Selective Somatostatin 5 (SST5) and Somatostatin 2 (SST2) Nonpeptide Agonists Potently Suppress Glucose- and Tolbutamide-Stimulated Dynamic Insulin Secretion From Isolated Human Islets

Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in newborns and infants and arises from dysregulated insulin secretion. Rapid recognition and treatment are vital to prevent seizures, permanent developmental delays, coma, or even death. Very few medical options exist to treat congenital HI patients: the KATP channel activator diazoxide, the injectable somatostatin receptor peptide agonists octreotide and lanreotide, or chronic glucose infusions. However, side effects and/or limited efficacy render these therapies inadequate for many patients. Somatostatin is a 14-amino acid peptide hormone with a broad spectrum of biological actions, which are regulated through five somatostatin receptor subtypes (SST1-SST5). Somatostatin’s common physiological role is to down-regulate secretion of other hormones in various tissues. Its role in the maintenance of euglycemia is to regulate insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Somatostatin regulates insulin secretion by decreasing the intracellular levels of cAMP, inhibition of voltage-gated calcium channels (VGCC), activation of the G protein-activated inward rectifier K+ channel (GIRK), and direct inhibition of insulin exocytosis. Several studies have evaluated the effect of somatostatin, somatostatin peptide analogs, and a limited number of nonpeptide somatostatin receptor agonists on insulin secretion in static assays using isolated human islets. However, the lack of highly selective agonists has made the interpretation of the contribution of SST receptor sub-types difficult to discern. Our programs for the treatment of hyperinsulinism, acromegaly, and other indications have led to the development of selective nonpeptide SST2, SST3, SST4, and SST5 agonists, possessing EC50s < 1 nM in cell-based assays of receptor activation and selectivity > 130 times over the other members of the family. The ability of these selective nonpeptide agonists to regulate glucose- and tolbutamide-stimulated dynamic insulin secretion from human islets was evaluated using a perifusion system (Biorep, FL). We found that selective SST2 and SST5 agonists potently suppressed dynamic insulin secretion in contrast to SST3 or SST4 selective agonists. Importantly, SST5 agonists were shown to have a greater effect than selective SST2 agonists or diazoxide, demonstrating their potential utility in human conditions such as congenital HI. In addition, SST5 activation is also known to have a smaller effect on glucagon secretion and is also less prone to agonist-driven desensitization than SST2 activation. Taken together, these studies support our program to identify, characterize, and develop potent, nonpeptide, orally-bioavailable, selective SST5 agonists with appropriate pharmaceutical and safety characteristics for the treatment of congenital HI.

Expression of Somatostatin Receptor Subtypes (SSTR-1–SSTR-5) in Pediatric Hematological and Oncological Disorders

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1–5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.

Evaluation of a New 177Lu-Labeled Somatostatin Analog for the Treatment of Tumors Expressing Somatostatin Receptor Subtypes 2 and 5

Targeted radionuclide therapy of somatostatin receptor (SST)-expressing tumors is only partially addressed by the established somatostatin analogs having an affinity for the SST subtype 2 (SST2). Aiming to target a broader spectrum of tumors, we evaluated the bis-iodo-substituted somatostatin analog ST8950 ((4-amino-3-iodo)-d-Phe-c[Cys-(3-iodo)-Tyr-d-Trp-Lys-Val-Cys]-Thr-NH2), having subnanomolar affinity for SST2 and SST5, labeled with [177Lu]Lu3+ via the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Human Embryonic Kidney (HEK) cells stably transfected with the human SST2 (HEK-SST2) and SST5 (HEK-SST5) were used for in vitro and in vivo evaluation on a dual SST2- and SST5-expressing xenografted mouse model. natLu-DOTA-ST8950 showed nanomolar affinity for both subtypes (IC50 (95% confidence interval): 0.37 (0.22–0.65) nM for SST2 and 3.4 (2.3–5.2) for SST5). The biodistribution of [177Lu]Lu-DOTA-ST8950 was influenced by the injected mass, with 100 pmol demonstrating lower background activity than 10 pmol. [177Lu]Lu-DOTA-ST8950 reached its maximal uptake on SST2- and SST5-tumors at 1 h p.i. (14.17 ± 1.78 and 1.78 ± 0.35%IA/g, respectively), remaining unchanged 4 h p.i., with a mean residence time of 8.6 and 0.79 h, respectively. Overall, [177Lu]Lu-DOTA-ST8950 targets SST2-, SST5-expressing tumors in vivo to a lower extent, and has an effective dose similar to clinically used radiolabeled somatostatin analogs. Its main drawbacks are the low uptake in SST5-tumors and the persistent kidney uptake.

Somatostatin receptor subtypes 1 and 4 redundantly regulate neprilysin, the major amyloid-beta degrading enzyme in brain

Alzheimer’s disease (AD) brains are characterized by increased levels of the pathogenic amyloid beta (Aβ) peptide, which accumulates into extracellular plaques. Finding a way to lower Aβ levels is fundamental for the prevention and treatment of AD. Neprilysin is the major Aβ degrading enzyme which is regulated by the neuropeptide somatostatin. Here we used a combination of in vitro and in vivo approaches to identify the subtype specificity of the five somatostatin receptors (SSTs) expressed in the brain, involved in the regulation of neprilysin. Using a battery of Sst double knockout (dKO) mice we show that neprilysin is regulated by SST1 and SST4 in a redundant manner. Sst1 and Sst4 dKO mice exhibit a specific decrease of presynaptic neprilysin in the Lacunosum molecular layer. Moreover, a genetic deficiency of Sst1 and Sst4 in amyloid beta precursor protein (App) knock-in mice, an AD mouse model, aggravates the Aβ pathology in the hippocampus. As a first proof of concept towards an Aβ-lowering strategy involving neprilysin, we demonstrate that treatment with an agonist selective for SST1 and SST4 ameliorates the Aβ pathology and improves cognition in the App knock-in AD mouse model.

SAT-305 Veldoreotide (COR005) Mechanisms of Action Studies: Comparison to Octreotide and Pasireotide

Stable somatostatin analogs (SSAs) are the first choice for medical treatment of pituitary adenomas and other neuroendocrine tumors. The somatostatin analogs octreotide, pasireotide, and veldoreotide primarily have been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (SSTs) has not been directly assessed so far. In this study, we assessed G-protein signaling in human embryonic kidney (HEK293) cells stably expressing G protein-coupled inwardly rectifying potassium (GIRK) channels and SSTs using a novel fluorescence-based membrane potential assay. Dose-response curves obtained for veldoreotide revealed high potency and efficacy in cells expressing SST2, SST4, and SST5. Veldoreotide also inhibited proliferation and chromogranin A secretion in SST4-transfected BON-1 cells. In addition, we assessed G-protein signaling in primary pituitary cultures from SST2 and SST5 knockout mice. Our results show that octreotide mediates its effects selectively via the SST2 receptor. Conversely, pasireotide mediates its effects selectively via the SST5 receptor. In contrast, veldoreotide can activate both SST2 and SST5 receptors under otherwise identical conditions. Thus, veldoreotide is a unique SSA with full agonistic activity at the SST2, SST4, and SST5 receptors.

Somatostatin receptor expression in parathyroid neoplasms

Introduction Parathyroid carcinoma represents a rare cause of primary hyperparathyroidism. Distinguishing carcinoma from the benign tumors underlying primary hyperparathyroidism remains challenging. The diagnostic criteria for parathyroid carcinoma are local and/or metastatic spreading. Atypical parathyroid adenomas share other histological features with carcinomas but lack invasive growth. Somatostatin receptors are commonly expressed in different neuroendocrine tumors, but whether this also holds for parathyroid tumors remains unknown. Aim Our aim is to examine the immunohistochemical expression of somatostatin receptor 1–5 in parathyroid typical adenomas, atypical adenomas and carcinomas. Methods We used a tissue microarray construct from a nationwide cohort of parathyroid carcinomas (n = 32), age- and gender-matched typical parathyroid adenomas (n = 72) and atypical parathyroid adenomas (n = 27) for immunohistochemistry of somatostatin receptor subtypes 1–5. We separately assessed cytoplasmic, membrane and nuclear expression and also investigated the associations with histological, biochemical and clinical characteristics. Results All parathyroid tumor subgroups expressed somatostatin receptors, although membrane expression appeared negligible. Except for somatostatin receptor 1, expression patterns differed between the three tumor types. Adenomas exhibited the weakest and carcinomas the strongest expression of somatostatin receptor 2, 3, 4 and 5. We observed the largest difference for cytoplasmic somatostatin receptor 5 expression. Conclusions Parathyroid adenomas, atypical adenomas and carcinomas all express somatostatin receptor subtypes 1–5. Somatostatin receptor 5 may serve as a potential tumor marker for malignancy. Studies exploring the role of somatostatin receptor imaging and receptor-specific therapies in patients with parathyroid carcinomas are needed.

Immunohistochemical Expression of Somatostatin Receptor Subtypes in a Panel of Neuroendocrine Neoplasias

Neuroendocrine neoplasias (NENs) are known to express somatostatin receptors (SSTRs) 1–5, which are G-protein-coupled cell membrane receptors. Somatostatin receptor imaging and therapy utilizes the SSTR expression. Synthetic somatostatin analogs with radioligands are used to detect primary tumors, metastases, and recurrent disease. Receptor analogs are also used for treating NENs. Furthermore, commercially available SSTR antibodies can be used for the immunohistochemical (IHC) detection of SSTRs. We investigated different SSTR antibody clones applying diverse IHC protocol settings to identify reliable clones and feasible protocols for NENs. A tissue microarray including NENs from 12 different primary sites were stained. Only UMB clones were able to localize SSTR on the cell membranes of NENs. SSTR2 (UMB1) emerged as the most common subtype followed by SSTR5 (UMB4) and SSTR1 (UMB7). SSTR3 (UMB5) expression was mainly cytoplasmic. Yet, SSTR4 expression was weak and located primarily in the cytoplasm. Thus, appropriate IHC protocols, including proper positive and negative controls, represent requirements for high-quality NEN diagnostics and for planning personalized therapy.