[212Pb]Pb-eSOMA-01: A Promising Radioligand for Targeted Alpha Therapy of Neuroendocrine Tumors

Peptide receptor radionuclide therapy (PRRT) has been applied to the treatment of neuroendocrine tumors (NETs) for over two decades. However, improvement is still needed, and targeted alpha therapy (TAT) with alpha emitters such as lead-212 (212Pb) represents a promising avenue. A series of ligands based on octreotate was developed. Lead-203 was used as an imaging surrogate for the selection of the best candidate for the studies with lead-212. 203/212Pb radiolabeling and in vitro assays were carried out, followed by SPECT/CT imaging and ex vivo biodistribution in NCI-H69 tumor-bearing mice. High radiochemical yields (≥99%) and purity (≥96%) were obtained for all ligands. [203Pb]Pb-eSOMA-01 and [203Pb]Pb-eSOMA-02 showed high stability in PBS and mouse serum up to 24 h, whereas [203Pb]Pb-eSOMA-03 was unstable in those conditions. All compounds exhibited a nanomolar affinity (2.5–3.1 nM) for SSTR2. SPECT/CT images revealed high tumor uptake at 1, 4, and 24 h post-injection of [203Pb]Pb-eSOMA-01/02. Ex vivo biodistribution studies confirmed that the highest uptake in tumors was observed with [212Pb]Pb-eSOMA-01. [212Pb]Pb-eESOMA-01 displayed the highest absorbed dose in the tumor (35.49 Gy/MBq) and the lowest absorbed dose in the kidneys (121.73 Gy/MBq) among the three tested radioligands. [212Pb]Pb-eSOMA-01 is a promising candidate for targeted alpha therapy of NETs. Further investigations are required to confirm its potential.


Introduction
Neuroendocrine tumors (NETs) are a rare class of tumors arising from neuroendocrine cells. NETs occur throughout the body, although the majority arise in the gastroenteropancreatic (GEP) tract or the lung. Surgical resection is the standard of care for localized NETs, while chemotherapy and targeted drug therapy are often administered to patients with metastatic disease. However, the slow-growing nature of NETs makes these treatments relatively inefficient. PRRT (Peptide receptor radionuclide therapy) represents an interesting therapeutic option for advanced metastatic NET patients. PRRT is a molecularly targeted therapy that makes use of a peptide linked to a radionuclide emitting ionizing radiation to induce tumor cell death. For NETs, the peptide is designed to bind selectively to the somatostatin receptor subtype 2 (SSTR2), which is highly upregulated in neuroendocrine tumor cells.
The first radiopeptide used for PRRT of NETs was [ 111 In]In-DTPA-octreotide (Octreoscan ® ). Octreotide (TOC) was chosen because this peptide was found to bind selectively to SSTR2 [1]. Indium-111 was used as an Auger emitter, although it is commonly used for gamma imaging. Octreoscan ® demonstrated therapeutic efficacy without significant toxicity due to the high linear energy transfer (LET) of indium-111 [2][3][4]. However, the short range of the auger electrons hampered its efficacy in large tumors and end-stage patients. Therefore, a new generation of radioligands, labeled with beta emitters, was developed. The two most common beta-emitting radionuclides in PRRT are yttrium-90 (T 1/2 = 64.05 h, E βmax = 2.28 MeV) and lutetium-177 (T 1/2 = 159.5 h, E βmax = 0.50 MeV). Two SSTR2-targeting peptides labeled with these radionuclides, [ 90 Y]Y-DOTA-TOC and [ 177 Lu]Lu-DOTA-TATE, have been extensively studied over the last two decades [5][6][7][8]. Similar to octreotide, octreotate (TATE) was designed to target SSTR2 [1]. Both radiopharmaceuticals showed encouraging clinical results by improving progression-free survival and the quality of life of NET patients. [ 177 Lu]Lu-DOTA-TATE (Lutathera ® ) was approved by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) in 2017 and 2018, respectively, for the treatment of GEP-NETs. However, the current PRRT of NETs requires improvement since the overall response rates remain insufficient and relapse often occurs 2-3 years after the first treatment [9].
Herein, we report the development of a new series of octreotate derivatives, dubbed eSOMA, containing either the DO3AM or p-Bn-SCN-TCMC chelator, two functionalized DOTAM derivatives, and an Amcha or Pip linker ( Figure 1). The SSTR2-targeting ligands have been labeled with 212 Pb and 203 Pb, an imaging surrogate of lead-212. In vitro assays and non-invasive SPECT/CT scans were performed with the 203 Pb-labeled analogs to determine the best candidate for further studies. Ex vivo biodistribution studies with the 212 Pb-labeled lead compound were carried out to determine the potential of the eSOMA derivatives for targeted alpha therapy of NETs.

Chemistry and Radiochemistry
The synthesis of Tyr 3 -octreotate was performed on solid support using a standard Fmoc-based strategy (Scheme 1). The amino acids were coupled to each other using conventional coupling reagents, such as HATU. On-resin cyclization was carried out by treating the linear peptidyl-resin with Tl (TFA)3 to generate the intramolecular disulfide bond. The Acm-protecting group was specifically chosen for the cysteine residues because it can be selectively deprotected during the cyclization step [17]. Analysis of an aliquot demonstrated that the cyclic-protected D-Phe-cyclo[Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys]-Thr(tBu)-resin (4) was obtained in quantitative yield. DOTAM-TATE was synthesized by coupling DO3AM acetic acid to 4 in the presence of PyBOP, followed by the deprotection of the side-chain protecting groups and cleavage from the solid support. DOTAM-TATE was obtained with a yield of 11% after semi-preparative HPLC purification. eSOMA-01 and eSOMA-02 were obtained via the same approach after attachment of the corresponding linkers (Amcha or Pip) to the resin-bound protected Tyr 3octreotate 4 and removal of the Fmoc group. eSOMA-01 and eSOMA-02 were obtained with 12% and 7% yields, respectively, after purification. Synthesis of D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(ivDde)-Thr(tBu)-Cys(Acm)-Thr(tBu)-resin, containing a lysine residue protected with an ivDde instead of the Boc protecting group used for 4, was required to obtain eSOMA-03 (Scheme 2). After elongation and cyclization, the peptide was cleaved from the resin and coupled to p-SCN-Bn-TCMC to yield intermediate 6.

Chemistry and Radiochemistry
The synthesis of Tyr 3 -octreotate was performed on solid support using a standard Fmoc-based strategy (Scheme 1). The amino acids were coupled to each other using conventional coupling reagents, such as HATU. On-resin cyclization was carried out by treating the linear peptidyl-resin with Tl (TFA) 3 to generate the intramolecular disulfide bond. The Acm-protecting group was specifically chosen for the cysteine residues because it can be selectively deprotected during the cyclization step [17]. Analysis of an aliquot demonstrated that the cyclic-protected D-Phe-cyclo[Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys]-Thr(tBu)-resin (4) was obtained in quantitative yield. DOTAM-TATE was synthesized by coupling DO3AM acetic acid to 4 in the presence of PyBOP, followed by the deprotection of the side-chain protecting groups and cleavage from the solid support. DOTAM-TATE was obtained with a yield of 11% after semi-preparative HPLC purification. eSOMA-01 and eSOMA-02 were obtained via the same approach after attachment of the corresponding linkers (Amcha or Pip) to the resin-bound protected Tyr 3 -octreotate 4 and removal of the Fmoc group. eSOMA-01 and eSOMA-02 were obtained with 12% and 7% yields, respectively, after purification. Synthesis of D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp(Boc)-Lys(ivDde)-Thr(tBu)-Cys(Acm)-Thr(tBu)-resin, containing a lysine residue protected with an ivDde instead of the Boc protecting group used for 4, was required to obtain eSOMA-03 (Scheme 2). After elongation and cyclization, the peptide was cleaved from the resin and coupled to p-SCN-Bn-TCMC to yield intermediate 6. Finally, deprotection of the ivDde group and semi-preparative HPLC purification afforded eSOMA-03 in 17% yield.
Labeling of DOTAM-TATE, eSOMA-01, eSOMA-02, and eSOMA-03 was performed with 203/212 PbCl 2 at room temperature in the presence of sodium acetate buffer and a mixture of ascorbic acid and gentisic acid to prevent radiolysis. The radiochemical yield (RCY) and radiochemical purity (RCP) were determined by iTLC and radio-HPLC, respectively ( Figure 2). All labeled compounds were obtained with a RCY ≥ 96% and a RCP ≥ 95%. Labeling of DOTAM-TATE, eSOMA-01, eSOMA-02, and eSOMA-03 was performed with 203/212 PbCl2 at room temperature in the presence of sodium acetate buffer and a mixture of ascorbic acid and gentisic acid to prevent radiolysis. The radiochemical yield (RCY) and radiochemical purity (RCP) were determined by iTLC and radio-HPLC, respectively ( Figure 2). All labeled compounds were obtained with a RCY ≥ 96% and a RCP ≥ 95%.  Labeling of DOTAM-TATE, eSOMA-01, eSOMA-02, and eSOMA-03 was performed with 203/212 PbCl2 at room temperature in the presence of sodium acetate buffer and a mixture of ascorbic acid and gentisic acid to prevent radiolysis. The radiochemical yield (RCY) and radiochemical purity (RCP) were determined by iTLC and radio-HPLC, respectively ( Figure 2). All labeled compounds were obtained with a RCY ≥ 96% and a RCP ≥ 95%.      IC 50 values were obtained by a competitive binding assay using [ 111 In]In-DOTA-TATE as radioligand and purified Chinese hamster ovary-K1 (CHO-K1) membranes overexpressing the somatostatin receptor subtype 2 ( Table 3). The four ligands exhibited nanomolar affinity for SSTR2. eSOMA-01, eSOMA-02, and eSOMA-03 showed IC 50 values relatively similar to the parent peptide DOTA-TATE but 2.8-to 3.5-fold higher than the IC 50 value of the reference DOTAM-TATE. However, after complexation with lead, the IC 50 values of eSOMA-01, eSOMA-02, and eSOMA-03 were 1.1-to 1.5-fold lower than the IC 50 value of [ nat Pb]Pb-DOTAM-TATE.

In Vivo Preclinical Evaluation
SPECT/CT imaging was performed in H69-xenografted Balb/c nu/nu mice at 1, 4, and 24 h after the administration of [ 203 Pb]Pb-eSOMA-01 or [ 203 Pb]Pb-eSOMA-02. Tumors were visible by SPECT/CT for both radioligands ( Figure 3A,B). As expected, high uptake was observed in the kidneys as a result of the renal excretion of the radioligands and partial reabsorption in proximal tubule cells. Analysis of the SPECT images showed a similar tumor uptake (~3-4% ID/mL, p > 0.05) for both 203 Pb-labeled compounds ( Figure 3C). However, we noticed that the tumor/kidney ratios were significantly different (p < 0.05) from 4 h to 24 h between the two radioligands ( Figure 3D  5.64 b a n = 2; b n = 1.

In Vivo Preclinical Evaluation
SPECT/CT imaging was performed in H69-xenografted Balb/c nu/nu mice at 1, 4, and 24 h after the administration of [ 203 Pb]Pb-eSOMA-01 or [ 203 Pb]Pb-eSOMA-02. Tumors were visible by SPECT/CT for both radioligands ( Figure 3A,B). As expected, high uptake was observed in the kidneys as a result of the renal excretion of the radioligands and partial reabsorption in proximal tubule cells. Analysis of the SPECT images showed a similar tumor uptake (~3-4% ID/mL, p > 0.05) for both 203 Pb-labeled compounds ( Figure 3C). However, we noticed that the tumor/kidney ratios were significantly different (p < 0.05) from 4 h to 24 h between the two radioligands ( Figure 3D    Uptakes in all the other non-target organs were below 1% ID/g for both radioligands, but slightly higher uptake in the spleen, pancreas, lung, liver, skin, and bone were observed for [ 203 Pb]Pb-eSOMA-02 than [ 203 Pb]Pb-eSOMA-01. ]Pb-eSOMA-02, respectively, p < 0.05), while in the pancreas they all displayed a similar accumulation (~6 to 8% ID/g, p > 0.05). However, radioactivity was rapidly cleared from those organs. In the other non-target organs, the uptake was constantly below 5% ID/g at all time points. Furthermore, [ 212 Pb]Pb-eSOMA-01 showed an increase in the tumor/kidney ratio over time (0.19 ± 0.03 at 1 h to 0.42 ± 0.10 at 24 h), whereas the ]Pb-eSOMA-02, respectively, p < 0.05), while in the pancreas they all displayed a similar accumulation (~6 to 8% ID/g, p > 0.05). However, radioactivity was rapidly cleared from those organs. In the other non-target organs, the uptake was constantly below 5% ID/g at all time points.    The tumor and organ doses absorbed for the different radioligands are indicated in

Discussion
Targeted alpha therapy is a promising therapeutic alternative to improve the outcome of radionuclide therapy for NETs. In this study, we focused on the development of SSTR2 radioligands that could be labeled with lead-212 and performed a head-to-head comparison to [ 212 Pb]Pb-DOTAM-TATE, a reference compound that is currently under evaluation in phase II clinical trials [18]. Our radioligands were based on the SSTR2 agonist octreotate (TATE) and contained TCMC or DO3AM, two chelators known to enhance the stability of lead radionuclide-chelator complexes in vivo compared to other commonly used chelators, such as DOTA [19]. Two linkers (4-(aminomethyl)cyclohexane-1-carbonyl (Amcha) and 4-amino-1-carboxymethyl-piperidinyl (Pip)) were inserted between the chelator and the peptide sequence to preserve the binding of the ligand to SSTR2 and influence the pharmacokinetic properties.  [21]. The Pip linker led to an improved biodistribution profile and better tumor visualization than the other linkers tested in this study. Therefore, both linkers were included in the design of our SSTR2 radioligands to

Discussion
Targeted alpha therapy is a promising therapeutic alternative to improve the outcome of radionuclide therapy for NETs. In this study, we focused on the development of SSTR2 radioligands that could be labeled with lead-212 and performed a head-to-head comparison to [ 212 Pb]Pb-DOTAM-TATE, a reference compound that is currently under evaluation in phase II clinical trials [18]. Our radioligands were based on the SSTR2 agonist octreotate (TATE) and contained TCMC or DO3AM, two chelators known to enhance the stability of lead radionuclide-chelator complexes in vivo compared to other commonly used chelators, such as DOTA [19]. Two linkers (4-(aminomethyl)cyclohexane-1-carbonyl (Amcha) and 4-amino-1-carboxymethyl-piperidinyl (Pip)) were inserted between the chelator and the peptide sequence to preserve the binding of the ligand to SSTR2 and influence the pharmacokinetic properties. The Amcha linker was previously reported by dos Santos [21]. The Pip linker led to an improved biodistribution profile and better tumor visualization than the other linkers tested in this study. Therefore, both linkers were included in the design of our SSTR2 radioligands to determine if they would have a similar beneficial effect on the biodistribution of DOTAM-TATE.
Radiolabeling of the ligands with 203/212 Pb was performed with a high radiochemical yield and purity at room temperature and at a pH maintained between 5 and 6. These radiolabeling conditions are well suited for the labeling of sensitive biomolecules [22]. We did not observe radiolytic degradation of the radiolabled compounds, except for eSOMA-03, despite the presence of quenchers (e.g., ascorbic acid and gentisic acid), which are preventing radiolysis in the final formulation [23]. Radiolysis is a form of decomposition occurring in the presence of radical species, such as hydroxyl radicals (•OH) or superoxide (•O2). We speculated that the instability of eSOMA-03 might originate from the hydrolysis of the thiourea bond [24][25][26][27]. Moreover, unlike eSOMA-03, all radiolabeled compounds were stable in mouse serum, demonstrating their inertness toward peptidase digestion. eSOMA-03 was therefore withheld from further investigations as it would fail during in vivo evaluation. The LogD 7 . 4 values obtained for our SSTR2-targeting ligands were negative, but we noticed a decreased hydrophilicity compared to previous octreotate analogs, such as [ 68 Ga]Ga-DOTA-TATE (LogD 7 . 4 = −3.69 ± 0.02) [28]. The lower hydrophilicity might be the result of the change of the chelator from DOTA containing three carboxylic acid groups to DO3AM bearing three amide groups. eSOMA-01 and eSOMA-02 showed a slightly lower LogD 7 . 4 value than DOTAM-TATE and eSOMA-03, which highlights the influence of the linker on the hydrophilicity of the radioligand. The three new ligands exhibited a binding affinity for SSTR2 that was slightly better than that of the gold standard, DOTA-TATE. However, their IC 50 values were 3-to 4-fold higher than the IC 50 value of DOTAM-TATE. This difference could be explained by the sensitivity of the binding of octreotate to SSTR2 when chemical modifications are performed at the N-terminus of the peptide [29,30]. The IC 50 value of [ nat Pb]Pb-DOTAM-TATE was comparable to the Kd value reported by Stallons and coworkers (7.6 nM vs. 12.9 nM, respectively) [18]. Complexation with lead had a slight effect on the binding affinity since all complexed compounds showed a 1.8-8.3-fold higher IC 50 value compared to the non-complexed analogs. However, after Pb-complexation, the eSOMA derivatives exhibited lower IC 50 values than [ nat Pb]Pb-DOTAM-TATE (5.2, 6.8, and 5.6 nM for [ nat Pb]Pb-eSOMA-01, -02, and -03, respectively, vs. 7.6 nM for [ nat Pb]Pb-DOTAM-TATE), demonstrating that metal complexation had more influence on DOTAM-TATE than the three new ligands. The presence of the linkers in the eSOMA derivatives reduced the effect on binding to the receptor caused by the change of the secondary and tertiary structures of the peptides due to metal complexation. Furthermore, it has been previously reported that the complexation of SSTR2 ligands with different metals influences more or less the binding to the receptor [30,31]. Therefore, a good chelate-metal combination must be found.
eSOMA-01 and eSOMA-02 were first labeled with 203 Pb, an imaging surrogate of 212 Pb, and evaluated in a human small-cell lung cancer (NCI-H69) xenograft model overexpressing SSTR2. Tumor uptake at 1 h post-injection was~4% ID/mL for both radioligands and slowly decreased to~2-3% ID/mL at 24 h. Tworowska et al. reported an uptake of 13% ID/g at 1 h post-injection in AR42J tumors for [ 203 Pb]Pb-DOTAM-TATE [32]. This tumor uptake is higher than the uptake observed with our compounds in H69 tumors. However, the difference could be explained by the fact that the AR42J cell line is known to have a 3-fold higher SSTR2 expression than the NCI-H69 cell line [33]. A high kidney uptake was also observed for [ 203 Pb]Pb-eSOMA-01 and [ 203 Pb]Pb-eSOMA-02. Kidney uptake was expected and is often reported in preclinical studies dealing with PRRT of NET due to the reabsorption of the peptide by the proximal tubules [34,35]. A similar uptake in kidneys (~13% ID/g) was reported by Tworowska  kidney uptake of [ 212 Pb]Pb-DOTAM-TATE (from 54 ± 5 at 1 h to 29 ± 6% ID/g at 24 h) was approximately 3-fold higher than the uptake reported by Tworowska et al. (from 21% ID/g at 1 h to 10% ID/g at 24 h). However, the pancreas uptake of [ 212 Pb]Pb-DOTAM-TATE was lower at 1 h and 4 h (5.19 ± 0.84% ID/g and 1.14 ± 0.13% ID/g, respectively) than the values reported by Tworowska et al. (32% ID/g at 1 h and 15% ID/g at 4 h). The difference observed can be explained by the fact that two different mouse models were used (Balb/c nude mice in our study and CD-1 mice for Tworowska et al.) [37]. A therapeutic index corresponding to the ratio of the absorbed dose to the tumor and the absorbed dose to the main limiting organ (i.e., the kidneys) was defined to better evaluate the potential of the radioligands for therapy. In our study, [ 212 Pb]Pb-eSOMA-01 presented a better therapeutic index than [ 212 Pb]Pb-DOTAM-TATE and [ 212 Pb]Pb-eSOMA-02 (1.6-and 3.0-fold lower, respectively). Thus, [ 212 Pb]Pb-eSOMA-01 was identified as the best candidate for TAT of NETs among the SSTR2-targeting radioligands tested.
For therapy, the dose activity injected is usually determined by the maximum tolerated absorbed dose (MTD) of the dose-limiting organs, typically the kidneys for PRRT of NETs [38]. A MTD to the kidneys of 18-23 Gy was used by Tworowska et al. for [ 212 Pb]Pb-DOTAM-TATE in mice [36,39]. Based on this limit, the maximum dose injected into mice would be around 0.2 MBq for [ 212 Pb]Pb-eSOMA-01. However, the dose calculation assumed homogeneous uptake in the kidneys, whereas specific uptake in the tubuli might lead to lower absorbed doses in the glomeruli, especially with the short radiation range of the α particles [40]. Consequently, the dose absorbed by the kidneys could be less than the dose calculated. Autoradiography of the kidneys could provide more accurate information about the exact location of the radionuclide in this organ to refine the estimation of the absorbed dose. As a result, an absorbed dose to the tumor of 7 Gy was estimated for an injected activity of 0.2 MBq. However, a minimum dose of 20 Gy is typically needed in mice to achieve optimal therapeutic efficacy [41]. Therefore, an effort to reduce the dose absorbed by the kidneys is required in order to enhance the dose administered while keeping nephrotoxicity as low as possible. Co-injection of a basic amino acid cocktail or plasma expander (e.g., Gelofusine) could be explored to reduce renal retention of [ 212 Pb]Pb-eSOMA-01 [42][43][44]. This strategy has already been used in preclinical and clinical studies with [ 212 Pb]Pb-DOTAM-TATE and showed a reduction in kidney uptake by 3-fold without affecting tumor uptake [16,18].

General Information
All chemicals and solvents were obtained from commercial suppliers and used without further purification, unless specified. DO3AM acetic acid and 2-chlorotrityl chloride resin were purchased from Chematech (Dijon, France) and Advanced Chemtech (Louisville, KY, USA), respectively. Chinese hamster ovary-K1 (CHO-K1) membranes overexpressing human SSTR2 were purchased from Perkin Elmer (Waltham, MA, USA). [ 111 In]InCl 3 was ordered from Curium (Petten, The Netherlands). The peptide sequence was synthesized manually using standard solid-phase synthesis protocols. Mass analyses were carried out using a Thermo Fischer Scientific LC/MS system with electrospray ionization (ESI) (Breda, The Netherlands). Quality control and purification of the synthesized ligands were performed by high performance liquid chromatography (HPLC) using a Waters 2695 system (Etten-Leur, The Netherlands) equipped with a diode array detector 2998 and a radioactivity detector from Canberra (Zadik, Belgium). The HPLC and LC/MS systems were controlled by Empower3 and Xcalibur softwares, respectively. Instant thin-layer chromatography plates (iTLC) were analyzed by a bSCAN radio-chromatography scanner from Brightspec (Antwerp, Belgium) equipped with a sodium iodide detector. Activity measurements were performed using a VDC-405 dose calibrator from Comecer (Joure, The Netherlands). The radioactive samples used for the determination of LogD 7 . 4 , in vitro assays, and in vivo uptake in tissues were counted using a Wizard 2480 gamma counter from Perkin Elmer (Waltham, MA, USA). It is noteworthy to mention that the activity of 212 Pb samples was counted using a window energy of 204-267 keV.

HPLC Conditions
The analysis of the products was performed by HPLC on an analytical column (Phenomenex Gemini C 18 , 5 µm, 250.0 × 4.6 mm) at a flow rate of 1 mL/min and with a mobile phase consisting of: A (0.1% TFA in water (v/v)) and B (0.1% TFA in acetonitrile (v/v)). The elution of the products was performed according to the following gradient: 0-3 min: 5% B, 3-23 min: 5-100% B, and 23-27 min: 100% B.

DO3AM-D-Phe-cyclo[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Thr-OH (DOTAM-TATE)
A fraction of the cyclic peptidyl resin 4 (40 µmol) was swollen in DMF. DO3AM acetic acid (50 mg, 120 µmol, 3 equiv.), PyBop (65 mg, 120 µmol, 3.0 equiv.), and DIPEA (70 µL, 400 µmol, 10.0 equiv.) in 1 mL of DMF were added to the resin, and the mixture was stirred overnight at rt. Conjugation of the chelator was confirmed by analytical HPLC after cleavage and deprotection of a small sample of peptide. After completion of the reaction, the resin was washed with DMF (5 × 3 mL) and dichloromethane (3 × 3 mL). A solution of TFA/H 2 O/TIPS (v/v/v = 95:2.5:2.5) was added to the resin, and the mixture was stirred for 2 h at rt. The solution was removed from the resin by filtration, and ice-cold diethyl ether was added to the filtrate. The precipitate, collected by centrifugation, was purified by semi-preparative HPLC (Condition 2) to give 6.3 mg of DOTAM-TATE as a white solid (11%). Purity > 99%. ESI-MS: m/z calc' for C 65

Radiolabeling with 203 Pb
Before labeling, the concentration of all the peptides was determined via titration according to a method previously described [45]. The mixture was incubated for 20 min at rt. The reaction was monitored by instant thin-layer chromatography on iTLC-SG sheets eluted with a solution of sodium citrate (0.1 M, pH 5.0), which were cut into 10 equal pieces and measured in a gamma counter. DTPA (5 µL, 3 mg/mL) was added to complex the remaining free lead-212. An aliquot was taken and injected into radio-HPLC using Method 2.

In Vitro Stability
The 203 Pb-labeled compounds (~2 MBq) or the 212 Pb-labeled compounds (~70 kBq) were incubated in 300 µL of phosphate buffered saline (PBS; 0.1 M, pH 7.4) or mouse serum at 37 • C. The stability of the radiolabeled peptides was monitored at 1, 4, and 24 h post-incubation. The samples in PBS were directly analyzed by radio-HPLC without any pretreatment. However, samples of mouse serum were mixed with an equal volume of acetonitrile to precipitate the proteins. The solution was centrifuged twice for 5 min at 13,000 rpm, and the supernatant was analyzed by radio-HPLC using Method 1 for lead-203 and Method 2 for lead-212.

LogD 7 . 4
Distribution coefficients (LogD 7 . 4 ) were determined by the shake-flask method. A sample containing the 203 Pb-labeled compound (~1 MBq) was added to a vial containing PBS (600 µL, pH 7.4) and n-octanol (600 µL). The vial was vortexed vigorously and then centrifuged at 10,000 rpm for 10 min for phase separation. Samples (100 µL) of the n-octanol and aqueous phases were taken and measured by a gamma counter. The LogD 7 . 4 value was calculated by using the equation: LogD 7 . 4 = log [(counts in the n-octanol phase)/(counts in the aqueous phase)]. Measurements were performed in triplicate.

Animal Models
Six-week-old male Balb/c nu/nu-specific and opportunistic pathogen-tree (SOPF) mice (Janvier Labs, Le Genest-Saint-Isle, France) were housed in individually ventilated cages, with four mice per cage. Upon arrival, the mice were acclimated for 1 week with access to food and water ad libitum. Mice were subcutaneously inoculated on the right shoulder with NCI-H69 cells (5 × 10 6 cells suspended in 100 µL of 1/3 Matrigel (Corning Inc.; Corning, NY, USA) and 2/3 Hank's balanced salt solution (Gibco; Paisley, UK). NCI-H69 xenografts were allowed to grow for 3 weeks. Tumor sizes were 391 ± 173 mm 3 at the start of the studies. Whole-body SPECT images were acquired over one frame of 20 min (for 1 and 4 h time points) and two frames of 20 min (for 24 h time points) using a spiral scan in normal scan mode, in list-mode acquisition. This was followed by a whole-body normal CT scan (angle step 0.75 degrees, 50 kV tube voltage, 0.21 mA tube current). Reconstruction of the SPECT images was performed using the similarityregulated SROSEM method (MILabs Rec 11.00 software), performing five iterations with a voxel size of 0.4 mm, using 72 keV ± 30% and 280 keV ± 8% energy windows for lead-203. Reconstructed volumes of SPECT scans were post-filtered with an isotropic three-dimensional Gaussian filter of 1 mm full width at half-maximum. Image processing and analyses of the reconstructed data were performed using the PMOD image analysis software version 3.9 (PMOD Technologies; Zurich, Switzerland) to calculate the percentage of injected dose per mL (% ID/mL). An Eppendorf tube filled with a solution of lead-203 of known activity was measured to determine the calibration factor.

Ex-Vivo Biodistribution with Lead-203
To determine the biodistribution of the compounds after imaging (1, 4, and 24 h p.i.), blood was collected via retro-orbital puncture under isoflurane/O 2 anesthesia, after which the mice were sacrificed via cervical dislocation. The tumor and organs of interest (heart, lungs, liver, spleen, stomach, large intestine, small intestine, pancreas, kidneys, muscle, skin, and bone) were excised, washed in PBS, blotted dry, and collected into tubes. The blood, tumor, and relevant organs were weighed and measured with a gamma counter. The percentage of injected dose per gram (% ID/g) was determined for each tissue sample and corrected for both the injected volume and the % ID present at the injection site (the tail).

Biodistribution Study with Lead-212
Biodistribution studies were performed to determine tumor and organ uptake of The tumor and organs of interest (heart, lungs, liver, spleen, stomach, large intestine, small intestine, pancreas, kidneys, muscle, skin, and bone) were excised, washed in PBS, and blotted dry. To confirm receptor specificity of radioligand uptake, mice (n = 3) were coinjected with an excess (25 nmol) of the respective unlabeled peptide, and uptake in the organs of interest was determined at 24 h p.i. To determine the total injected radioactivity per animal, a calibration curve with lead-212 was established using the gamma counter. The percentage of injected dose per gram (% ID/g) was determined for each tissue sample and corrected for both the injected volume and the % ID present at the injection site (the tail).

Statistical Analysis
Statistical analysis and nonlinear regression were performed using GraphPad Prism 5 (GraphPad software, San Diego, CA, USA), and a Grubbs' test (α = 0.05) was used to compare medians between groups. The Akaike information criterion was used to decide if a residual plateau needed to be included in the exponential fit. Data were reported as mean ± SEM (standard error of mean) for at least three independent replicates. The data sets were analyzed for significance using a one-way ANOVA using SigmaPlot 15.0 software. A p-value lower than 0.05 was considered statistically significant.

Dosimetry
Single exponential curves were fitted through the organ biodistribution time-activity data using Graphpad Prism. The number of disintegrations per injected activity per gram of tissue ([TIAC]) was calculated by integrating the single exponential curve folded with the 212 Pb decay function with a half-life of 10.64 h. The TIAC per gram of tissue by the progenies of 212 Pb: 212 Bi, 212 Po (64.06%), and 208 Tl (35.94%) were assumed to be equal to 212 Pb, as they are in equilibrium. The absorbed doses to the organs were calculated by using the 212 Pb and its progenies' S-values for the 25 g RADAR mouse phantom [46]. The absorbed doses were calculated with the MIRD equation D = ∑ s m s × [TI AC s ] × S(t ← s), with m s being the source organ mass. A relative biological effect (RBE) of 5 was assumed for the alpha-radiation [47]. The uncertainty propagation in the dosimetry result was calculated [48].

Conclusions
In this study, we successfully developed and evaluated in preclinical models three new SSTR2-targeting ligands labeled with 203/212 Pb for imaging and treatment of NETs. We demonstrated that eSOMA-01 is a promising theragnostic candidate. eSOMA-01 showed a superior biodistribution profile and therapeutic index than the actual reference, DOTAM-TATE. However, further investigations are required to reduce the absorbed dose to the kidneys, the dose-limiting organ for radionuclide therapy of NETs. Therapy studies will be conducted to compare the efficacy of [ 212 Pb]Pb-eSOMA-01 with the clinical reference [ 212 Pb]Pb-DOTAM-TATE.

Patents
D.C. and Y.S. have a pending patent application for the aforementioned compounds.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ph16070985/s1. The following supporting information can be downloaded: Figure

Informed Consent Statement:
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Data Availability Statement: Data is contained within the article and Supplementary Material.