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‐
3]. Somatostatin receptors comprise five subtypes (sst
1-5) and are also found in many human tumors where they are expressed alone or in various combinations [
4‐
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
1-5, 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].
111In-DTPA]octreotide (OctreoScan®) is the first approved sst
2-avid peptide radiopharmaceutical. When administered in patients, it localizes in primary and metastatic sst
2
+-lesions, which can be efficiently visualized with the aid of an external imaging device [
14]. Following OctreoScan®, several other sst
2-seeking radiopeptides, suitable for SPECT (
99mTc-,
111In-,
67 Ga-labeled), PET (
68 Ga-,
64Cu-labeled), or radionuclide therapy (
90Y- or
177Lu-labeled) have been evaluated in animal models and in patients with neuroendocrine tumors (NETs) [
15‐
19].
In all above instances, the sst2 subtype prevails in incidence and density of expression allowing the successful application of sst2-preferring radioligands. However, it should be stressed that, despite the predominance of sst2 expression in many human tumors, co-expression of sst2 with other sst1-5 subtypes is frequent enough. Thus, sst2 and sst5 are expressed often together in GH-secreting pituitary adenomas, and various combinations of ssts, such as sst2 and sst1, are expressed in gastroenteropancreatic (GEP)-NETs.
Moreover, a number of human tumors devoid of sst
2 may instead express one or more of the other sst
1-5[
6,
7,
15,
20‐
22]. For example, ductal pancreatic carcinomas or primary hormone-sensitive prostate cancers are reported to often express sst
1[
23‐
26]. Hence, the use of pansomatostatin-like agents will broaden the clinical indications and will increase the diagnostic/therapeutic efficacy of currently available sst
2-preferring (radio)peptides.
SOM230 and KE108 are two multi-somatostatin receptor ligands that have been developed to improve somatostatin analog-based therapy. SOM230 has high affinity for sst
1-3 and sst
5[
27], while KE108 has high affinity for all five sst
1-5[
28]. However, the absence of sst
2 internalization may turn out to be a serious disadvantage of SOM230- or KE108-based radioligands compromising their accumulation in target cells, in the most frequent cases where sst
2 expression prevails [
29‐
32]. On the other hand, well sst
2-internalizing and multi-sst
2-/sst
3-/sst
5-binding analogs, such as DOTA-NOC [
33], will miss sst
1-expressing tumors. Thus, a pansomatostatin affinity profile and the preservation of important pharmacological traits, especially sst
2 internalization, seem to represent an advantageous combination for enhancing the efficacy of sst
1-5-targeting radioligands.
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,
111In-[DTPA,DAla
1,DTrp
8,Tyr
11SS14 showed specific and comparable to OctreoScan® 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
111In 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
d Trp
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
1-5-expressing cells, metabolic studies, and biodistribution of
111In-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.
Discussion
The success of OctreoScan® 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‐
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‐
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‐
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‐
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,
111In-[DTPA,DAla
1,DTrp
8,Tyr
11SS14, was previously studied in healthy mice and compared to OctreoScan® [
34]. This analog displayed a pansomatostatin-like profile and showed equivalent to OctreoScan® 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 N-terminus 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
111In 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 sst1-5 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 sst1 and sst5. A similar trend was also observed for [DTPA,DAla1,DTrp8,Tyr11]SS14. Of particular interest is the ability of AT2S to induce sst2 and sst3 internalization in vitro, as evidenced by immunofluorescence microscopy. This agonistic behavior for both, sst2 and sst3, subtypes is similar to native SS14 as it is elicited at comparable concentration levels (≈10 nM). In agreement to this finding, [111In]AT1S and [111In]AT2S internalized in AR4-2J cells by a sst2-mediated process. Within 30 min at 37 °C, ≈80% of cell bound activity was found within the cells. It is interesting to note that [111In]AT2S showed faster internalization of total-added radioactivity as compared with [111In]AT1S. This difference in internalization rates is reflected in dissimilar uptake of the two radioligands in sst2
+ organs after injection in mice (vide infra).
The metabolic fate of [111In]AT1S and [111In]AT2S was followed 5 min after entry in the bloodstream of mice and revealed their susceptibility to enzymatic degradation. [111In]AT1S was almost totally degraded within this period, despite the N-terminal capping conveyed by the 111In-DOTA moiety, as compared with native SS14. By Trp8/DTrp8 substitution in [111In]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 [111In]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, [111In]AT1S failed to show any measurable specific uptake, most probably as a result of its slower sst2-mediated internalization combined with its poorer in vivo stability. Accordingly, further evaluation in tumor-bearing mice was focused on [111In]AT2S.
In mice bearing AR4-2J tumors spontaneously expressing the rat sst2, [111In]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 sst2 blockade with excess of sst2-selective Tate. Similarly high and specific uptake was observed in HEK-hsst2A
+ and HEK-sst3
+ tumors at 4 h pi, although the affinity of AT2S for the hsst2A was slightly higher as for the hsst3, and AT2S showed a similar agonistic capacity in triggering the internalization of both subtypes in vitro. On the other hand, [111In]AT2S showed a much lower, although still specific, uptake in the HEK-hsst5
+ implants. This decrease may be attributed to its ≈ 10-fold lower affinity for hsst5. 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 DTrp8 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 DTrp8 derivative, AT2S, were labeled with 111In, 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 pansomatostatin-like affinity profile, and AT2S displays a clear agonistic character for hsst2 and hsst3
in vitro. In contrast with previously reported pansomatostatin-like radioligands showing poor sst2-related internalization, [111In]AT1S and [111In]AT2S do internalize in AR4-2J cells via a sst2-mediated mechanism. This parameter is promising for in vivo application, and it was more pronounced for [111In]AT2S. Furthermore, after injection in mice, [111In]AT2S survived longer in circulation to effectively target physiological somatostatin binding sites, such as the pancreas. Likewise, [111In]AT2S specifically localized in experimental tumors in SCID mice which selectively expressed one of sst2 (both of rat and human origin), hsst3, or hsst5. 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 sst2 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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AT was actively engaged in peptide synthesis, radiolabeling, and biological evaluation and assisted in writing the manuscript (ms). TM performed animal studies and drafted most parts of the ms. RC, BW, and JCR were engaged in sst1-5 affinity profile determination of AT1/2 S and sst2/sst3 internalization studies and drafted the corresponding ms sections. PC supervised peptide synthesis and participated in the design of analogs. EPK, MdJ, and JCR edited the ms. BAN designed the overall study and supervised radiochemical work, as well as the generation and final editing of this ms. All authors read and approved the final manuscript.