Selection of the First 99mTc-Labelled Somatostatin Receptor Subtype 2 Antagonist for Clinical Translation—Preclinical Assessment of Two Optimized Candidates

Recently, radiolabelled antagonists targeting somatostatin receptors subtype 2 (SST2) in neuroendocrine neoplasms demonstrated certain superior properties over agonists. Within the ERA-PerMED project “TECANT” two 99mTc-Tetramine (N4)-derivatized SST2 antagonists (TECANT-1 and TECANT-2) were studied for the selection of the best candidate for clinical translation. Receptor-affinity, internalization and dissociation studies were performed in human embryonic kidney-293 (HEK293) cells transfected with the human SST2 (HEK-SST2). Log D, protein binding and stability in human serum were assessed. Biodistribution and SPECT/CT studies were carried out in nude mice bearing HEK-SST2 xenografts, together with dosimetric estimations from mouse-to-man. [99mTc]Tc-TECANT-1 showed higher hydrophilicity and lower protein binding than [99mTc]-TECANT-2, while stability was comparable. Both radiotracers revealed similar binding affinity, while [99mTc]Tc-TECANT-1 had higher cellular uptake (>50%, at 2 h/37 °C) and lower dissociation rate (<30%, at 2 h/37 °C). In vivo, [99mTc]Tc-TECANT-1 showed lower blood values, kidney and muscles uptake, whereas tumour uptake was comparable to [99mTc]Tc-TECANT-2. SPECT/CT imaging confirmed the biodistribution results, providing the best tumour-to-background image contrast for [99mTc]Tc-TECANT-1 at 4 h post-injection (p.i.). The estimated radiation dose amounted to approximately 6 µSv/MBq for both radiotracers. This preclinical study provided the basis of selection of [99mTc]Tc-TECANT-1 for clinical translation of the first 99mTc-based SST2 antagonist.


Introduction
Combination of molecular and anatomical imaging (hybrid imaging, SPECT/CT and PET/CT) is currently the most sensitive approach for visualization of somatostatin receptor (SST)-positive tumours, in particular neuroendocrine neoplasms (NEN), utilizing radiolabelled somatostatin analogues [1][2][3]. These analogues were constructed focusing on their agonistic behaviour, based on their internalization after SST activation and consequent retention within the tumour cell, believed to be crucial for efficient molecular imaging and therapy. Over the last few years, it has been shown that novel molecular probes, SST antagonists, recognize more binding sites and hence improve the diagnostic efficacy, especially when the density of SST is low [4][5][6][7].
Preclinical data and subsequent clinical evaluation demonstrated higher tumour uptake of an indium-111 ( 111 In) labelled antagonist [ 111 In]In-DOTA-SST2-ANT ([ 111 In]In-DOTA-BASS) compared to the agonist [ 111 In]In-DTPA 0 -octreotide or [ 111 In]In-DTPA 0octreotate, as well as superior tumour-to-background ratios [5,8]. One of the first reports describing the SST2 antagonist LM3 indicates the high potential of gallium-68 ( 68 Ga) and copper-64 ( 64 Cu) radiolabelled LM3 (p-Cl-Phe-cyclo(D-Cys-Tyr-D-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)-D-Tyr-NH 2 ) in PET/CT [6]. The authors have demonstrated strong dependence of the affinity and pharmacokinetics of the somatostatin-based radiolabelled antagonists on the chelator and radiometal, also confirmed using another SST2 antagonist, namely JR11 [9,10]. Superiority of an SST antagonist versus agonist was demonstrated in a phase I/II clinical study using the PET tracer [ 68 Ga]Ga-NODAGA-JR11 [7,11] and in a pilot study conducted with the beta-emitting, therapeutic radionuclide (lutetium-177, 177 Lu) labelled SST2 antagonist [ 177 Lu]Lu-DOTA-JR11 [12]. As a result, research in the field is currently strongly focused on radiolabelled SST2 antagonists. 68 Ga-labelled somatostatin analogues are established for PET imaging as a unique tool for personalizing treatment of NEN. Nevertheless, single-photon emitting radiopharmaceuticals still represent the cornerstone of molecular imaging, particularly those based on technetium-99m ( 99m Tc). Its physical properties (half-life of 6 h, optimal energy of 140 keV for imaging and lowest radiation exposure), widest on-site availability and costeffectiveness are of major importance for routine clinical applications. Medical diagnostic imaging techniques using 99m Tc account for approximately 80% of all nuclear medicine procedures. In a recent study [13], the SST2 antagonist SS01 (p-Cl-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH 2 ), based on the first radiolabelled SST2 antagonist applied in humans (BASS, bearing a p-NO 2 -Phe in position 1 instead of p-Cl-Phe), was conjugated to two different chelating systems, both suitable for radiolabelling with 99m Tc. In contrast to the hydrazinonicotinamide/ethylenediaminediacetic acid (HYNIC/EDDA) conjugate that lost its affinity for SST2, the tetramine-chelator N4 conjugate revealed high cellular uptake in SST2-expressing cells, and was clearly superior, again confirming the importance of the chelator's choice in the development of radiolabelled SST2 antagonists. 99m Tc-N4-SS01 was evaluated in vivo in nude mice bearing SST2-expressing xenografts, showing an impressive tumour uptake of 47% IA/g at 4 h post-injection (p.i.) and high tumour to normal organ ratios.
The main aim of this work was to study head-to-head the two 99m Tc-labelled SST2 antagonists (see Scheme 1), one based on the LM3 structure ("TECANT-1" [6]), the other based on SS01 structure ("TECANT-2" [13]), both conjugated with the N4-chelator, providing a positively charged dioxo-99m Tc(V)-complex. These two candidates differ only on the amino acid in position four of the peptide sequence; D-4-amino-Phe(carbamoyl) in TECANT-1 vs. Trp in TECANT-2. The goal was to select the best 99m Tc-labelled SST2 antagonist for clinical translation in NEN imaging.

Lipophilicity and Protein Binding
The results of the lipophilicity and protein binding are presented in Table 1

Binding Affinity Studies
The results of competition assays are presented in Figure 3. The mean indicative half maximal inhibitory concentration (IC 50 ) values did not differ significantly and were for TECANT-1: 4.14 ± 0.83 nM, for TECANT-2: 5.00 ± 1.14 nM, comparable to DOTA 0 -Tyr 3 -Octreotate (DOTA-TATE) with 5.18 ± 1.53 nM, in the same assay. The corresponding Recomplexes revealed no alteration of the binding affinity, as compared to the non-metallated peptides, with IC 50 values of 5.01 ± 0.93 nM and 4.47 ± 0.89 nM for nat Re-TECANT-1 and nat Re-TECANT-2, respectively.

Dosimetry
To account for various possible locations of the tumour, we used two S-values. One was the value for the liver and is representative for the liver lesions. Other was the "average abdominal S-value", which was the average S-value for the following organs: stomach, liver, gallbladder, spleen, pancreas, small intestine, kidneys, large intestine, and adrenal glands. Estimated dose to various organs, average total body dose and effective dose are summarized in Table 3 for both radiotracers and two typical tumour locations.

Dosimetry
To account for various possible locations of the tumour, we used two S-values. One was the value for the liver and is representative for the liver lesions. Other was the "average abdominal S-value", which was the average S-value for the following organs: stomach, liver, gallbladder, spleen, pancreas, small intestine, kidneys, large intestine, and adrenal glands. Estimated dose to various organs, average total body dose and effective dose are summarized in Table 3 for both radiotracers and two typical tumour locations.

Discussion
Somatostatin analogues, labelled with γ-emitters (e.g., 111 In, 99m Tc) or β + -emitters (e.g., 68 Ga, 64 Cu), are nowadays widely used for the detection of the primary tumour, staging, restaging and assessment of treatment response in patients with NENs [2]. Despite the recent and increasing use of PET-radiopharmaceuticals due to the higher sensitivity and resolution of the PET images as compared to SPECT, 99m Tc remains a backbone for diagnostic procedures in nuclear medicine. The combination of its optimal nuclear properties and the diverse coordination chemistry render it highly versatile, able to be incorporated in different chelate systems at different oxidation states, and thus modulating the biological and chemical properties of the radiopharmaceutical. Even though the 99m Tc-based somatostatin agonist hydrazinonicotinamide/ethylenediamine N,N -diacetic acid-Tyr 3octreotide (HYNIC/EDDA-TOC) is used in several European countries ( 99m Tc-Tektrotyd ® ), few preclinical studies have reported the development of somatostatin receptor antagonists radiolabelled with 99m Tc [13][14][15][16].
In this work, the two SST2 antagonists [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 were assessed comparatively to determine the best candidate for potential clinical translation within the international ERA-PerMED project "TECANT" [17]. 99m Tc-labelling was simple for both peptides and resulted in reproducible high yields. Details on radiolabelling results, analytics related to all potential impurities and the development of a kit-formulation will be described in a separate paper. No remarkable differences were found in the stability and the competition assays. Each radiotracer remained intact in serum at 37 • C over a period of 24 h. The affinity for SST2 was comparable for both (IC 50 = 4.1 vs. 5.0 nM for TECANT-1 and TECANT-2, respectively). In addition, metal complexing did not alter SST2 affinity significantly, which could be shown by using the respective rhenium complexes as surrogates for 99m Tc.
The in vitro studies on HEK-SST2 intact cells depicted a similar, but not identical, behaviour of both radiotracers. [ 99m Tc]Tc-TECANT-1 showed higher cell surface binding and slower dissociation rate, indicating a stronger receptor-peptide interaction as compared to [ 99m Tc]Tc-TECANT-2. The low internalization rate is indicative of their antagonistic behaviour. For [ 99m Tc]Tc-TECANT-1, no direct comparison with literature data is possible since it is the first time that the peptide LM3 coupled to the chelator N4 has been evaluated. Interestingly, our data revealed that this peptide-known to be highly susceptible to diverse chelate systems [9]-in this conjugated form (N4-LM3) retained high affinity and binding properties on HEK-SST2 expressing cells, in vitro and in vivo. In addition, our data regarding the in vitro performance of [ 99m Tc]Tc-TECANT-2 on HEK-SST2 cells are in agreement with Abiraj et al. [13].
Pharmacokinetic studies showed clear differences between the two radiotracers. [ 99m Tc]Tc-TECANT-1 presented 2.9 and 2.5 higher uptake in the pancreas and stomach, respectively, (both being SST-positive) as compared to [ 99m Tc]Tc-TECANT-2, but 2.7 lower kidney uptake at 1 h p.i.. The highest tumour uptake was reached for both radiotracers at 4 h p.i. being very similar (26.01 ± 1.33% IA/g and 28.41 ± 4.84% IA/g for [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2, respectively). Nevertheless, [ 99m Tc]Tc-TECANT-1 showed longer tumour retention with 19.02 ± 2.00% IA/g uptake at 24 h versus the 10.98 ± 4.69% IA/g of the [ 99m Tc]Tc-TECANT-2, even though not statistically significant. Imaging studies performed at 1 h and 4 h p.i. for both radiotracers reflected the biodistribution data: [ 99m Tc]Tc-TECANT-1 showed higher abdomen uptake at 1 h, already washed out at 4 h p.i., while [ 99m Tc]Tc-TECANT-2 presented higher and more persistent kidney uptake. The lower kidney uptake and fast washout of the radioactivity from the target tissues compared to the accumulation in the tumour led to better tumour-to-background contrast for [ 99m Tc]Tc-TECANT-1 at 4 h p.i., as shown in the SPECT/CT images.
The biodistribution profile of [ 99m Tc]Tc-TECANT-2 reflected the results published by Abiraj et al. on this radiotracer ([ 99m Tc]Tc-SS04 in [13], with similar distribution pattern in target tissues as well as in excretory organs over time. The lower hydrophilicity of this Few attempts to develop 99m Tc-based somatostatin antagonistic radiotracers have been published until now, despite the wide availability and low cost of this radionuclide. A critical parameter is the choice of the chelator that needs to fulfil certain properties, including stable complexation while maintaining high affinity and specificity of the conjugate for the targeted receptor. The work of Abiraj et al. [13] illustrated the influence of the chelator on the affinity and properties of 99m Tc-based SST2 antagonists. The HYNIC monodentate ligand-which is used in combination with the somatostatin agonist TOC in patients ([ 99m Tc]Tc-HYNIC/EDDA-TOC)-was not a suitable chelating system for the SST2 antagonist SS01, as it led to loss of affinity. SS01 restored its affinity only when the chelator N4 was used for 99m Tc-labelling. The Hennkens group chose, alternatively, the  [15] and the polycarboxylate-derivatives of 1,4,7-triazacyclononane (TACN), such as NODAGA and NOTA [16]. Among all [ 99m Tc]Tc(CO) 3 -labelled sst2-ANT complexes, the [ 99m Tc]Tc-NOTA-sst2-ANT and [ 99m Tc]Tc-NODAGA-sst2-ANT showed better biodistribution patterns, compared to the other complexes, presumably due to their higher hydrophilicity. More precisely, [ 99m Tc]Tc-NOTA-sst2-ANT demonstrated the highest tumour uptake (16.70 ± 3.32% IA/g at 1 h p.i.) in AR4-2J tumour bearing mice, but also higher accumulation in the liver and the abdomen, compared to [ 99m Tc]Tc-NODAGA-sst2-ANT. On the other hand, the most hydrophilic [ 99m Tc]Tc-NODAGA-sst2-ANT showed significantly lower tumour uptake (2.78 ± 0.27% IA/g vs. 16.70 ± 3.32% IA/g at 1 h p.i., respectively), leading to reduced tumour-to-background ratios (i.e., tumour-to-blood and tumour-to-kidneys) [18]. The chelator of choice in this work, namely the 6-carboxy-1,4,8,11tetraazaundecane (N4), forms a [O=Tc=O] + core that exhibits high kinetic stability and hydrophilicity, while allowing easy and convenient labelling at room temperature. These features, taken together with the high tumour uptake and favourable in vivo distribution, render [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 suitable candidates for clinical translation.
The dosimetry study predicts low radiation dose to the human for [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2, independent of the exact location of the tumour in the abdomen. The estimated radiation dose amounted to approximately 6 µSv/MBq for both radiotracers. For typical injected activity of 740 MBq, the effective dose is 4.9 mSv and 4.3 mSv for [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2, respectively. The dose to some organs is several times higher, especially the dose to kidneys, stomach, liver, and pancreas, which is not surprising, based on the biodistribution results. The dose to particular organs is also substantially different for [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2, reflecting the biodistribution results. The resulting effective dose for both radiotracers only differs to a minor extent (12% difference), while the average total body dose is almost the same for both radiotracers (within 3%). The total body dose is two times lower than the effective dose because lower dose to the extremities (not listed in the table) affects the average dose considerably, while having little effect to the effective dose. The estimated dose to the bladder is slightly higher than the effective dose and almost the same for both radiotracersit obviously depends considerably on the assumed bladder voiding interval and will be much higher when a longer bladder voiding interval is considered.

Lipophilicity
Determination of the distribution coefficient (log D) was performed by the shakeflask method in 1:1 mixture of octanol/Phosphate buffered saline (PBS). Then, 50 µL of [ 99m Tc]Tc-TECANT-1 or [ 99m Tc]Tc-TECANT-2 solution was diluted up to 500 µL with PBS (pH 7.4; 8 mM phosphate). Next, 20 µL of this solution was added to six eppendorf tubes, each containing 500 µL octanol and 480 µL PBS. The samples were vortexed for 15 min at 1400 rpm followed by centrifugation for 2 min at 4500 rpm. Aliquots of 100 µL from each phase were taken and measured in a γ-counter. The log D was calculated as the average of the logarithmic values (n = 3, six technical replicates) of the ratio between the radioactivity in the octanol and the PBS phase.

Stability in Human Serum
The stability of [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 was tested in human serum, mixed as described above, at 37 • C for 0, 30, 60, 120, 240 min, and 24 h. At each preselected time-point, equal parts of the incubation solution and methanol were mixed in eppendorf tubes and centrifuged for 2 min at 14,000 rpm. Fifty µL of the supernatant was transferred in a separate eppendorf tube, containing the same amount of water, and analysed by radio-RP-HPLC (2 replicates).

Cell Culture
The human embryonic kidney-293 (HEK293) cell line expressing the T7-epitope tagged human SST 2 receptor (HEK-SST2) was provided by Professor Stefan Schulz (Institute of Pharmacology and Toxicology, Jena University Hospital, Jena, Germany). The cells were cultured at 37 • C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplement with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (10,000 units-10 mg/mL), 2% L-Glutamin (200 mM) and 1% G418 (50 mg/mL). For all cell experiments, HEK-SST2 were seeded at a density of 10 6 cells/well in 6-well plates and incubated over night with culture medium, containing 1% fetal bovine serum (FBS) to obtain a good cell adherence. The plates were pre-treated with a solution of 10% poly-lysine to promote cell adherence. Non-transfected HEK cells (SST2-negative) were used as negative control.

Internalization
On the day of the experiment, the medium was removed from the plates, the cells were washed with PBS and were incubated with fresh medium (DMEM with 1% FBS) for 1 h at 37 • C/5% CO 2 . [ 99m Tc]Tc-TECANT-1 or [ 99m Tc]Tc-TECANT-2 (final concentration 0.25 nM) was added to the medium and the cells were incubated (in triplicates) for 0.5, 1, 2 and 4 h at 37 • C/5% CO 2 . The internalization process was stopped by removing the medium and washing the cells twice with ice-cold PBS. The cells were then treated twice for 5 min with ice-cold glycine solution (0.05 M, pH 2.8), to distinguish between cell surface-bound (acid releasable) and internalized (acid resistant) radiotracer. Finally, the cells were detached with NaOH 1 M at 37 • C and washed twice with NaOH 1 M. To determine non-specific cellular uptake, selected wells were incubated with the [ 99m Tc]Tc-TECANT-1 or [ 99m Tc]Tc-TECANT-2 in the presence of 0.25 µM TECANT-1 or TECANT-2, respectively. The results are expressed as percentage of the total radioactivity added (% of surface bound radioactivity for the glycine fraction and % of internalized for the NaOH fraction).

Dissociation of the Cell-Surface Bound [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 with or without Competitors
The dissociation of [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 was studied by replacing the media at each time point, with or without the addition of high excess of the competitors TECANT-1 and TECANT-2, respectively. HEK-SST2 cells were seeded on 6-well plates (10 6 cells/well) that were placed on ice for 30 min, before starting the experiment. The radiotracers were added to the medium (final concentration 0.25 nM) and allowed to bind to the cells at 4 • C, in order to prevent internalization. After incubation for 2 h, the cells were quickly washed twice with ice-cold PBS to remove the unbound radiotracer, followed by the addition of 1 mL pre-warm medium (with or without the competitor) and incubated at 37 • C for 10, 20, 30, 60, 120, and 240 min. At the pre-selected time points, the medium was removed for quantification and it was replaced by fresh prewarmed (37 • C) medium (with or without 2.5 µM concentration of the respective TECANT analogue, as competitor). At the end of the experiment, the cells were solubilized with NaOH 1 M, and collected for quantification of the remaining cell-associated radiotracer (cell-surface bound and internalized fractions).

Comparative Apparent Binding Affinity Studies (IC 50 )
The receptor binding assay was performed on 96-well MultiScreen Filter Plates HTS (1 µm glass fibre filter, Merck Millipore, Darmstadt, Germany), using [ 177 Lu]Lu-DOTA-TATE as radioligand and TECANT-1, TECANT-2, nat Re-TECANT-1, nat Re-TECANT-2 or DOTA-TATE as competitors. After moistening the plate with TRIS buffer, 5 × 10 5 HEK-SST 2 cells (100 µL), 50 µL of the competitor in 8-10 different concentrations (ranging from 0.004 to 4000 nM) and 50 µL of [ 177 Lu]Lu-DOTA-TATE (1 nM) were added to each well. The plate was incubated for 2 h at 4 • C. The supernatant was then removed by vacuum and the plate was washed twice with ice-cold PBS/0.1% bovine serum albumin (BSA). The filters were collected, and the remaining activities were measured in a gamma counter. The apparent IC 50 values were calculated using a nonlinear curve fitting in OriginPro 6.1 software (Northampton, MA, USA) from 3 replicates.

Biodistribution Studies of [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2
Animal experiments were approved by the authorities in accordance with the Swiss regulations for animal treatment (approval no. 2799). Female athymic nude-Foxn1 nu /Foxn1 + mice (Envigo, The Netherlands), 4-6 weeks old, were inoculated subcutaneously with 10 7 HEK-SST2 cells on the shoulder, freshly suspended in 100 µL sterile PBS. One group of mice was inoculated, in addition, with 10 7 HEK cells (SST2-negative) on the other shoulder. The tumours were allowed to grow for approximately 3 weeks, until they reached an average weight of approximately 250-300 mg.

Dosimetric Calculations
Dosimetric calculations were based on the biodistribution of [ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 (Table 3). In order to account for the differences in metabolic rate among mice and humans due to significantly different body mass, time-scaling of biodistribution data was completed according to [19]: where t m is the time at which a measurement was made in mice, t h is the corresponding time assumed for the human data, and m m and m h are the total body masses of the experimental mice (on average) and of the human phantom that is then used for the dosimetry, respectively. Percent of injected activity per organ in a human model was evaluated according to the animal data extrapolation model [19]: Here m is the organ mass and M is the total body weight of mouse or human. Average total weight of the mouse was measured, while human phantom has the total weight of 73 kg [20]. The %IA/g and %IA/organ is the percentage of the injected activity concentration (i.e., per gram of tissue) and per organ, respectively. For the tumour, we assumed that the percent of injected activity uptake into the tumour is the same for mouse and human.
Subsequently, percentage of the injected activity per organ was folded with the decay curve of 99m Tc and interpolated with the exponential function. The interpolation function was used to estimate initial %ID/organ and the time-integrated activity concentration (TIAC). Absorbed dose per injected activity for various organs was calculated with the TIAC factors and S-values for a standard man from the OpenDose project [21]. Effective dose was evaluated according to the ICRP 103 recommendations.
It was assumed that all remaining activity, i.e., the activity that does not go to the organs or tumour included in the biodistribution study, is uniformly distributed over the rest of the body and cleared to the bladder with the biological half-life of 9.7 h for [ 99m Tc]Tc-TECANT-1 and 13.3 h for [ 99m Tc]Tc-TECANT-2 (estimate from the remaining activity in experimental mice, time-scaled to humans according to Equation (1)). We also assumed that the remaining activity from the organs and tumour ends up in the bladder. For the bladder, a biological half-life of 1.2 h, which roughly corresponds to voiding every 2.4 h, was assumed. Radiotracer biological pathway is presented in Figure A1 (Appendix A).

Conclusions
[ 99m Tc]Tc-TECANT-1 and [ 99m Tc]Tc-TECANT-2 showed a similar behaviour regarding their stability in human serum and their affinity for the human SST2 receptor. Nevertheless, the cellular uptake of [ 99m Tc]Tc-TECANT-1 was higher and its dissociation slower, compared with [ 99m Tc]Tc-TECANT-2. The in vivo comparison indicates better performance of [ 99m Tc]Tc-TECANT-1 due to the longer tumour residence time and the lower kidney and liver uptake, whereas estimated human radiation dose was similar for both radiotracers. Overall, this head-to-head preclinical evaluation provided the basis for selection of [ 99m Tc]-TECANT-1 for clinical translation as the first 99m Tc-labelled SST2 antagonist. Toxicity studies are planned to complete the preclinical characterisation of this radiotracer and the development of a kit formulation (to be reported separately) is on-going for the translation of [ 99m Tc]Tc-TECANT-1 into a clinical feasibility study in NEN patients.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Swiss Federal Food Safety and Veterinary Office in accordance with the Swiss Regulations for animal treatment (approval no. 2799).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this article.

Conflicts of Interest:
The authors declare no conflict of interest. Appendix A Figure A1. Biological pathway of the radiotracer. Biodistribution data are used to estimate the dose contribution from the organs included in the biodistribution study. Excreted radiotracers Figure A1. Biological pathway of the radiotracer. Biodistribution data are used to estimate the dose contribution from the organs included in the biodistribution study. Excreted radiotracers from these organs are supposed to go into the urinary bladder. The remaining activity is uniformly distributed over the rest of the body and cleared to the bladder with the biological half-life of 9.