Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5μg/kg dose administered 1h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P<0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y(1), a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y(1) receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y(1) found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells.

Radiolabeled bicyclic somatostatin-based analogs: a novel class of potential radiotracers for SPECT/PET of neuroendocrine tumors

A variety of radiolabeled somatostatin analogs have been developed for targeting of somatostatin receptor (sst)-positive tumors. Bicyclic somatostatin-based radiopeptides have not been studied yet. Hypothesizing that the introduction of conformational constraints may lead to receptor subtype selectivity or may help to delineate structural features determining pansomatostatin potency, we developed and evaluated first examples of this new class of potential radiotracers for imaging or therapy of neuroendocrine tumors.

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as (111)In and (68)Ga.

Absence of somatostatin SST(2) receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model

Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst(2)) internalization is a potent mechanism for tumor targeting with sst(2) affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst(2) internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst(2) receptors were injected intravenously with SOM230 or with the [Tyr(3), Thr(8)]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst(2) receptors. SOM230-induced sst(2) internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst(2) internalization triggered by intravenous [Tyr(3), Thr(8)]-octreotide, intravenous SOM230 did not elicit sst(2) internalization: immunohistochemically stained sst(2) in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5min, 10min, 1h, 6h, or 24h after SOM230 injection while sst(2) were found intracellularly after [Tyr(3), Thr(8)]-octreotide injection. The inability of stimulating sst(2) internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst(2) internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst(2) internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst(2) binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230.

Switch from antagonist to agonist after addition of a DOTA chelator to a somatostatin analog

Peptide receptor targeting has become an increasingly attractive method to target tumors diagnostically and radiotherapeutically. Peptides linked to a variety of chelators have been developed for this purpose. They have, however, rarely been tested for their agonistic or antagonistic properties. We report here on a somatostatin antagonist that switched to an agonist upon coupling to a DOTA chelator.

Tetraamine-derived bifunctional chelators for technetium-99m labelling: synthesis, bioconjugation and evaluation as targeted SPECT imaging probes for GRP-receptor-positive tumours

Owing to its optimal nuclear properties, ready availability, low cost and favourable dosimetry, (99m)Tc continues to be the ideal radioisotope for medical-imaging applications. Bifunctional chelators based on a tetraamine framework exhibit facile complexation with Tc(V)O(2) to form monocationic species with high in vivo stability and significant hydrophilicity, which leads to favourable pharmacokinetics. The synthesis of a series of 1,4,8,11-tetraazaundecane derivatives (01-06) containing different functional groups at the 6-position for the conjugation of biomolecules and subsequent labelling with (99m)Tc is described herein. The chelator 01 was used as a starting material for the facile synthesis of chelators functionalised with OH (02), N(3) (04) and O-succinyl ester (05) groups. A straightforward and easy synthesis of carboxyl-functionalised tetraamine-based chelator 06 was achieved by using inexpensive and commercially available starting materials. Conjugation of 06 to a potent bombesin-antagonist peptide and subsequent labelling with (99m)Tc afforded the radiotracer (99m)Tc-N4-BB-ANT, with radiolabelling yields of >97% at a specific activity of 37 GBq micromol(-1). An IC(50) value of (3.7+/-1.3) nM was obtained, which confirmed the high affinity of the conjugate to the gastrin-releasing-peptide receptor (GRPr). Immunofluorescence and calcium mobilisation assays confirmed the strong antagonist properties of the conjugate. In vivo pharmacokinetic studies of (99m)Tc-N4-BB-ANT showed high and specific uptake in PC3 xenografts and in other GRPr-positive organs. The tumour uptake was (22.5+/-2.6)% injected activity per gram (% IA g(-1)) at 1 h post injection (p.i.). and increased to (29.9+/-4.0)% IA g(-1) at 4 h p.i. The SPECT/computed tomography (CT) images showed high tumour uptake, clear background and negligible radioactivity in the abdomen. The promising preclinical results of (99m)Tc-N4-BB-ANT warrant its potential candidature for clinical translation.

Agonist-biased signaling at the sst2A receptor: the multi-somatostatin analogs KE108 and SOM230 activate and antagonize distinct signaling pathways

Somatostatin analogs that activate the somatostatin subtype 2A (sst2A) receptor are used to treat neuroendocrine cancers because they inhibit tumor secretion and growth. Recently, new analogs capable of activating multiple somatostatin receptor subtypes have been developed to increase tumor responsiveness. We tested two such multi-somatostatin analogs for functional selectivity at the sst2A receptor: SOM230, which activates sst1, sst2, sst3, and sst5 receptors, and KE108, which activates all sst receptor subtypes. Both compounds are reported to act as full agonists at their target sst receptors. In sst2A-expressing HEK293 cells, somatostatin inhibited cAMP production, stimulated intracellular calcium accumulation, and increased ERK phosphorylation. SOM230 and KE108 were also potent inhibitors of cAMP accumulation, as expected. However, they antagonized somatostatin stimulation of intracellular calcium and behaved as partial agonists/antagonists for ERK phosphorylation. In pancreatic AR42J cells, which express sst2A receptors endogenously, SOM230 and KE108 were both full agonists for cAMP inhibition. However, although somatostatin increased intracellular calcium and ERK phosphorylation, SOM230 and KE108 again antagonized these effects. Distinct mechanisms were involved in sst2A receptor signaling in AR42J cells; pertussis toxin pretreatment blocked somatostatin inhibition of cAMP accumulation but not the stimulation of intracellular calcium and ERK phosphorylation. Our results demonstrate that SOM230 and KE108 behave as agonists for inhibition of adenylyl cyclase but antagonize somatostatin's actions on intracellular calcium and ERK phosphorylation. Thus, SOM230 and KE108 are not somatostatin mimics, and their functional selectivity at sst2A receptors must be considered in clinical applications where it may have important consequences for therapy.

Glucagonlike peptide-1 receptor: an example of translational research in insulinomas: a review

Glucagonlike peptide-1 receptors (GLP-1R) play an increasingly important role in endocrine gastrointestinal tumor management. In particular, virtually all benign insulinomas express GLP-1R in high density. Exendin-4 is a GLP-1 analog that has a longer half-life than GLP-1. Targeting GLP-1R by (111)In-DOTA-exendin-4 or (111)In-DPTA-exendin-4 offers a new approach that permits the successful localization of small benign insulinomas. It is likely that this new noninvasive technique has the potential to replace the invasive localization by selective arterial stimulation and venous sampling.

Distribution pattern of 68Ga-DOTATATE in disease-free patients

68Ga-DOTA-DPhe1,Tyr3-octreotate (68Ga-DOTATATE) is a somatostatin analogue that shows high affinity for somatostatin receptor subtype 2 (sst2) and has been used for imaging neuroendocrine tumours. However, normal uptake patterns and potential pitfalls have not been described with this high-sensitivity radiotracer. The aim of this study was therefore to outline the normal distribution pattern of 68Ga-DOTATATE in disease-free patients, to provide standardized uptake values (SUVs) of various organs and to compare our results with the current knowledge on sst2 receptor expression in vitro.

Novel octreotide dicarba-analogues with high affinity and different selectivity for somatostatin receptors

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

Role of somatostatins in gastroenteropancreatic neuroendocrine tumor development and therapy

The incidence and prevalence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have increased in the past 20 years. GEP-NETs are heterogeneous tumors, in terms of clinical and biological features, that originate from the pancreas or the intestinal tract. Some GEP-NETs grow very slowly, some grow rapidly and do not cause symptoms, and others cause hormone hypersecretion and associated symptoms. Most GEP-NETs overexpress receptors for somatostatins. Somatostatins inhibit the release of many hormones and other secretory proteins; their effects are mediated by G protein-coupled receptors that are expressed in a tissue-specific manner. Most GEP-NETs overexpress the somatostatin receptor SSTR2; somatostatin analogues are the best therapeutic option for functional neuroendocrine tumors because they reduce hormone-related symptoms and also have antitumor effects. Long-acting formulations of somatostatin analogues stabilize tumor growth over long periods. The development of radioactive analogues for imaging and peptide receptor radiotherapy has improved the management of GEP-NETs. Peptide receptor radiotherapy has significant antitumor effects, increasing overall survival times of patients with tumors that express a high density of SSTRs, particularly SSTR2 and SSTR5. The multi-receptor somatostatin analogue SOM230 (pasireotide) and chimeric molecules that bind SSTR2 and the dopamine receptor D2 are also being developed to treat patients with GEP-NETs. Combinations of radioactive labeled and unlabeled somatostatin analogues and therapeutics that inhibit other signaling pathways, such as mammalian target of rapamycin (mTOR) and vascular endothelial growth factor, might be the most effective therapeutics for GEP-NETs.

One-Step (18)F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac?AcNH-?-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/?mol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC?? = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-?-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹?F]SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

Nerve root sedimentation sign: evaluation of a new radiological sign in lumbar spinal stenosis

Retrospective case-referent study.

Desmosomal gene analysis in arrhythmogenic right ventricular dysplasia/cardiomyopathy: spectrum of mutations and clinical impact in practice

Five desmosomal genes have been recently implicated in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) but the clinical impact of genetics remains poorly understood. We wanted to address the potential impact of genotyping.