Bis(Disulfide)-Bridged Somatostatin-14 Analogs and Their [111In]In-Radioligands: Synthesis and Preclinical Profile

The overexpression of one or more somatostatin receptors (SST1–5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of “pansomatostatin” analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST2R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ser13-Cys14]) and AT6S (DOTA-Ala1-Gly2-c[Cys3-Lys4-c[Cys5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-Cys12]-Ser13-Cys14]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [111In]In-AT5S and [111In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST1–5R and agonistic properties at the SST2R, whereas AT5S lost all affinity to SST1–5R. Both [111In]In-AT5S and [111In]In-AT6S remained stable in the peripheral blood of mice, while [111In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST3R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [111In]In-AT6S targeted SST2R-/SST3R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.


Synthesis of AT5S
For the synthesis of AT5S, the linear protected sequence DOTA(tris( t Bu))-Ala 1 -Gly 2 -Cys(Acm) 3 -Lys(Boc) 4 -Asn(Trt) 5 -Cys(Trt) 6 -Phe 7 -DTrp(Boc) 8 -Lys(Boc) 9 -Thr( t Bu) 10 -Cys(Trt) 11 -Thr( t Bu) 12 -Ser( t Bu) 13 -Cys(Acm) 14 -was built on the H-Cys(Acm)-2-Cl-Trt resin adopting Fmoc/ t Bu methodologies (Figure 2a; and detailed in Sections 4.1 and 4.2 in Section 4).Couplings were successful, but the DOTA(tris( t Bu)) chelator precursor required two rounds to full attachment.Release from the resin with concomitant deprotection of lateral groups, except for Acm, was achieved by a TFA/TIS/EDT/anisole/H2O (93:3:2:1:1) solution.Next, the first disulfide bond formation between Cys 6 and Cys 11 and the generation of the respective 6-mer ring followed, using a mixture of 0.01 M phosphate, pH 7.5, and 1% DMSO.This step turned out to be slow (27 h) as monitored by the Ellman test and reverse-phase high performance liquid chromatography (RP-HPLC).The product was purified by RP-HPLC twice and its formation was confirmed by electrospray-ionization mass-spectroscopy (ESI-MS).The second oxidation conducted by I 2 in aqueous acetic acid was completed rapidly (25 min) under concomitant Acm removal and formation of the second disulfide Cys 3 -Cys 14 bond (12-mer ring) [38].The bicyclic peptide-conjugate was isolated by RP-HPLC to 94% purity and the ESI-MS result was in agreement with the expected structure (Table 1) in a 27% yield; the latter was lower from the one reported for other monocyclic ATXS products [37].This was attributed to the high number of by-products from the first oxidation step, necessitating repeated purification by RP-HPLC prior to moving further to the second oxidation and the closure of the 12-mer ring.
linear sequence DOTA(tris( Bu))-Ala -Gly -Cys(Acm) -Lys(Boc) -Cys(Trt) -Phe -Phe -DTrp(Boc) 8 -Lys(Boc) 9 -Thr( t Bu) 10 -Phe 11 -Cys(Trt) 12 -Ser( t Bu) 13 -Cys(Acm) 14 was built on the same resin (Figure 2b; and detailed in Sections 4.1.and 4.2. in Section 4), but all coupling reactions were straightforward, not requiring repetition.The two disulfide bonds, Cys 5 -Cys 12 (8-mer ring) and Cys 3 -Cys 14 (12-mer ring), were consecutively formed following the same protocol used for AT5S.Again, the first cyclization using DMSO required longer reaction times (24 h), careful monitoring, and isolation of the product by RP-HPLC prior to starting the second cyclization step, which lasted only 25 min.The final compound was isolated by RP-HPLC to >93% purity in an overall yield of 27% and the ESI-MS result was consistent with the expected structure (  Synthesis of (a) bicyclic 6/12-member-ring AT5S and (b) bicyclic 8/12-member-ring AT6S, conducted on a solid support.The assembly of the respective protected peptide chains on the resin by Fmoc/ t Bu methodology was followed by the coupling of the protected DOTA-chelator at the N-terminus, release from the resin, and removal of lateral protecting groups by TFA treatment.The formation of the first disulfide bridge (Cys 6 -Cys 11 for AT5S and Cys 5 -Cys 12 for AT6S) was achieved with 1% DMSO in phosphate buffer and the second disulfide bridge (Cys 3 -Cys 14 ) by I 2 oxidation in an aqueous acetic acid solution, concomitantly removing the Acm protecting groups.

Synthesis of AT6S
A similar process was followed for the synthesis of AT6S.This time, the protected linear sequence DOTA(tris( t Bu))-Ala 1 -Gly 2 -Cys(Acm) 3 -Lys(Boc) 4 -Cys(Trt) 5 -Phe 6 -Phe 7 -DTrp(Boc) 8 -Lys(Boc) 9 -Thr( t Bu) 10 -Phe 11 -Cys(Trt) 12 -Ser( t Bu) 13 -Cys(Acm) 14 was built on the same resin (Figure 2b; and detailed in Sections 4.1 and 4.2 in Section 4), but all coupling reactions were straightforward, not requiring repetition.The two disulfide bonds, Cys 5 -Cys 12 (8-mer ring) and Cys 3 -Cys 14 (12-mer ring), were consecutively formed following the same protocol used for AT5S.Again, the first cyclization using DMSO required longer reaction times (24 h), careful monitoring, and isolation of the product by RP-HPLC prior to starting the second cyclization step, which lasted only 25 min.The final compound was isolated by RP-HPLC to >93% purity in an overall yield of 27% and the ESI-MS result was consistent with the expected structure (Table 1 and Figures S1-S4 in Supplementary File).

Radiochemistry
Radiolabeling of AT5S and AT6S with In-111-Quality Control Labeling of the two bioconjugates with In-111 was achieved by heating at 95 • C for 20 min at pH 4.6 to facilitate the full incorporation of the radiometal without formation of undesirable hydroxides.Radiochemical purities of >94% were achieved at a molar activity of 3.7-7.4MBq/nmol.As verified by RP-HPLC analysis, a single radiopeptide species was obtained each time and therefore, the forming [ 111 In]In-AT5S and [ 111 In]In-AT6S radioligands were used without further purification in all subsequent biological tests.Moreover, the preservation of the radiochemical purity of both [ 111 In]In-AT5S and [ 111 In]In-AT5S was confirmed prior to and at the end of the assays by RP-HPLC.The binding affinities of AT5S and AT6S to each of the five human SST 1-5 R (hSST 1-5 R) were evaluated in CHO (hSST 1 R), CCL39 (hSST 2 R, hSST 3 R, or hSST 4 R), or HEK293 (hSST 5 R) cells transfected to stably express one of the above subtypes.Competition binding assays against the pansomatostatin radioligand [ 125 I][Leu 8 ,DTrp 22 ,Tyr 25 ]SS28 ([ 125 I]I-[LTT]SS28) were performed in suitably prepared frozen slices of the cells.Receptor autoradiography methods were applied to determine half-maximum inhibitory constants (IC 50 ), using SS14 and AT2S as reference compounds [32,37].The results summarized in Table 2 represent mean IC 50 values ± SEM (standard error of the mean) in nM.

Ligand-Induced Internalization of the hSST 2 R
An immunofluorescence microscopy-based internalization assay was performed in HEK293 cells transfected to stably express the hSST 2 R tagged with the T7-epitope (HEK293-SST 2 R) to determine the agonistic/antagonistic properties of AT5S and AT6S, using native SS14, TOC, and AT1S as reference agonists [32].As shown in Figure 3, in the absence of a hSST 2 R-ligand, the receptor remains on the cell membrane of the cells (a), but the known agonists SS14 (at 10 nM, (b)), TOC (at 10 nM-(c); 1 µM-(d)), and AT1S (at 1 µM, (e)) were clearly able to trigger the internalization of hSST 2 R. On the other hand, the 6/12-member ring AT5S failed to trigger the hSST 2 R internalization at 1 µM (f) or even at 10 µM (g).It furthermore failed to prevent the hSST 2 R internalization induced by 10 nM SS14 in the same cells, even at a concentration of 10 µM (h).Therefore, it behaves neither as an agonist nor as an antagonist in this assay, a finding attributed to its poor affinity for the hSST 2 R. In contrast, the 8/12-member ring bicyclic AT6S, exhibiting a single-digit binding affinity for the hSST 2 R, behaved as a potent agonist triggering the internalization of the receptor at 1 µM (i).

Ligand-Induced Internalization of the hSST2R
An immunofluorescence microscopy-based internalization assay was performed in HEK293 cells transfected to stably express the hSST2R tagged with the T7-epitope (HEK293-SST2R) to determine the agonistic/antagonistic properties of AT5S and AT6S, using native SS14, TOC, and AT1S as reference agonists [32].As shown in Figure 3, in the absence of a hSST2R-ligand, the receptor remains on the cell membrane of the cells (a), but the known agonists SS14 (at 10 nM, (b)), TOC (at 10 nM-(c); 1 µM-(d)), and AT1S (at 1 µM, (e)) were clearly able to trigger the internalization of hSST2R.On the other hand, the 6/12-member ring AT5S failed to trigger the hSST2R internalization at 1 µM (f) or even at 10 µM (g).It furthermore failed to prevent the hSST2R internalization induced by 10 nM SS14 in the same cells, even at a concentration of 10 µM (h).Therefore, it behaves neither as an agonist nor as an antagonist in this assay, a finding attributed to its poor affinity for the hSST2R.In contrast, the 8/12-member ring bicyclic AT6S, exhibiting a single-digit binding affinity for the hSST2R, behaved as a potent agonist triggering the internalization of the receptor at 1 µM (i).cells were then processed for immunofluorescence microscopy.Compared to SS14, TOC, and AT1S, ΑΤ6S is a strong agonist, while AT5S is not an agonist, since it is not able to stimulate hSST2R-internalization up to 1 and 10 µM.Moreover, AT5S is not an antagonist either, since it cannot inhibit the SS14-induced internalization of the receptor even in high molar excess over the native hormone (10 µM to 10 nM).

Internalization of [ 111 In]In-AT6S in AR4-2J and HEK293-hSST3R Cells
The internalization of [ 111 In]In-AT6S was studied in AR4-2J cells that endogenously express the rat SST2R (rSST2R) [39,40] and in HEK293 cells transfected to stably express the hSST3R (HEK293-hSST3R).The [ 111 In]In-AT5S was not included in this study, in view of its lack of affinity for any of the hSST1-5R, as well as its lack of agonist/antagonist behavior at the hSST2R during the immunofluorescence microscopy-based analysis.For [ 111 In]In-AT6S, assays were conducted by a 1 h incubation at 37 °C at 1-2 nM without or in the presence of 1 µM blocker to determine non-specific internalization (TATE for rSST2R  (c,d) 10 nM and 1 µM TOC, respectively (positive controls), (e) 1 µM AT1S (positive control), (lower row) (f,g) 1 µM and 10 µM AT5S, respectively, (h) 10 nM SS14 + 10 µM AT5S, and (i) 1 µM AT6S; cells were then processed for immunofluorescence microscopy.Compared to SS14, TOC, and AT1S, AT6S is a strong agonist, while AT5S is not an agonist, since it is not able to stimulate hSST 2 R-internalization up to 1 and 10 µM.Moreover, AT5S is not an antagonist either, since it cannot inhibit the SS14-induced internalization of the receptor even in high molar excess over the native hormone (10 µM to 10 nM).

Internalization of [ 111 In]In-AT6S in AR4-2J and HEK293-hSST 3 R Cells
The internalization of [ 111 In]In-AT6S was studied in AR4-2J cells that endogenously express the rat SST 2 R (rSST 2 R) [39,40] and in HEK293 cells transfected to stably express the hSST 3 R (HEK293-hSST 3 R).The [ 111 In]In-AT5S was not included in this study, in view of its lack of affinity for any of the hSST 1-5 R, as well as its lack of agonist/antagonist behavior at the hSST 2 R during the immunofluorescence microscopy-based analysis.For [ 111 In]In-AT6S, assays were conducted by a 1 h incubation at 37 • C at 1-2 nM without or in the presence of 1 µM blocker to determine non-specific internalization (TATE for rSST 2 R blockade in AR4-2J cells) and KE108 (for hSST 3 R blockade in HEK293-hSST 3 R cells) [28].The results are summarized in Figure 4, revealing low internalization (0.63 ± 0.10%) and overall uptake (1.17 ± 0.39%) of the radioligand in the AR4-2J cells, with these values turning out to be clearly higher in the HEK293-hSST 3 R cells (2.35 ± 0.63% and 4.76 ± 0.67%, respectively).

Biodistribution of [ 111 In]In-AT6S in Mice
The bicycle 6/12-member ring [ 111 In]In-AT5S was not included in the biodistribution studies, due to its inability to interact with any of the five hSST1-5R.The biodistribution of [ 111 In]In-AT6S was assessed in rSST2R-positive AR4-2J tumors raised in severe combined immunodeficiency disease (SCID) mice at 4 and 24 h pi and during in vivo SST2R-blockade at 4 h pi induced by co-injection of excess TATE [32].In addition, [ 111 In]In-AT6S was studied in HEK293-hSST3R xenograft-bearing SCID mice at 4 h pi and during in vivo SST3Rblockade at 4 h pi by co-injection of excess KE108 [28].The results, calculated as percent injected activity per gram tissue (%IA/g), are summarized in Table 3 and represent mean values ± sd (n = 4).Moreover, the comparative uptake of [ 111 In]In-AT6S and [ 111 In]In-AT2S for the AR4-2J and the HEK293-hSST3R tumors, as well as in mice kidneys at 4 h pi [32] is included in Figure 6.The radioligand exhibited low levels in the blood at 4 h pi and showed visible uptake in the liver, spleen, intestines, and especially in the kidneys, but uptake declined further at 24 h pi.Uptake in the AR4-2J tumors was low at 4 h pi (1.9 ± 0.1%IA/g) and significantly The radioligand exhibited low levels in the blood at 4 h pi and showed visible uptake in the liver, spleen, intestines, and especially in the kidneys, but uptake declined further at 24 h pi.Uptake in the AR4-2J tumors was low at 4 h pi (1.9 ± 0.1%IA/g) and significantly dropped with the co-injection of excess TATE (0.8 ± 0.05%IA/g; p < 0.001).The AR4-2J tumor values of [ 111 In]In-AT6S declined at 24 h pi (0.8 ± 0.1%IA/g).In the animals bearing the HEK293-hSST 3 R xenografts, a similar pattern was observed in healthy organs and tissues at 4 h pi, but [ 111 In]In-AT6S achieved a markedly higher uptake in the HEK293-hSST 3 R tumors (3.7 ± 0.4%IA/g), in line with its higher uptake in the HEK293-hSST 3 R cells.Tumor uptake could be significantly reduced during hSST 3 R-blockade by co-injection of excess KE108 (0.3 ± 0.05%IA/g; p < 0.001), confirming a hSST 3 R-mediated process.It is interesting to observe the drop of renal uptake in both groups of blocked animals by co-injection of either TATE (from 86.7 ± 10.9%IA/g to 49.5 ± 10.5%IA/g; p < 0.001) or KE108 (from 61.1 ± 10.6%IA/g to 26.8 ± 5.4%IA/g; p < 0.001).Both peptide analogs carry pendant primary amines (Lys 5 in TATE; DDab 1 /Lys 6 in KE108), which may partially saturate the cubilin/megalin system in the endocytic apparatus of the renal proximal tubule of the kidneys [41,42], thereby facilitating the excretion of radioactivity into urine.
Compared with the quickly biodegradable [ 111 In]In-AT2S (≈6% intact at 5 min pi in the peripheral blood of mice) [32], [ 111 In]In-AT6S displayed similar uptake in the AR4-2J tumors but a notably higher uptake in the HEK293-hSST 3 R xenografts (Figure 6a,b, respectively).Of particular interest are the elevated renal values of [ 111 In]In-AT6S compared to [ 111 In]In-AT2S, tentatively attributed to their different metabolic fates and further processing of [ 111 In]In-AT2S-derived radiometabolites (Figure 6c).

Discussion
Previous efforts to develop metabolically robust pansomatostatin-like radioligands for cancer theranostics were driven by the co-expression of SST 1-5 Rs in different combinations in NET and other tumor lesions [8,[13][14][15][16][17][18]27,28,31,43].The accomplishment of this goal has been hitherto impeded by two major challenges.On one hand, SS14 and SS28 and their radiolabeled pansomatostatin-like analogs, such as [ 111 In]In-AT1S and [ 111 In]In-AT2S, were shown to undergo fast enzymatic degradation after entering the circulation [32,34,36].On the other hand, less biodegradable synthetic analogs of smaller ring size ended up with partial or total loss of SST 1-5 R affinity.In addition, such analogs have been often linked with the absence of SST 2 R-internalization as well as unfavorable pharmacokinetics in mice, compromising their clinical applicability [29][30][31]33,37,44].It should be stressed that the currently applied octreotide-based radioligands, like [ 68 Ga]Ga/[ 177 Lu]Lu-DOTA-TOC and [ 68 Ga]Ga/[ 177 Lu]Lu-DOTA-TATE, are actually SST 2 R-preferring [5,6,8,9,11].Their clinical success relies on the 6-member ring octapeptide structure leading to high metabolic stability and to enhanced internalization in cancer cells [6,8,31].These features, in combination with the prevailing SST 2 R-expression in NETs and other human tumors, have synergistically contributed to the high diagnostic accuracy and therapeutic efficacy achieved in patients [5,7,15].
In contrast to the above, we have observed that 6-member ring analogs containing a total of 14 amino acids, like AT3S, unexpectedly lost affinity to all SST 1-5 Rs, while the respective [ 111 In]In-AT3S remained stable in mouse circulation [37].Furthermore, the 8-member ring 14-peptide analog, AT4S, showed a pansomatostatin affinity profile and the corresponding [ 111 In]In-AT4S was notably more stable compared to the 12-mer ring analogs [ 111 In]In-AT1S and [ 111 In]In-AT2S [32,37].In a continuation of this work, we have now synthesized two bicyclic 6/12-mer and 8/12-mer ring 14-peptide analogs by introducing two disulfide bridges in the SS14 scaffold, namely, Cys 6 -Cys 11 /Cys 3 -Cys 14 for AT5S and Cys 5 -Cys 12 /Cys 3 -Cys 14 for AT6S (Figure 1).In this way, the issue of metabolic stability is vigorously addressed for both.Concurrently, the effects of enhanced molecule rigidity on SST 1-5 R affinity, internalization, and tumor uptake could be compared with their respective monocyclic counterparts (AT3S and AT4S) [37], as well as with the AT1S/AT2S prototypes [32].
The first results on SST 1-5 R affinity, obtained by receptor autoradiography on cells expressing one of the five receptors are summarized in Table 1.The introduction of the second Cys 3 -Cys 14 disulfide bridge in AT5S failed to improve the affinity to any of the five SST 1-5 R when compared with monocyclic AT3S [37].However, the same intervention (introduction of a second Cys 3 -Cys 14 disulfide bridge) in AT6S led to affinity enhancement in all five receptor subtypes with the exception of SST 4 R, compared with the monocyclic 8-mer ring AT4S [37], revealing the positive influence of the increased rigidity of the bicyclic molecule.The unexpected lack of SST 1-5 R affinity of AT3S and AT5S is truly intriguing, in view of the high SST 2 R-affinity of related 6-mer monocyclic octapeptide analogs, such as octreotide, having identical 6 amino acid rings.Dedicated studies using NMR methodology may reveal conformational changes between 6-mer monocyclic and 6/12-mer bicyclic tetradecapeptide analogs compared to their octapeptide counterparts, causing this discrepancy in affinity, especially on the SST 2 R. The agonist/antagonist properties of AT5S and AT6S were studied next by an immunofluorescence microscopy-based internalization assay in HEK293-SST 2 R cells and the results are summarized in Figure 3.As expected by its poor SST 2 R-affinity, AT5S failed not only to trigger SST 2 R-internalization in concentrations up to 10 µM, but also to inhibit the SS14-induced internalization of the receptor even in high molar excess (10 µM to 10 nM).Hence, AT5S behaved neither as an agonist nor as an antagonist at the SST 2 R. On the other hand, the 8/12-mer bicyclic AT6S, similarly to its monocyclic AT4S counterpart [37], behaved as a strong agonist in the same assay by stimulating the SST 2 R-internalization at 1 µM.Lastly, the internalization of [ 111 In]In-AT6S was investigated in AR4-2J and HEK293-SST 3 R cells (Figure 4).Internalization in AR4-2J cells was comparable to that of the monocyclic 8-mer ring [ 111 In]In-AT4S [37], but clearly inferior to the internalization of the monocyclic 12-mer ring [ 111 In]In-AT1S and [ 111 In]In-AT2S [32].Interestingly, the internalization of [ 111 In]In-AT6S was more pronounced in the HEK293-SST 3 R cells.A similar higher internalization in HEK293-SST 3 R cells than in HEK293-SST 2 R cells was reported for [ 111 In]In-DOTA-LTT-SS28 [33].The latter internalized much more efficiently compared to [ 111 In]In-AT6S in both cell lines, most probably due to the higher affinity of DOTA-LTT-SS28 and [ nat In]In-DOTA-LTT-SS28 for SST 2 R and SST 3 R [33].
The bicyclic 6/12-mer [ 111 In]In-AT5S and 8/12-mer [ 111 In]In-AT6S showed >96% stability in the peripheral blood of mice, in contrast to the monocyclic 12-mer ring [32].The rapid cleavage of peptide bonds in the SS14 motif could be previously assigned to NEP by monitoring the formation of (radio)metabolites without or in the presence of potent NEP-inhibitors, like PA and thiorphan [34][35][36]45].It is interesting to note that increased stability was already observed for the respective 6-mer ring [ 111 In]In-AT3S (96%) and 8-mer ring [ 111 In]In-AT4S (>68%) 14-peptide analogs.Furthermore, the DCys 5 -substituted 8-mer ring [ 111 In]In-AT9S showing moderate affinities to all SST 1-5 R was >92% stable in the same assay.These findings confirm previous observations that molecule rigidity imposed by the two disulfide bridges, along with ring size and conformation play a critical role in the in vivo stability of SS14-based radioligands.
The biodistribution of pansomatostatin-like and in vivo robust [ 111 In]In-AT6S was studied in mice bearing either AR4-2J (rSST 2 R-positive) or HEK293-hSST 3 R xenografts with the aim to evaluate its suitability for clinical use (Table 3 and Figure 6).Compared with the quickly biodegradable but more SST 2 R-affine [ 111 In]In-AT2S, [ 111 In]In-AT6S showed similar levels of uptake in AR4-2J tumors, but superior uptake in HEK293-hSST 3 R tumors, emphasizing the importance of metabolic stability for good tumor localization [32,37].This assumption was further supported by the high values attained by the fairly stable and highly SST 1-5 R-affine [ 111 In]In-DOTA-LTT-SS28 in the same tumor models [33].Unluckily, by increasing the stability of SS14-based analogs, an unfavorably increasing kidney accumulation was most often observed, which was found to be overwhelming in the case of [ 111 In]In-DOTA-LTT-SS28 [32,33,36,37].Administration of positive amino acid solutions alone or together with plasma expanders, such as Gelofusine, have been previously proposed to mitigate this problem [41,42].It is interesting to compare our findings with another class of bicyclic somatostatin-like peptides comprising an octreotide ring and a head-to-tailcoupled Arg-Dab(DOTA) 8-mer ring cycle, such as AM3 (DOTA-Tyr-c{[Dab-Arg-c[Cys-Phe-DTrp-Lys-Thr-Cys]}) [46].These analogs were SST 2 R/SST 3 R-preferring, having reduced affinities for SST 1 R and SST 5 R.However, their [ 68 Ga]Ga/ 177 Lu]Lu-radioligands were likewise metabolically robust.They displayed superior internalization in SST 2 R/SST 3 R-positive cells and hence in respective tumors xenografted in mice.Kidney values were moderate and were reduced by half by pre-treatment of mice with a Lys solution [46].

Cyclization and Isolation of DOTA-Conjugates
For the formation of the first disulfide bridge, Cys 6 -Cys 11 (AT5S) or Cys 5 -Cys 12 (AT6S), the linear peptide was dissolved in 0.01 M phosphate buffer, pH 7.5 (final concentration 1 mg/mL) and 1% DMSO was added.The mixture was stirred at room temperature and the formation of the monocyclic product was monitored by HPLC and MS.For the formation of the second disulfide bridge (Cys 3 -Cys 14 , 12 member ring), oxidation with iodine in a AcOH/H 2 O 4:1 (v/v) solution after in situ removal of the Acm groups was pursued, as previously described [38].The bicyclic analogs were isolated by semi-preparative HPLC and lyophilized.Analytical HPLC confirmed the high purity of peptide conjugates and ESI-MS spectra were consistent with the expected formula (Table 1 and Figures S1-S4 in Supplementary File).

Radiolabeling with In-111 and Quality Control
Labeling of the peptide-conjugates with In-111 was conducted by adding 10 nmol peptide analog per 37 to 74 MBq of [ 111 In]InCl 3 in 0.1 M sodium acetate buffer and 10 mM sodium ascorbate.The typical end pH was 4.6.Labeling was completed by incubation in a boiling water bath for 20 min.Prior to HPLC quality control, EDTA in 0.1 M acetate buffer was added to a final concentration of 1 mM to the labeling reaction mixture as a "free" [ 111 In]In 3+ scavenger.Analyses were performed on a RP18 column using system 2.

Cell Lines and Cell Culture
For in vitro assays, CHO cells transfected to stably express the hSST 1 R were kindly offered by Drs.T. Reisine and G. Singh (University of Pennsylvania, Philadelphia, PA, USA), whereas the CCL39 cells transfected to stably express one of the hSST 2 R, hSST 3 R, or hSST 4 R were a kind gift of Dr. D. Hoyer (Novartis Pharma, Basel, Switzerland).Furthermore, HEK293 cells, transfected to stably express one of the expressing the T7-epitope-tagged hSST 2 R, hSST 3 R or hSST 5 R, were given by Dr. S. Schultz (Institute of Pharmacology and Toxicology, Friedrich Schiller University of Iena, Jena, Germany).CHO cells were cultured in Ham's F-12 medium, CCL39 cells in a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium in a 1:1 v/v ratio.HEK293 cells were grown in DMEM GLUTAMAX-I.Media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin along with 400-500 µg/mL G418-sulfate.The rat pancreatic tumor cell line AR4-2J, endogenously expressing the SST 2 R, was provided by Prof. S. Mather (St.Bartholomew's Hospital, London, UK) and cultured in F-12 K medium supplemented as above, but without addition of G418.All culture reagents were obtained from Gibco BRL, Life Technologies (Grand Island, NY, USA) or from Biochrom KG Seromed (Berlin, Germany).Cells were cultured at 37 • C and a humidified 5% CO 2 atmosphere and weekly passages were performed using a Trypsin/EDTA (0.05%/0.02% w/v) solution.

Receptor Autoradiography
The hSST 1-5 R affinity profiles of monocyclic AT2S and of the two bicyclic AT5S and AT6S analogs were determined by receptor autoradiography [43,47].Cell membrane pellets were prepared from CHO-hSST 1 R cells, CCL39-hSST 2 R, CCL39-hSST 3 R, and CCL39-hSST 4 R cells, and HEK293-hSST 2 R cells and stored at −80 • C. Receptor autoradiography was performed on 20-µm-thick cryostat (Microm HM 500, Walldorf, Germany) sections of the membrane pellets mounted on microscope slides, and then stored at −20 • C, as previously described [33].For each of the tested compounds, complete displacement experiments were performed with the universal SS28 radioligand [ 125 I][Leu 8 ,DTrp 22 ,I-Tyr 25 ]SS28 ([ 125 I]I-[LTT]SS28) (74 GBq/mmol; Anawa, Wangen, Switzerland) using 15,000 cpm/100 µL and increasing concentrations of the unlabeled peptide ranging from 0.1 to 1000 nM.Native SS14 was run in parallel as an internal control using the same increasing concentrations.The sections were incubated with [ 125 I]I-[LTT]SS28 for 2 h at room temperature in 170 mmol/L Tris-HCl buffer (pH 8.2), containing 1% bovine serum albumin (BSA), 40 mg/L bacitracin, and 10 mmol/L MgCl 2 to inhibit endogenous proteases.The incubated sections were washed twice for 5 min in cold 170 mmol/L Tris-HCl (pH 8.2) containing 0.25% BSA.After a brief dip in 170 mmol/L Tris-HCl (pH 8.2), the sections were dried quickly and exposed for 1 week to Kodak BioMax MR film (Rochester, NY, USA).IC 50 values were calculated after quantification of the data using a computer-assisted image processing system as described previously [33].Tissue standards (Autoradiographic [ 125 I] and/or [ 14 C] microscales, GE Healthcare; Little Chalfont, UK) that contain known amounts of isotope, cross-calibrated to tissue-equivalent ligand concentrations, were used for quantification.

Immunofluorescence Microscopy-Based Internalization Assay
An immunofluorescence microscopy-based internalization assay for hSST 2 R was performed in HEK293-hSST 2 R cells, grown on poly-DLys (20 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) coated 35-mm four-well plates (Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany) [44,48].Cells were treated for 30 min at 37 • C in growth medium containing either the compounds to be tested (AT5S: 1 µM, 10 µM or 10 µM + 10 nM SS14; AT6S: 1 µM) or TOC (10 nM and 1 µM) and SS14 (10 nM), serving as positive controls.HEK293-hSST 2 R cells treated with vehicle alone were used as a negative control.The cells were then rinsed twice with PS (100 mM phosphate buffer containing 0.15 M sucrose), fixed and permeabilized for 7 min with cold methanol (−20 • C), rinsed twice with PS, and then blocked for 60 min at room temperature with PS containing 0.1% BSA.Next, cells were incubated for 60 min at room temperature with the hSST 2 R-specific primary antibody R2-88 (provided by Dr. A. Schönbrunn, Houston, TX, USA) diluted 1:1000 in PS and washed 3 × 5 min with PS containing 0.1% BSA.The cells were incubated for 60 min at room temperature in the dark with the secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H+ L) (Molecular Probes, Inc., Eugene, OR, USA) diluted in PS (1:600), washed 3 × 5 min with PS containing 0.1% BSA, embedded with PS/glycerol 1:1, and covered with a glass cover slip.The cells were imaged using a Leica DM RB immunofluorescence microscope (Leica, Deerfield, IL, USA) and an Olympus DP10 camera (Olympus Corporation, Shinjuku, Tokyo, Japan).

Radioligand Internalization in AR4-2J and HEK293-hSST 3 R Cells
Radioligand internalization experiments were performed using the rSST 2 R-positive AR4-2J cells and HEK293-hSST 3 R cells [32].Cells were grown to confluence in six-well plates 24 h prior to the experiment.The following day, the cells were washed twice with ice-cold internalization medium (F-12 K with 1% FBS for the AR4-2J cells and DMEM GLUTAMAX-I with 1% FBS for the HEK293-hSST 3 R cells).They were supplied with fresh medium (1.2 mL) and [ 111 In]In-AT6S (150 µL, ≈300,000 cpm/2 pmol peptide) were added per well, followed by 0.5% BSA phosphate buffered saline (PBS) alone (150 µL, total series) or by a 1 µM TATE (AR4-2J cells) or a KE108 (HEK293-hSST 3 R cells) solution (0.5% BSA-PBS; 150 µL, nonspecific series).Cells were incubated at 37 • C for 60 min and incubation was interrupted by the removal of the medium and rapid rinsing with ice-cold 0.5% BSA-PBS.Cells were incubated (2 × 5 min) at ambient temperature in acid wash buffer (50 mM glycine in 0.1 M NaCl, pH 2.8).The supernatants were collected (membrane-bound radioligand fraction).The cells were rinsed with 0.5% BSA-PBS, lysed with 1 N NaOH and collected (internalized radioligand fraction).Collected fractions were measured for their radioactivity content in the γ-counter and the percentage of internalized activity was calculated versus the total added activity per well.Experiments were performed twice in triplicate.The radioligand, [ 111 In]In-AT5S or [ 111 In]In-AT6S, was injected as a 150 µL bolus (11-22 MBq, 3 nmol total peptide) in the tail vein of male Swiss albino mice (30 ± 5 g, NCSR "Demokritos" Animal House, Athens, Greece) [32,36].At 5 min pi blood (~1mL) was collected from the heart of mice in a prechilled insulin syringe and swiftly placed in an ice-cold polypropylene tube containing EDTA on ice.Blood samples were centrifuged at 2000× g at 4 • C for 10 min.The supernatant (>90% radioactivity recovered) was collected and an equal volume of MeCN was added.The mixture was centrifuged for 10 min at 15,000× g at 4 • C. The supernatant (>90% recovery of radioactivity) was collected, and the organic solvent was removed under a mild N 2 -stream; the residue was redissolved in physiological saline, passed through a 0.22-µm Millex-GV filter (Millipore, Milford, CT, USA) (>90% recovery of radioactivity), and analyzed by RP-HPLC (>96% radioactivity recovered).An RP18 Symmetry Shield cartridge column (5 µm, 3.9 mm × 20 mm, Waters, Vienna, Austria) was eluted at a 1 mL/min flow rate with the following gradient: 100%A/0%B to 60%A/40%B in 40 min, whereby A = 0.1% aqueous TFA and B = MeCN (system 3).Co-injection of blood samples with aliquots of the original labeling reaction on the HPLC column helped to identify intact [ 111 In]In-AT5S or [ 111 In]In-AT6S by co-elution in this system (identical t R ).

Biodistribution of [ 111 In]In-AT6S in AR4-2J Tumor-Bearing SCID Mice
For biodistribution experiments, male SCID mice of 7 weeks of age (15-20 g) on arrival day (NCSR "Demokritos" Animal House, Athens, Greece) were subcutaneously injected in their flanks with a suspension of AR4-2J cells (150 µL inocula of ~1 × 10 7 cells in PBS) [32].The animals were kept under aseptic conditions for 12 days until well-palpable tumors were grown at the inoculation site.On the day of the biodistribution experiment, animals were injected in the tail vein in groups of four with [ 111 In]In-AT6S (100 µL, 37 to 74 kBq, 10 pmol total peptide) and were sacrificed at 4 and 24 h pi.In an additional group, animals were co-injected with excess TATE (100 nmol) together with the radioligand (blocked animals) and were sacrificed at 4 h pi.Blood was immediately collected, and the organs of interest were excised and weighed.Sample radioactivity was measured in the γ-counter using proper standards of the injected activity.Data were calculated as %IA/g with the aid of standard solutions and represent mean values ± sd.For comparisons, a Two-way ANOVA with Tukey's post hoc analysis was applied (PRISM TM GraphPad-6 Software, San Diego, CA, USA).p values < 0.05 were considered statistically significant.For the second biodistribution experiments, male SCID mice of 7 weeks of age (15-20 g) on arrival day (NCSR "Demokritos" Animal House, Athens, Greece) were subcutaneously injected in their flanks with a suspension of HEK293-hSST 3 R cells (150 µL inocula of ~3 × 10 7 cells in PBS) [32].The animals were kept under aseptic conditions for 3-4 weeks until well-palpable tumors were grown at the inoculation site.On the day of the biodistribu-tion experiment, animals were injected in the tail vein in groups of four with [ 111 In]In-AT6S (100 µL, 37 to 74 kBq, 10 pmol total peptide) alone, or co-injected with excess KE108 (80 nmol) (blocked animals).Mice were sacrificed at 4 h pi, and biodistribution analyses were conducted, as described above.
Mice experiments complied with European and national regulations and study protocols were approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (#440448, 01-06-2021 for the stability studies and #440451, 01-06-2021 for the biodistribution and imaging studies).

Conclusions
This work on bicyclic 6/12-mer and 8/12-mer ring SS14 analogs and their [ 111 In]Inlabeled versions, has shown that the introduction of two rings was a successful strategy to ensure the metabolic stability of the radioligands in circulation.On the other hand, it revealed that other factors are equally important for effective tumor targeting.Thus, changes in molecule rigidity, ring(s) size and conformation induced by the double ring system directly impacted the SST 1-5 R affinity profile, internalization capacity, and pharmacokinetics.Accordingly, the bicyclic 6/12-mer AT5S/[ 111 In]In-AT5S failed to interact with any of the SST 1-5 R, despite its high stability and presence of the "-Phe 7 -DTrp 8 -Lys 9 -Thr 10 -" pharmacophore sequence.In contrast, the bicyclic 8/12-mer AT6S/[ 111 In]In-AT6S behaved like a pansomatostatin and showed superior biological features compared with a previously reported monocyclic 8-mer counterpart (AT4S/[ 111 In]In-AT4S).It is interesting to note that unlike other bicyclic analogs with reduced overall number of amino acids, the tetradecapeptide AT6S/[ 111 In]In-AT6S retained a pansomatostatin character, albeit showing inferior internalization at the SST 2 R and lower SST 2 R-specific tumor uptake in mice.More studies are warranted to better explore and smartly exploit structural interventions leading to the most advantageous combination of all of the above factors in order to make available true pansomatostatin radiopharmaceuticals for successful application in tumor cancer theranostics.
Figure2.Synthesis of (a) bicyclic 6/12-member-ring AT5S and (b) bicyclic 8/12-member-ring AT6S, conducted on a solid support.The assembly of the respective protected peptide chains on the resin by Fmoc/ t Bu methodology was followed by the coupling of the protected DOTA-chelator at the N-terminus, release from the resin, and removal of lateral protecting groups by TFA treatment.The formation of the first disulfide bridge (Cys 6 -Cys 11 for AT5S and Cys 5 -Cys 12 for AT6S) was achieved with 1% DMSO in phosphate buffer and the second disulfide bridge (Cys 3 -Cys14 ) by I 2 oxidation in an aqueous acetic acid solution, concomitantly removing the Acm protecting groups.

Figure 4 .
Figure 4. Internalization and cell uptake of bicyclic 8/12-member-ring AT6S [ 111 In]In-AT6S in (a) AR4-2J cells and (b) in HEK293-hSST3R cells.Blue bars correspond to the results in the absence of blocker and gray bars to the results in the presence of 1 µM blocker; the blocker is TATE for AR4-2J cells and KE108 for HEK293-hSST3R cells.The results represent the mean percentages of total-added activity per well ± sd of at least two experiments performed in triplicate.

2. 4 . 1 .
Metabolic Stability of [ 111 In]In-AT5S and [ 111 In]In-AT6S in Mice The metabolic stability of [ 111 In]In-AT5S and [ 111 In]In-AT6S after their entry into the circulation of mice was studied by the analysis of blood samples collected 5 min postinjection (pi) by radio-HPLC.Representative radiochromatograms are included in Figure 5, revealing the high metabolic stability of the bicyclic analogs.

Figure 5 .
Figure 5. Representative radiochromatograms of blood samples collected 5 min pi of (a) bicyclic 6/12-member-ring [ 111 In]In-AT5S and (b) bicyclic 8/12-member-ring [ 111 In]In-AT6S (system 3).The tR of the intact radioligands was established after co-injection with radiolabeled samples not administered in mice in the HPLC-column and are indicated with the arrows.

Figure 4 .
Figure 4. Internalization and cell uptake of bicyclic 8/12-member-ring AT6S [ 111 In]In-AT6S in (a) AR4-2J cells and (b) in HEK293-hSST 3 R cells.Blue bars correspond to the results in the absence of blocker and gray bars to the results in the presence of 1 µM blocker; the blocker is TATE for AR4-2J cells and KE108 for HEK293-hSST 3 R cells.The results represent the mean percentages of total-added activity per well ± sd of at least two experiments performed in triplicate.

2. 4 .
Animal Studies 2.4.1.Metabolic Stability of [ 111 In]In-AT5S and [ 111 In]In-AT6S in Mice The metabolic stability of [ 111 In]In-AT5S and [ 111 In]In-AT6S after their entry into the circulation of mice was studied by the analysis of blood samples collected 5 min postinjection (pi) by radio-HPLC.Representative radiochromatograms are included in Figure 5, revealing the high metabolic stability of the bicyclic analogs.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 18

Figure 4 .
Figure 4. Internalization and cell uptake of bicyclic 8/12-member-ring AT6S [ 111 In]In-AT6S in (a) AR4-2J cells and (b) in HEK293-hSST3R cells.Blue bars correspond to the results in the absence of blocker and gray bars to the results in the presence of 1 µM blocker; the blocker is TATE for AR4-2J cells and KE108 for HEK293-hSST3R cells.The results represent the mean percentages of total-added activity per well ± sd of at least two experiments performed in triplicate.

2. 4 . 1 .
Metabolic Stability of [ 111 In]In-AT5S and [ 111 In]In-AT6S in Mice The metabolic stability of [ 111 In]In-AT5S and [ 111 In]In-AT6S after their entry into the circulation of mice was studied by the analysis of blood samples collected 5 min postinjection (pi) by radio-HPLC.Representative radiochromatograms are included in Figure 5, revealing the high metabolic stability of the bicyclic analogs.

Figure 5 .
Figure 5. Representative radiochromatograms of blood samples collected 5 min pi of (a) bicyclic 6/12-member-ring [ 111 In]In-AT5S and (b) bicyclic 8/12-member-ring [ 111 In]In-AT6S (system 3).The tR of the intact radioligands was established after co-injection with radiolabeled samples not administered in mice in the HPLC-column and are indicated with the arrows.

Figure 5 .
Figure 5. Representative radiochromatograms of blood samples collected 5 min pi of (a) bicyclic 6/12member-ring [ 111 In]In-AT5S and (b) bicyclic 8/12-member-ring [ 111 In]In-AT6S (system 3).The t R of the intact radioligands was established after co-injection with radiolabeled samples not administered in mice in the HPLC-column and are indicated with the arrows.

xH + /x] c Found/Calcd HPLC t R (min), UV Trace System 1 d System 2 e
Cys-residues participating in the ring closures with number of amino acids in the rings in parenthesis.b Purity was determined by HPLC system 2. c Fragment ion peaks found by ESI-MS with the calculated mass included. a