Mihaela Ginj

Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH2-CO-c(DCys-Phe-Tyr-DAgl8(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst3-ODN-8) and a sst2-selective antagonist (sst2-ANT)], for labeling with 111/natIn. 111/natIn-DOTA-sst3-ODN-8 and 111/natIn-DOTA–[4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2] (111/natIn-DOTA-sst2-ANT) showed high sst3- and sst2-binding affinity, respectively. They did not trigger sst3 or sst2 internalization but prevented agonist-stimulated internalization. 111In-DOTA-sst3-ODN-8 and 111In-DOTA-sst2-ANT were injected intravenously into mice bearing sst3- and sst2-expressing tumors, and their biodistribution was monitored. In the sst3-expressing tumors, strong accumulation of 111In-DOTA-sst3-ODN-8 was observed, peaking at 1 h with 60% injected radioactivity per gram of tissue and remaining at a high level for >72 h. Excess of sst3-ODN-8 blocked uptake. As a control, the potent agonist 111In-DOTA–[1-Nal3]-octreotide, with strong sst3-binding and internalization properties showed a much lower and shorter-lasting uptake in sst3-expressing tumors. Similarly, 111In-DOTA-sst2-ANT was injected into mice bearing sst2-expressing tumors. Tumor uptake was considerably higher than with the highly potent sst2-selective agonist 111In-diethylenetriaminepentaacetic acid–[Tyr3,Thr8]-octreotide (111In-DTPA-TATE). Scatchard plots showed that antagonists labeled many more sites than agonists. Somatostatin antagonist radiotracers therefore are preferable over agonists for the in vivo targeting of sst3- or sst2-expressing tumors. Antagonist radioligands for other peptide receptors need to be evaluated in nuclear oncology as a result of this paradigm shift.

68Ga-DOTANOC: a first compound for PET imaging with high affinity for somatostatin receptor subtypes 2 and 5

Existing somatostatin-based radiotracers (e.g. In-DOTA TOC) have sole affinity for somatostatin receptor subtype 2 (sst2). This represents a drawback, given that sst1–5 have been shown to be over-expressed in different tumours, alone or concomitantly [1]. Our goal, therefore, was to develop radiopeptides with broader receptor subtype profiles. Ga-DOTANOC is a first compound for PET imaging with high affinity for sst2 and sst5 [2]. Its affinity profile (IC50 nM) for human sst1–5 is, respectively, >10,000, 1.9± 0.4, 40±5.8, 260±74 and 7.2±1.6. For comparison, the values for the standard compound, In-DOTATOC, are >10,000, 4.6±0.2, 120±26, 230±82 and 130±17. Here we present the 60 min p.i. Ga-DOTANOC PET images and the 21 h p.i. In-DOTATOC planar images of a 52-year-old patient with an advanced neuroendocrine tumour. The two examinations were performed within 4 weeks. During this time interval the patient received bisphosphonates. Preparation and application of Ga-DOTANOC PET and Ga-DOTATOC PET are comparable [3]. In the reported case study, the Ga-DOTANOC PET scan shows high radioligand uptake in the liver and bone metastases. Although many bone metastases appeared visually similar in the two scans, the right sixth rib and left occipital bone metastases (arrows) are much more visible on the Ga-DOTANOC PET scan. This selective difference cannot be explained simply by the advantages of the PET technique. The possible predominance of sst5 in these two bone metastases and the high sst5 affinity of GaDOTANOC are in fact the probable reasons for the high Ga-DOTANOC and low In-DOTATOC uptake. The enlarged liver and somatostatin receptor-positive organs such as the spleen (high uptake) and pituitary gland and thyroid (moderate uptake) are also visible. These normal organs, known to express more sst than just sst2, are better visualised with Ga-DOTANOC (see in particular the spleen). We conclude that Ga-DOTANOC is an excellent candidate for primary diagnostic and follow-up investigations in patients with suspected or proven somatostatin receptorpositive tumours. Furthermore, in this case, predictive imaging indicates that Yor Lu-DOTANOC has greater potential for treatment of this patient than Yor LuDOTATOC.

Pasireotide and Octreotide Stimulate Distinct Patterns of sst2A Somatostatin Receptor Phosphorylation

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushings disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.

New Pansomatostatin Ligands and Their Chelated Versions: Affinity Profile, Agonist Activity, Internalization, and Tumor Targeting

Purpose: Somatostatin receptor (sst) targeting is an established method to image and treat sst-positive tumors. Particularly, neuroendocrine tumors express the receptor subtype 2 in high density, but sst1, sst3, sst4, and sst5 are also expressed to some extent in different human tumors. Currently used targeting peptides mainly have sst2 affinity. We aimed at developing (radio)peptides that bind with high affinity to all receptor subtypes. Experimental Design: Carbocyclic octapeptides were coupled with macrocyclic chelators for radiometal labeling. Affinity, internalization, and agonist potencies were determined on sst1- to sst5-expressing cell lines. Biodistribution was determined on nude mice bearing HEK-sst2 or AR4-2J and HEK-sst3 tumors. Results: High affinity to all receptor subtypes was found. YIII-KE88 showed agonistic properties at all five sst receptor subtypes as it inhibits forskolin-stimulated cyclic AMP production. Surprisingly, very low or even absent sst2 receptor internalization was found compared with currently clinically established octapeptides, whereas the sst3 internalization was very efficient. Biodistribution studies of [111In]KE88 and [67Ga]KE88/[68Ga]KE88 reflected the in vitro data. In nude mice with s.c. implanted sst2 (HEK-sst2, AR4-2J)-expressing and sst3 (HEK-sst3)-expressing tumors, high and persistent uptake was found in sst3-expressing tumors, whereas the uptake in the sst2-expressing tumors was lower and showed fast washout. The kidney uptake was high but blockable by coinjection of lysine. Conclusion: This peptide family shows pansomatostatin potency. As radiopeptides, they are the first to show a full pansomatostatin profile. Despite some drawback, they should be useful for imaging sst2-expressing tumors with short-lived radiometals, such as 68Ga, at early time points and for sst3-expressing tumors at later time points with longer-lived radiometals, such as 64Cu or 86Y.