The success of OctreoScan® and related cyclic octapeptide sst2-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 sst2 expression in these tumors [11–19]. Soon, it became apparent that sst2-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 sst1-5 subtype in tumors already expressing the sst2, but also in tumors devoid of sst2 expression [6, 7, 15, 20–26]. This finding provides the opportunity to use radiolabeled somatostatin analogs with an extended sst1-5 affinity profile, which will consequently interact with more binding sites on the tumor than those limited to sst2. 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 sst2
+-cancer cells should not be compromised in order to promote accumulation in most sst1-5
+-human tumors whereby sst2 expression is dominant [27–30]. It is interesting to note that pansomatostatin-like radioligands failing to internalize after binding to sst2
in vivo indeed showed poor uptake in sst2
+ tissues in mouse models [31, 32]. On the other hand, multi-sst affine and well sst2-internalizing radioligands, such as radiolabeled DOTA-NOC , are expected to miss sst1-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,DAla1,DTrp8,Tyr11SS14, was previously studied in healthy mice and compared to OctreoScan® . 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 sst2. 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 Ala1 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, Trp8 was further substituted by DTrp8 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 sst2[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.