Figure 1). JR11 was coupled to HYNIC and HYNIC-Ahx ( Figure 1). In addition, both peptides were coupled to the chelator N4 to form the conjugates N4-SS01 and N4-JR11 ( Figure 1). The purity of all conjugates was confirmed Pharmaceuticals 2021, 14, 300 3 of 16 by RP-HPLC analysis (∼97%) and characterized by ESI-MS (Table 1). All conjugates were  labeled with 99m Tc with labeling yields of >97% at a maximum apparent molar activity of  130 MBq/nmol. The N4 conjugates were labeled with 99m Tc at room temperature within  30 min in the presence of Tin(II)chloride and citrate as an intermediate supporting Tc(V) oxidation state. The HYNIC conjugates were labeled with 99m Tc at 95 • C within 15 min in the presence of Tin(II)chloride and EDDA as a co-ligand. For the sake of simplicity, EDDA is omitted from the abbreviated names of the HYNIC-based radiotracers.
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 17 different spacers, namely, β-Ala, Ahx, Aun and PEG4 ( Figure 1). JR11 was coupled to HYNIC and HYNIC-Ahx ( Figure 1). In addition, both peptides were coupled to the chelator N4 to form the conjugates N4-SS01 and N4-JR11 ( Figure 1). The purity of all conjugates was confirmed by RP-HPLC analysis (∼97%) and characterized by ESI-MS (Table 1).  All conjugates were labeled with 99m Tc with labeling yields of >97% at a maximum apparent molar activity of 130 MBq/nmol. The N4 conjugates were labeled with 99m Tc at room  temperature within 30 min in the presence of Tin(II)chloride and citrate as an intermediate  supporting Tc(V) oxidation state. The HYNIC conjugates were labeled with 99m Tc at 95 °C  within 15 min in the presence of Tin(II)chloride and EDDA as a co-ligand. For the sake of simplicity, EDDA is omitted from the abbreviated names of the HYNIC-based radiotracers. The quality control of the radiotracers was repeated after 2 h in their labeling solutions, at pH 7, as described in the materials and methods section. The labeling solutions were kept at room temperature and at activity concentrations in the range of 500-1000 MB/mL for this period. The quality control was performed by radio-HPLC to assess the time period after radiolabeling that the radiotracer can be used safely for evaluation. Most of the radiotracers were found to be stable at room temperature over 2 h, with 95% of intact radiotracer, with the exception of [ 99m Tc]Tc-N4-SS01, [ 99m Tc]Tc-N4-JR11 and [ 99m Tc]Tc-HYNIC-JR11, which were 88 ± 2%, 92 ± 3% and 92 ± 1%, respectively (n = 2-3). The radiochemical species found after 2 h were peptide related (radiolytic products). No released 99m Tc was detected, while no radical scavengers were added or tested. Based on these results, special care was taken on the use of the three above-mentioned radiotracers immediately after radiolabeling.
The log D of the radiotracers was determined by their distribution in phosphate-buffered saline (pH 7.4) and 1-octanol, as described in the experimental section. The results are summarized in Table 1. [ 99m Tc]Tc-HYNIC-Ahx-JR11 exhibited the highest hydrophilicity among all radiotracers (log D = −3.15 ± 0.06). In general, all JR11-based radiotracers were more hydrophilic than the corresponding SS01-based radiotracers. The high hydrophobicity of [ 99m Tc]Tc-HYNIC-Aun-SS01 and its adsorption to materials (e.g., tubes, tips The quality control of the radiotracers was repeated after 2 h in their labeling solutions, at pH 7, as described in the materials and methods section. The labeling solutions were kept at room temperature and at activity concentrations in the range of 500-1000 MB/mL for this period. The quality control was performed by radio-HPLC to assess the time period after radiolabeling that the radiotracer can be used safely for evaluation. Most of the radiotracers were found to be stable at room temperature over 2 h, with 95% of intact radiotracer, with the exception of [ 99m Tc]Tc-N4-SS01, [ 99m Tc]Tc-N4-JR11 and [ 99m Tc]Tc-HYNIC-JR11, which were 88 ± 2%, 92 ± 3% and 92 ± 1%, respectively (n = 2-3). The radiochemical species found after 2 h were peptide related (radiolytic products). No released 99m Tc was detected, while no radical scavengers were added or tested. Based on these results, special care was taken on the use of the three above-mentioned radiotracers immediately after radiolabeling.
The log D of the radiotracers was determined by their distribution in phosphatebuffered saline (pH 7.4) and 1-octanol, as described in the experimental section. The results are summarized in Table 1. [ 99m Tc]Tc-HYNIC-Ahx-JR11 exhibited the highest hydrophilicity among all radiotracers (log D = −3.15 ± 0.06). In general, all JR11-based radiotracers were more hydrophilic than the corresponding SS01-based radiotracers. The high hydrophobicity of [ 99m Tc]Tc-HYNIC-Aun-SS01 and its adsorption to materials (e.g., tubes, tips and plates) did not enable the reliable determination of its partition coefficient or further evaluation in vitro. [ 99m Tc]Tc-HYNIC-Aun-SS01 was excluded from further studies.

SPECT/CT Imaging
The SPECT/CT images were acquired head-to-head for all radiotracers at 1 and 4 h post injection (p.i.) ( Figure 5). Imaging studies were not performed for [ 99m Tc]Tc-HYNIC-SS01 or [ 99m Tc]Tc-HYNIC-JR11, as the in vitro data revealed very low cellular uptake, nor for [ 99m Tc]Tc-HYNIC-Aun-SS01, which was excluded for the reasons described above. On the basis of the SPECT/CT images, and of the in vitro cellular uptake, the radiotracers [ 99m Tc]Tc-HYNIC-Ahx-SS01 and [ 99m Tc]Tc-HYNIC-Ahx-JR11 were selected for further in vivo evaluation, in comparison to their [ 99m Tc]Tc-N4 counterparts.
In the case of the SS01 analogs (  On the basis of the SPECT/CT images, and of the in vitro cellular uptake, the radiotracers [ 99m Tc]Tc-HYNIC-Ahx-SS01 and [ 99m Tc]Tc-HYNIC-Ahx-JR11 were selected for further in vivo evaluation, in comparison to their [ 99m Tc]Tc-N4 counterparts.
In the case of the SS01 analogs ( ± 1.59 %IA/g; p = 0.002). The difference between the two JR11-based radiotracers in total body distribution also followed the same trend, with lower background activity for [ 99m Tc]Tc-HYNIC-Ahx-JR11 but not as profound as in the case of the SS01-based radiotracers. Consequently, superiority was not observed in one vs. another radiotracer in terms of tumor-to-non-tumor ratios.

Discussion
A number of preclinical and initial clinical studies (summarized in [17,18]) suggested improvements in both diagnostic sensitivity and therapeutic efficacy by the use of radiolabeled SST2 antagonists (i.e., DOTA-JR11) instead of agonists (i.e., DOTA-TATE or DOTA-TOC). This was attributed to different reasons, including but not limited to, the following: (a) a higher number of binding sites recognized by the antagonists on the surface of SST-expressing cells [19,20], with consequently higher accumulation; (b) lower uptake of the antagonists in organs that were the primary sites of metastasis (i.e., liver), enabling better lesion-to-background contrast, and as such better image sensitivity [4]; (c) longer retention of the antagonists in the tumor, leading to higher tumor radiation doses [3,21]; and (d) a higher number of DNA double strand breaks caused by the antagonists on SST-expressing cells [22], suggesting more effective treatment. All these findings indicate that internalization, which is induced upon the binding of an agonist to the receptor but not by an antagonist, is not a prerequisite for high image contrast or for effective treatment, as it was initially assumed.
The choice of the somatostatin analog, i.e., agonist or antagonist; the presence of spacers; the chelating system; and the radiometal play an important role in modulating receptor binding affinity and in vivo distribution [2,[23][24][25]. A remarkable example was reported by Abiraj et al. [15] on the somatostatin antagonist SS01, showing drastically different binding properties when conjugated to two chelating systems widely used for radiolabeling with 99m Tc. The complete loss of affinity of [ 99m Tc]Tc-HYNIC-SS01 for SST2, which could be restored using the acyclic tetramine N4 ([ 99m Tc]Tc-N4-SS01), warranted further investigation. This work intended to investigate the role of the HYNIC as a monodentate ligand for 99m Tc-labeled SST2 antagonists and the influence of different spacers on the modulation of the binding affinity of the HYNIC conjugates. In addition, we aimed to determine the suitability of known chelating systems for the development of 99m Tc-labeled SST2 antagonists.
The introduction of spacers between the chelator and the peptide moiety is a common strategy to prevent steric influence and to retain the affinity of the peptide for its recep-tor [26]. This was not required when SS01 was conjugated to N4 ([ 99m Tc]Tc-N4-SS01) or to DOTA ([ 177 Lu]Lu-DOTA-SS01) [15]. However, it was shown that SST2 antagonists are very sensitive to N-terminus modifications, with different chelating systems and radiometals having unexpected impacts on affinity, biodistribution and pharmacokinetics [2,27]. This seemed to be the case for the HYNIC vs. N4 conjugates of the SST2 antagonists. A significant difference between the two is the nature of the 99m Tc-complex. N4 is labeled via the Tc-dioxo core forming the positively charged [ 99m Tc(O) 2 (N 4 )] +1 moiety (Figure 1). HYNIC is labeled via the Tc(V)-hydrazino metal fragment ([ 99m Tc=N=N-R] 2+ ), where it can be coordinated as a diazene (neutral) or diazenido (anionic) ligand, with EDDA acting as a co-ligand to stabilize the coordination geometry (Figure 1). The structure of the Tc primary coordination sphere in the case of HYNIC is uncertain. [ 99m Tc]Tc-HYNIC complexes have different isomeric forms, and the corresponding radiotracers adapt various conformations, without well-defined chemical structures [28][29][30]. We hypothesized that certain conformations of the HYNIC conjugates [ 99m Tc]Tc-HYNIC-SS01 and [ 99m Tc]Tc-HYNIC-JR11 were responsible for the loss of affinity and that they may be altered when introducing spacers. Unfortunately, the unclear chemistry around [ 99m Tc]Tc-HYNIC complexes and the difficulty to obtain a non-radioactive chemically identical compound (e.g., 99g Tc-HYNIC/EDDA-SS01) limited the options of investigating this hypothesis by docking or NMR studies.
We confirmed the low cellular uptake of [ 99m Tc]Tc-HYNIC-SS01, reported by Abiraj et al. [15], and were able to identify that introducing a spacer restores, to different extents depending on the spacer's length and physicochemical properties, the binding affinity of SS01 toward SST2. The neutral C 3 β-Ala and C 6 Ahx and the polar and hydrophilic C 11 -type PEG 4 led to a significant improvement in the SST2-mediated cellular uptake in vitro, with the C 6 distance allowing the best binding to the receptor. The aliphatic C 10 Aun was proven to be an unsuitable spacer due to its hydrophobicity and adsorption to materials. Therefore, [ 99m Tc]Tc-HYNIC-Aun-SS01 was excluded from the study. More specifically, the introduction of a C 3 spacer ([ 99m Tc]Tc-HYNIC-β-Ala-SS01) improved the cellular uptake of [ 99m Tc]Tc-HYNIC-SS01 by a factor of six (approx. 15 vs. 2.5%, respectively, at 4 h). Increasing the length to C 6 ([ 99m Tc]Tc-HYNIC-Ahx-SS01) increased the cellular uptake nearly to the levels of [ 99m Tc]Tc-HYNIC-TOC (approx. 40 vs. 50%, respectively, at 4 h). Further increasing the length, while introducing polar hydrophilic properties ([ 99m Tc]Tc-HYNIC-PEG 4 -SS01), did not lead to any further improvement. On the contrary, cellular uptake was at the level of the β-Ala conjugate (approx. 15%). In summary, [ 99m Tc]Tc-HYNIC-Ahx-SS01 increased the cellular uptake of [ 99m Tc]Tc-HYNIC-SS01 by 16-fold, though it remained significantly lower than the uptake achieved with [ 99m Tc]Tc-N4-SS01 (38.1 ± 0.2 vs. 78.5 ± 0.3%, respectively, at 4 h).
The influence of the spacers was also illustrated in the SPECT/CT images: the β-Ala conjugate showed high tumor and low kidney uptake at 1 h p.i., yet very fast washout from the tumor from 1 to 4 h p.i. The PEG 4 conjugate was characterized by high kidney uptake and rather low accumulation in the tumor, compared to all other derivatives, despite the similar cellular uptake to the β-Ala conjugate in vitro. We observed that the in vivo tumor uptake was better correlated to the affinity (K D ) and B max in vitro data (measured at concentrations at saturation level) than to the cellular uptake determined at sub-saturation levels. The Ahx conjugate showed high and persistent accumulation in the tumor, lower, however, than [ 99m Tc]Tc-N4-SS01 (14.94 ± 5.15 and 12.82 ± 3.09 %IA/g at 1 and 4 h p.i., respectively, for the HYNIC-Ahx conjugate vs. 19.12 ± 4.47 and 28.41 ± 4.84 %IA/g at 1 and 4 h p.i., respectively, for the N4 conjugate), as confirmed by quantitative biodistribution studies. The difference between these two conjugates may be attributed to the lower number of binding sites recognized by the HYNIC-Ahx vs. the N4 conjugate (B max = 0.06 ± 0.01 vs. 0.17 ± 0.02 nM, respectively) and/or to the slightly lower affinity (K D = 1.60 ± 0.46 vs. 0.92 ± 0.16 nM, respectively). On the other hand, the HYNIC-Ahx conjugate showed significantly lower kidney retention (9.47 ± 1.74 and 5.60 ± 0.47 %IA/g at 1 and 4 h p.i.) compared to the N4 conjugate (43.63 ± 11.37 and 25.85 ± 5.23 %IA/g at 1 and 4 h p.i.). The kidney uptake was washed out faster than the tumor, leading to a tumor-to-kidney ratio of 2.3 at 4 h p.i. for [ 99m Tc]Tc-HYNIC-Ahx-SS01, which was higher than that achieved with [ 99m Tc]Tc-N4-SS01 (T/K = 1.1 at 4 h p.i., recently published as [ 99m Tc]Tc-TECANT-2 [16]). Interestingly, the lower kidney and background activity of [ 99m Tc]Tc-HYNIC-Ahx-SS01, compared to [ 99m Tc]Tc-N4-SS01, favored [ 99m Tc]Tc-HYNIC-Ahx-SS01 in terms of tumor-to-background contrast, despite the significantly higher tumor uptake of [ 99m Tc]Tc-N4-SS01. The image contrast of [ 99m Tc]Tc-HYNIC-Ahx-SS01 compared well with [ 99m Tc]Tc-HYNIC-TOC in SPECT/CT imaging at 1 h p.i.
The choice of JR11 as an alternative SST2 antagonist has been dictated by the presence of innumerable data on this peptide, conjugated with different chelators and radiolabeled with various radiometals. It is currently being evaluated in clinical trials (known as OPS201 or [ 177 Lu]Lu-DOTA-JR11 and as OPS202 or [ 68 Ga]Ga-NODAGA-JR11) (summarized in [18]). The trend observed in the SS01 family was the same for the conjugates of the JR11 family, although [ 99m Tc]Tc-HYNIC-JR11 showed non-negligible cellular uptake (approx. 15% at 4 h), which was not the case for [ 99m Tc]Tc-HYNIC-SS01. The introduction of Ahx increased the cellular uptake by a factor of four (approx. 55%), while once more, the highest cellular uptake was found for [ 99m Tc]Tc-N4-JR11 (approx. 70%).
In summary, although we could not elucidate the mechanism behind the influence of the spacer in HYNIC conjugates of SST2 antagonists, we could show that the introduction of spacers has a significant impact on this class of radiotracers. This is clinically relevant, first because there are indications that the 99m Tc-based SST2 antagonist may improve NET diagnostics with SPECT, similarly to 68 Ga-based SST2 antagonists for PET [4], and second because there is already an approved 99m Tc-radiopharmaceutical based on the HYNIC strategy ([ 99m Tc]Tc-HYNIC-TOC, Tektrotyd), while there is no approved radiopharmaceutical based on N4.

Synthesis of the Chelator-Peptide Conjugates
The peptide SS01 was assembled on the automated microwave peptide synthesizer Liberty Blue (CEM, Charlotte, NC, USA) using Rink-Amide methylbenzylhydryl (MBHA) resin. The spacers, Fmoc-Ahx-OH, Fmoc-Aun-OH and Fmoc-PEG 4 -OH, and the succinimidyl-N-Boc-HYNIC were then coupled manually to afford the desired peptide conjugate derivatives. The conjugate HYNIC-Ahx-JR11 was synthesized following the same protocol as above. The peptides were finally purified on a Bischoff chromatography system by semi-preparative RP-HPLC using a waters XBridge C-18 column (10 mm × 150 mm, 5 µm particle size). Eluents: A = H 2 O (0.1% TFA) and B = acetonitrile (0.1% TFA); gradient: 5 to 50% B in 15 min; flow rate: 2 mL/min. The conjugates HYNIC-β-Ala-SS01, N4-SS01, HYNIC-JR11 and N4-JR11 were purchased from PiChem (Austria). HYNIC-TOC was kindly provided by POLATOM (Poland). Purity and identity were confirmed by LC-MS. The analytical data of the peptide conjugates are reported in Table 1. The stability of all radiotracers at room temperature was assessed up to 2 h by radio-HPLC.

Log D Measurements
Determination of log D (pH = 7.4) was performed by the shake-flask method. In a prelubricated Eppendorf tube, a pre-saturated mixture of 500 µL 1-octanol and 500 µL of phosphate-buffered saline (PBS) at pH 7.4 was added. An amount of 10 µL of 1 µM radiotracer solution was added to this mixture, followed by vortexing of the tube at room temperature for 15 min to reach equilibrium. Centrifugation for 15 min at 3000 rpm was performed for separation of the phases. Aliquots of 100 µL were removed from the octanol and from the PBS phase, and the activity measured in a γ-counter. The partition coefficient was calculated as the average log ratio value of the radioactivity in the organic fraction and PBS fraction.

Cell Cultures and Cell Membranes
The HEK-293 cell line expressing the T7-epitope tagged human SST2 receptor (HEK-SST2) was provided by Prof. Stefan Schulz (Institute of Pharmacology and Toxicology, Jena University Hospital, Jena, Germany). HEK-SST2 cells were cultured at 37 • C and 5% CO 2 in DMEM containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 200 µmol/mL L-Glutamin and 500 µg/mL G418. For all the in vitro assays on intact cells, HEK-SST2 cells were seeded into six-well plates (about 1.0 × 10 6 cells) 24 h before starting the experiment, incubated with 1% FBS containing medium at 37 • C/5% CO 2 . On the day of the experiment, the cells were washed and incubated with the adjusted medium volume for 1 h prior to starting the experiment. The plates were pre-treated with a solution of 10% poly-lysine to promote cell attachment.
For cell membrane preparation, the HEK-SST2 cells were grown to confluence, mechanically disaggregated, washed with PBS buffer (pH 7.0) and re-suspended in homogenization buffer (Tris 20 mM; ethylenediaminetetraacetic acid (EDTA) 1 mM; sucrose 0.25 M; bacitracin 1 mg/mL; soybean trypsin inhibitor 0.1 mg/mL; and phenylmethylsulfonyl fluoride (PMSF) 0.125 mg/mL, pH 7.5). Cells were homogenized using Ultra-Turrax, and the homogenized suspension was centrifuged at 500 g for 10 min at 4 • C. The supernatant was collected in centrifuge tubes (Beckman Coulter Inc., Brea, CA, USA), and this procedure was repeated five times. The collected supernatant was centrifuged in an ultra-centrifuge (Beckman) at 4 • C for 55 min at 20,000 RPM. Then, the pellet was re-suspended in icecold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM, pH 7.5), aliquoted and stored at −80 • C. The protein concentration of the membrane solution was determined using the Bradford method with bovine serum albumin as the standard.

In Vitro Internalization
The cell surface-bound and internalization rates of the radiotracers were studied in HEK-SST2 cells seeded in six-well plates, as described previously [27]. Briefly, the radiotracer (2.5 nM) was added, and the cells were incubated at 37 • C. At different time points (0.5, 1, 2 and 4 h), cellular uptake was stopped by washing twice with ice-cold PBS. A membrane-bound radiotracer was obtained by washing cells twice with ice-cold glycine buffer (pH 2.8), followed by a collection of the internalized fraction with 1 M NaOH. The activity in each fraction was measured in a γ-counter (Cobra II). Non-specific uptake was determined in the presence of 1000-fold excess of the corresponding conjugate. The results were expressed as a percentage of the applied radioactivity, after subtracting the non-specific uptake.  The Veterinary Office (Department of Health) of the Cantonal Basel-Stadt approved the animal experiments (approval no. 30515) in accordance with the Swiss regulations for animal treatment. Female athymic nude-Foxn1nu/Foxn1+ mice (Envigo, The Netherlands), 4-6 weeks old, were injected subcutaneously with 10 7 HEK-SST2 cells in the right shoulder, freshly suspended in 100 µL sterile phosphate-buffered saline. The tumors were allowed to grow for 2-3 weeks.

SPECT/CT Imaging
Mice bearing HEK-SST2 tumors were euthanized 1 or 4 h after intravenous injection of 4-6 MBq (200 pmol) of the 99m Tc-labeled conjugates and imaged supine, head first, using a SPECT/CT system dedicated to imaging small animals (NanoSPECT/CT TM Bioscan Inc.). Topograms and helical CT scans of the whole mouse were first acquired using the following parameters: X-ray tube current: 177 µA, X-ray tube voltage 45 kVp, 90 s and 180 frames per rotation, pitch 1. The helical SPECT scan was then acquired from head to toe using multi-purpose pinhole collimators (APT1). The energy window width was 20%, centered symmetrically over the energy peak of 99m Tc at 140 keV. Twenty-four projections (200 s per projection) were used, allowing acquisition of at least 50 kilocounts/projection. SPECT images were reconstructed iteratively and filtered using the HiSPECT software package (version 1.4.1876, SciVis GmbH, Goettingen, Germany) and the manufacturer's algorithm (3 subsets, 9 iterations, 35% postfiltering, 128 × 128 matrix, zoom 1, 30 × 20 mm transaxial field of view, resulting in a pixel size of 0.3 mm). CT images were reconstructed using CTReco (version r1.146), with a standard filtered back projection algorithm (exact cone beam) and postfiltered (RamLak, 100% frequency cut-off), resulting in a pixel size of 0.2 mm. Co-registered images were visualized in the three orthogonal planes using maximum intensity projection with InVivoScope (version 1.43, Bioscan Inc.).

Biodistribution and Pharmacokinetics
The radiotracers [ 99m Tc]Tc-N4-SS01, [ 99m Tc]Tc-HYNIC-Ahx-SS01, [ 99m Tc]Tc-N4-JR11 and [ 99m Tc]Tc-HYNIC-Ahx-JR11 were injected via the tail vein (100 µL, 20 pmol, 0.6-0.8 MBq) and sacrificed at 1, 4 and 24 h p.i. Organs of interest and blood were collected, rinsed of excess blood, blotted dry, weighed and counted in a γ-counter. The percentage of injected activity per gram (%IA/g) was calculated for each tissue. The total counts injected per animal were determined by extrapolation from counts of an aliquot taken from the injected solution as a standard.

Conclusions
Two highly potent SST2 antagonists, SS01 and JR11, were selected and conjugated to HYNIC, with and without different spacers, and to N4 as chelating systems for 99m Tclabeling and studied in vitro and in vivo. The conjugation of HYNIC significantly hindered the binding properties of both SS01 and JR11, but the introduction of a spacer remedied this obstacle. HYNIC proved to be a suitable chelating system for the development of 99m Tclabeled SST2 antagonists when a spacer of appropriate length, such as Ahx, is used, and the suitability of N4 was confirmed. When comparing HYNIC-Ahx to the N4 conjugates, while the HYNIC-Ahx conjugates showed faster washout from the tumor, they were either better (in the case of SS01) or equivalent (in the case of JR11) to the N4 conjugates, in terms of tumor-to-background contrast.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.