[111In-DOTA]Somatostatin-14 analogs as potential pansomatostatin-like radiotracers - first results of a preclinical study

Background In this study, we report on the synthesis, radiolabeling, and biological evaluation of two new somatostatin-14 (SS14) analogs, modified with the universal chelator DOTA. We were interested to investigate if and to what extent such radiotracer prototypes may be useful for targeting sst1-5-expressing tumors in man but, most importantly, to outline potential drawbacks and benefits associated with their use. Methods AT1S and AT2S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-Phe6-Phe7-Trp8/DTrp8-Lys9-Thr10-Phe11-Thr12-Ser13-Cys14-OH], respectively) were synthesized on the solid support and labeled with 111In. The sst1-5 affinity profile of AT1S/AT2S was determined by receptor autoradiography using [Leu8,dTrp22,125I-Tyr25]SS28 as radioligand. The ability of AT2S to stimulate sst2 or sst3 internalization was qualitatively analyzed by an immunofluorescence-based internalization assay using hsst2- or hsst3-expressing HEK293 cells. Furthermore, the internalization of the radioligands [111In]AT1S and [111In]AT2S was studied at 37 °C in AR4-2J cells endogenously expressing sst2. The in vivo stability of [111In]AT1S and [111In]AT2S was tested by high-performance liquid chromatography analysis of mouse blood collected 5 min after radioligand injection, and biodistribution was studied in normal mice. Selectively for [111In]AT2S, biodistribution was further studied in SCID mice bearing AR4-2J, HEK293-hsst2A+, -hsst3+ or -hsst5+ tumors. Results The new SS14-derived analogs were obtained by solid phase peptide synthesis and were easily labeled with 111In. Both SS14 conjugates, AT1S, and its DTrp8 counterpart, AT2S, showed a pansomatostatin affinity profile with the respective hsst1-5 IC50 values in the lower nanomolar range. In addition, AT2S behaved as an agonist for sst2 and sst3 since it stimulated receptor internalization. The 111In radioligands effectively and specifically internalized into rsst2A-expressing AR4-2J cells with [111In]AT2S internalizing faster than [111In]AT1S. Ex vivo mouse blood analysis revealed a rapid degradation of both radiopeptides in the bloodstream with the DTrp8 analog showing higher stability. Biodistribution results in healthy mice were consistent with these findings with only [111In]AT2S showing specific uptake in the sst2-rich pancreas. Biodistribution of [111In]AT2S in tumor-bearing mice revealed receptor-mediated uptake in the AR4-2J (1.82 ± 0.36 %ID/g - block 0.21 ± 0.17 %ID/g at 4 h post injection (pi)), the HEK293-hsst2A+ (1.49 ± 0.2 %ID/g - block 0.27 ± 0.20 %ID/g at 4 h pi), the HEK293-hsst3+ (1.24 ± 0.27 %ID/g - block 0.32 ± 0.06 %ID/g at 4 h pi), and the HEK293-hsst5+ tumors (0.41 ± 0.12 %ID/g - block 0.22 ± 0.006 %ID/g at 4 h pi). Radioactivity washed out from blood and background tissues via the kidneys. Conclusions This study has revealed that the native SS14 structure can indeed serve as a motif for the development of promising pansomatostatin-like radiotracers. Further peptide stabilization is required to increase in vivo stability and, consequently, to enhance in vivo delivery and tumor targeting.


Background
Somatostatin-14 (SS14) is a native peptide hormone exerting a variety of physiological actions in the brain and in peripheral tissues after binding to high affinity receptors on the cell membrane of target cells [1][2][3]. Somatostatin receptors comprise five subtypes (sst [1][2][3][4][5] and are also found in many human tumors where they are expressed alone or in various combinations [4][5][6][7]. Accordingly, they can serve as molecular targets for therapeutic interventions with somatostatin analogs. Native SS14 binds with nanomolar affinity to all five human receptor subtypes, hsst [1][2][3][4][5] , but its use for drug development is prevented by its poor in vivo stability [8]. This problem has been competently addressed by the advent of synthetic somatostatin analogs tailored to withstand enzymatic attack in vivo, such as octreotide (SMS 201-995, Sandostatin) [9] or Lanreotide (BIM 23014, Somatuline) [10]. Despite their higher potency and longer duration of action, these cyclic-octapeptide analogs have inadvertently become sst 2 -preferring and have lost most of somatostatin's affinity for the other subtypes. Yet, they have been used with success in the treatment of acromegaly and sst 2 -expressing tumors [11,12].
In a rather recent approach, metabolically stabilized somatostatin analogs have been functionalized with metal chelators to accommodate radiometals useful for diagnostic imaging and radionuclide therapy [13].
In all above instances, the sst 2 subtype prevails in incidence and density of expression allowing the successful application of sst 2 -preferring radioligands. However, it should be stressed that, despite the predominance of sst 2 expression in many human tumors, co-expression of sst 2 with other sst [1][2][3][4][5] subtypes is frequent enough. Thus, sst 2 and sst 5 are expressed often together in GH-secreting pituitary adenomas, and various combinations of ssts, such as sst 2 and sst 1 , are expressed in gastroenteropancreatic (GEP)-NETs.
In this respect, the parent SS14 motif has drawn our attention despite its suboptimal metabolic stability [8]. In fact, not much is reported on the in vivo performance of radiopeptides based on SS14. In a previous study, 111 In-[DTPA, DAla 1 ,DTrp 8 ,Tyr 11 ]SS14 showed specific and comparable to OctreoScan W accumulation in physiological sst 2 -rich tissues in mice [34], implying that SS14-based radioligands may indeed possess sufficient in vivo stability to successfully reach their target while still able to internalize via the sst 2 .
In this study, we have coupled the universal chelator DOTA to Ala 1 of SS14 (AT1S). In this way, labeling options beyond 111 In are feasible while N-terminal capping of SS14 is also achieved, a method known to prolong the biological half-life of peptides. In the second analog, AT2S, Trp 8 was replaced by DTrp 8 to further enhance stability [35]. This modification is also reported to improve sst 2 affinity by favoring the β-turn structure for several cyclic somatostatin analogs [36]. Detailed biological characterization of the AT1S prototype and its DTrp 8 analog, AT2S, is presented herein encompassing in vitro binding affinity and functional assays in sst 1-5expressing cells, metabolic studies, and biodistribution of 111 In-radioligands in mice bearing sst 2 + , sst 3 + , and sst 5 + tumors. This comprehensive study will provide the basis for structural interventions on the AT1S motif towards improved pansomatostatin-like radiopeptides with advantageous key pharmacological features, such as a preserved sst 2 -internalization capacity.

Biology Reagents
All reagents were of best grade available and were purchased from common suppliers. The sst 2 -specific antibody R2-88 was from Agnes Schönbrunn (Houston, TX, USA), and the sst 3 -specific antibody (SS-850) was purchased from Gramsch Laboratories, Schwabhausen, Germany. The secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (H + L), was from Molecular Probes, Inc.

Cell lines and animal experiments
The HEK293 cell line expressing the human T7-epitopetagged sst 2 receptor (HEK-sst 2 ) or the human sst 3 or sst 5 receptor (HEK-sst 3 , HEK-sst 5 ) were kindly provided by S. Schultz (Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University Jena, Germany) and cultured as previously described [38,39].
The rat pancreatic tumor cell line AR4-2J endogenously expressing sst 2 was kindly provided by Prof. S. Mather (St. Bartholomew's Hospital, London, UK) and cultured as previously described [37]. All culture reagents were from Gibco BRL, Life Technologies (Grand Island, NY, USA) or from Biochrom KG Seromed (Berlin, Germany). Animal experiments were carried out in compliance with European and national regulations and were approved by national authorities. For biodistribution experiments, in-house male Swiss albino mice (30 ± 5 g) were used. For experimental tumor models, in-house SCID mice of 7 weeks of age were used, and the animals were kept under aseptic conditions until biodistribution was performed.

Sst 2 -and sst 3 -internalization assay
Immunofluorescence microscopy-based internalization assay for sst 2 and sst 3 was performed as previously described [38,43]. HEK-sst 2 and HEK-sst 3   AT1S, X: AT2S, X:  cold 0.5% BSA PBS. Cells were then incubated twice for 5 min at ambient temperature in acid wash buffer (50 mM glycine buffer with pH 2.8, 0.1 M NaCl). The supernatant was collected (membrane-bound radioligand fraction) each time and pooled, and the cells were rinsed with 0.5% BSA PBS. Cells were lysed by treatment in 1 N NaOH, and cell radioactivity was collected (internalized radioligand fraction). Considering that total activity comprises membrane-bound plus internalized activity, the percent internalized activity versus the selected 5-, 15-, 30-, 60-, and 120-min time intervals could be calculated applying the Microsoft Excel program.

Metabolism in blood
A Animals were sacrificed in groups of four at 4-and 24-h time points post injection (pi). Blood and urine were immediately collected, and the organs of interest were excised and weighed; their radioactivity content was measured in an automatic gamma counter using proper standards of the injected dose. Tissue distribution data were calculated as percent injected dose per gram (%ID/g) applying a suitable algorithm.

In vivo distribution experiments in AR4-2J tumor-bearing mice
In the flanks of each of female SCID mice, inocula (150 μL) containing a suspension of 0.8 × 10 7 AR4-2J cells in PBS buffer were subcutaneously injected. Tumors of substantial size were grown within 12 days, whereupon biodistribution experiments were performed selectively for [ 111 In]AT2S. Animals were injected in the tail vein , and biodistribution was conducted as described above.

Statistical analysis
The in vivo data presented as mean %ID/g ± SD (n ≥ 4) were statistically analyzed with Student's t test (Prism TM 2.01, GraphPad Software, San Diego, CA, USA). Analyses were 2-tailed, and a P value < 0.05 was considered statistically significant.

Synthesis of conjugates
The linear amino acid AT1S and AT2S sequences were assembled on the solid support applying the Fmoc/ t Bu methodology, and the DOTA-protected chelator was coupled at the N-terminus. The DOTA-peptide conjugates were cleaved from the solid support, and the lateral protecting groups were removed by TFA treatment. Cyclization was conducted with iodine oxidation in solution and was monitored by analytical HPLC. The cyclized products (Figure 1) were isolated by semi-preparative HPLC and lyophilized. Product purity was assessed by analytical HPLC; ES-MS data were consistent with the expected formula (Table 1).

Radiolabeling
Labeling of ATIS and AT2S with 111 In (Figure 1) was achieved by a 20-min incubation of the analogs in acidic medium at 90°C in the presence of 111 InCl 3 , according to published protocols [13,44]. A >96% radiometal incorporation was typically shown by HPLC analysis on an RP column; a representative radiochromatogram of [ 111 In]AT2S quality control is shown in Figure 2.

Determination of hsst 1-5 profile
The IC 50 values of ATIS and AT2S for all five somatostatin receptor subtypes are summarized in Table 2. Data were acquired by receptor autoradiography assays in cells selectively expressing one of the five hsst 1-5 ; [Leu 8 , DTrp 22 , 125 I-Tyr 25 ]SS28 was used as pansomatostatin radioligand and SS14 and SS28 as controls. Both analogs, AT1S and AT2S, exhibit a clear pansomatostatin profile with a high affinity binding to all five hsst 1-5 . However, AT1S and AT2S show a slightly lower affinity for sst 1 and sst 5 compared with the natural somatostatins, SS14 and SS28.

Sst 2 -and sst 3 -internalization assay
The ability of AT2S to stimulate sst 2 or sst 3 internalization in HEK-sst 2 and HEK-sst 3 cells was analyzed using an immunofluorescent-based internalization assay. Figure 3 illustrates that AT2S exhibits similar agonistic properties as the natural SS14 for sst 2 ( Figure 3A) and for sst 3 ( Figure 3B) in respect of stimulating receptor internalization.

Internalization of radioligands
The internalization properties of the radiopeptides [ 111 In] AT1S and [ 111 In]AT2S were studied in rsst 2A + AR4-2J cells at 37°C with or without excess Tate. The internalization of both analogs was rapid and rsst 2A -mediated, as shown by the significant decrease of internalization levels manifested in the presence of excess Tate ( Figure 4A,B). More than 75% of cell-associated activity internalized within 30 min remaining at this level up to 2 h for both radiopeptides ( Figure 4A). At 120 min, 5% and 10% of the total added radioactivity internalized for [ 111 In]AT1S and [ 111 In]AT2S, respectively, revealing a significantly more efficient internalization process for the DTrp 8 analog ( Figure 4B).

Metabolism in blood
Both [ 111 In]AT1S and [ 111 In]AT2S showed suboptimal stability in the blood stream of healthy mice. As evidenced by HPLC analysis of murine blood collected 5 min after injection of the radioligands, both analogs degraded to at least one major radiometabolite eluting with the solvent front. Traces of additional metabolites having different elution patterns for the two analogs were also observed ( Figure 5A

Biodistribution in healthy mice
Tissue distribution data of [ 111 In]AT1S and [ 111 In]AT2S in healthy male Swiss albino mice for the 4-and 24-h time points are summarized as %ID/g in Figure 6A,B, respectively. Both analogs showed a rapid clearance from blood and background tissues with minimal residual activity in pool organs.  Table 3, as %ID/g at 1 and 4 h pi. Biodistribution data in SCID mice were consistent to the findings of healthy mice tissue distribution with specific uptake shown in the pancreas (5.13%ID/g versus 0.15%ID/g + excess Tate at 4 h pi) and the gastrointestinal tract. Uptake in the rsst 2A + tumor was shown to be specific as well, as suggested by the significantly reduced tumor values found during co-injection of excess Tate (1.82%ID/g at 4 h versus 0.21%ID/g + excess Tate at 4 h pi).

Discussion
The success of OctreoScan W and related cyclic octapeptide sst 2 -seeking radioligands in the diagnosis and treatment of certain human tumors relies both on their high metabolic stability and on the prevalence and high density of sst 2 expression in these tumors [11][12][13][14][15][16][17][18][19]. Soon, it became apparent that sst 2 -mediated internalization of radioligands into cancer cells represents a key element for the success of this strategy. Intracellular accumulation of the radiolabel has translated into higher contrast images and to better tumoricidal responses. On the other hand, recent studies have reported not only on the concomitant expression of at least one alternative sst 1-5 subtype in tumors already expressing the sst 2 , but also in tumors devoid of sst 2 expression [6,7,15,[20][21][22][23][24][25][26]. This finding provides the opportunity to use radiolabeled somatostatin analogs with an extended sst 1-5 affinity profile, which will consequently interact with more binding sites on the tumor than those limited to sst 2 . In this way, the diagnostic and therapeutic indications will be broadened to include more tumor types, while diagnostic sensitivity and therapeutic efficacy will improve. Such 'pansomatostatin-like' radioligands should possess sufficient metabolic stability to be able to reach their target after entry into the bloodstream. At the same time, their capacity to internalize in sst 2 + -cancer cells should not be compromised in order to promote accumulation in most sst 1-5 + -human tumors whereby sst 2 expression is dominant [27][28][29][30]. It is interesting to note that pansomatostatin-like radioligands failing to internalize after binding to sst 2 in vivo indeed showed poor uptake in sst 2 + tissues in mouse models [31,32]. On the other hand, multi-sst affine and well sst 2 -internalizing radioligands, such as radiolabeled DOTA-NOC [33], are expected to miss sst 1 -expressing tumors in patients [23][24][25][26].
The above requirements prompted us to consider the use of native SS14 for radioligand development. It is interesting to note that a SS14-derived radiopeptide, 111 In-[DTPA,DAla 1 ,DTrp 8 ,Tyr 11 ]SS14, was previously studied in healthy mice and compared to OctreoScan W [34]. This analog displayed a pansomatostatin-like profile and showed equivalent to OctreoScan W levels of specific uptake in key target organs, such as the pituitary, the pancreas, and the adrenals, implying that SS14-based radioligands do have opportunities of good sst-targeting in vivo, including the sst 2 . No other information on similar SS14-based radiopeptides is available.
Therefore, we have decided to couple DOTA to the Nterminus of native and non-modified SS14. In this way, AT1S was first generated with the purpose to serve as a lead compound to future structurally modified pansomatostatin-like radiopeptides and as a landmark for their biological evaluation. The universal chelator DOTA has been selected over DTPA with the aim to broaden labeling options beyond 111 In to numerous other medically attractive bi-and trivalent radiometals. Coupling of DOTA on the Ala 1 primary amine of SS14 inadvertently leads to N-terminal capping of the peptide chain as well, a strategy often pursued to increase metabolic stability of peptides. In the second analog, AT2S, Trp 8 was further substituted by DTrp 8 in our AT1S motif to convey additional metabolic stability. This modification is reported to also facilitate the β-turn conformation of several cyclic somatostatin analogs leading to enriched affinity for the sst 2 [35,36].
Both AT1S and AT2S exhibited a pansomatostatin-like in vitro profile, binding to all five sst [1][2][3][4][5] with affinities in the lower nanomolar range. The presence of DOTA at the N-terminus has caused minor affinity losses for all subtypes, which were more pronounced for sst 1 and sst 5 . A similar trend was also observed for [DTPA,DAla 1 , DTrp 8 ,Tyr 11 ]SS14. Of particular interest is the ability of AT2S to induce sst 2 and sst 3 internalization in vitro, as evidenced by immunofluorescence microscopy. This agonistic behavior for both, sst 2 and sst 3 , subtypes is similar to native SS14 as it is elicited at comparable concentration levels (10 nM showed faster internalization of total-added radioactivity as compared with [ 111 In]AT1S. This difference in internalization rates is reflected in dissimilar uptake of the two radioligands in sst 2 + organs after injection in mice (vide infra).
The metabolic fate of [ 111 In]AT1S and [ 111 In]AT2S was followed 5 min after entry in the bloodstream of mice and revealed their susceptibility to enzymatic degradation. [ 111 In]AT1S was almost totally degraded within this period, despite the N-terminal capping conveyed by the 111 In-DOTA moiety, as compared with native SS14. By Trp 8 /DTrp 8 substitution in [ 111 In]AT2S, the percentage of integer radiopeptide increased threefold while the pattern of detected metabolites changed. These differences, albeit small, may have a significant impact on biodistribution in the case where blood clearance and target delivery rates are fast enough to compensate, at least in part, rapid degradation rates. It is interesting to note that after injection in healthy mice, only [ 111 In]AT2S achieves to specifically target sst-binding organs, such as the pancreas, as revealed by co-injection of excess Tate. Pancreatic values remained unchanged from 1 to 24 h pi, whereas renal values substantially declined during this period. In contrast, [ 111 In]AT1S failed to show any measurable specific uptake, most probably as a result of its slower sst 2 -mediated internalization combined with its poorer in vivo stability. Accordingly, further evaluation in tumor-bearing mice was focused on [ 111 In] AT2S.
In mice bearing AR4-2J tumors spontaneously expressing the rat sst 2 , [ 111 In]AT2S showed clear specific uptake both in the experimental tumor and in the gut, including the pancreas, stomach, and intestines, as confirmed by suitable in vivo sst 2 blockade with excess of sst 2 -selective Tate. Similarly high and specific uptake was observed in HEK-hsst 2A + and HEK-sst 3 + tumors at 4 h pi, although the affinity of AT2S for the hsst 2A was slightly higher as for the hsst 3 , and AT2S showed a similar agonistic capacity in triggering the internalization of both subtypes in vitro. On the other hand, [ 111 In]AT2S showed a much lower, although still specific, uptake in the HEK-hsst 5 + implants. This decrease may be attributed to its 10-fold lower affinity for hsst 5 . It should be stressed, however, that individual hsst-expression levels on transfected HEK cells may be different, thereby affecting radioligand uptake.

Conclusions
In summary, native SS14 and its DTrp 8 analog were functionalized with the universal chelator DOTA to allow for labeling with most interesting diagnostic and therapeutic radiometals. The respective AT1S prototype and its DTrp 8 derivative, AT2S, were labeled with 111 In, and several in vitro and in vivo properties of resulting (radio)ligands were investigated. According to the data obtained, both AT1S and AT2S show a pansomatostatinlike affinity profile, and AT2S displays a clear agonistic character for hsst 2   AT2S survived longer in circulation to effectively target physiological somatostatin binding sites, such as the pancreas. Likewise, [ 111 In]AT2S specifically localized in experimental tumors in SCID mice which selectively expressed one of sst 2 (both of rat and human origin), hsst 3 , or hsst 5 . To our knowledge, this is the first comprehensive study that systematically explores strengths and weaknesses of employing native SS14-derived radioligands for nuclear oncology applications. It has demonstrated that the AT1S lead structure is promising for radioligand development owing to its pansomatostatin character and its preserved agonistic properties, especially regarding sst 2 internalization. Furthermore, it has revealed the feasibility of structural modifications to enhance metabolic stability in order to achieve higher tumor uptake. The body of data so far acquired will serve as a landmark in the evaluation of innovative structural interventions on the AT1S lead structure, such as key amino acid replacements and/or changes of ring size, which are currently pursued.