Gastrin-releasing Peptide Receptor Research Articles

The Effects of the Spacer on Radiochemical and Biological Properties of New Radiolabeled Bombesin(7-14) Derivative.

The aim of this study was to develop 99mTc-[HYNIC-X-D-Phe13]-BBN(7-14)NH2 derivatives using two different tripeptidic spacer groups (X=GGG and X=SSS) in order to improve its pharmacokinetics, in vitro stability, specific binding, and affinity. Bombesin (BBN), a 14-aminoacid amphibian peptide homolog of mammalian gastrinreleasing peptide (GRP), has demonstrated the ability to bind with high affinity and specificity to GRP receptor, which is overexpressed on a variety of human cancers. Peptide conjugates labeled with 99mTc using tricine-EDDA and radiochemical purity was assessed by TLC and HPLC. The stability of radio conjugates was evaluated in the presence of saline and human serum. Affinity, internalization, and also dissociation Constant was evaluated using MDAMB- 231 and PC-3 cell line. Biodistribution study was performed in BALB/C mice. Labeling yield of ˃95% was obtained. The change introduced in the BBN sequence increased plasma stability. In vitro blocking studies showed that binding and internalization of both radiolabeled peptides are mediated by their receptors on the surface of MDA-MB-231 and PC-3 cells. Biodistribution results demonstrated a rapid blood clearance, with predominantly renal excretion. Specific binding in GRP receptor-positive tissues, such as pancreas was confirmed with a blocking study. The introduction of the spacer sequence between chelator and BBN(7-14) led to improved bidistribution. Analog with tri-Gly spacer is the more promising radiopeptide for targeting GRP receptors than Ser conjugates. Therefore, these analogs can be considered as a candidate for the identification of bombesin-positive tumors.

Nanoparticles for Targeting of Prostate Cancer.

Prostate cancer (PCa) is the leading cause of death by cancer in men. Because of the drastic decline in the survival rate of PCa patients with advanced/metastatic disease, early diagnosis of disease and therapy without toxic side effects is crucial. Chemotherapy is widely used to control the progression of PCa at the later stages; however, it is associated with off-target toxicities and severe adverse effects due to the lack of specificity. Delivery of therapeutic or diagnostic agents by using targeted nanoparticles is a promising strategy to enhance accuracy and sensitivity of diagnosis of PCa and to increase efficacy and specificity of therapeutic agents. Numerous efforts have been made in past decades to create nanoparticles with different architectural bases for specific delivery payloads to prostate tumors. Major PCa associated cell membrane protein markers identified as targets for such purposes include folate receptor, sigma receptors, transferrin receptor, gastrin-releasing peptide receptor, urokinase plasminogen activator receptor, and prostate specific membrane antigen. Among these markers, prostate specific membrane antigen has emerged as an extremely specific and sensitive targetable marker for designing targeted nanoparticle-based delivery systems for PCa. In this article, we review contemporary advances in design, specificity, and efficacy of nanoparticles functionalized against PCa. Whenever feasible, both diagnostic as well as therapeutic applications are discussed.

Gastrin releasing peptide receptor targeted nano-graphene oxide for near-infrared fluorescence imaging of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor that occurs in the oral mucosa. Pathological biopsy is still the current gold standard for OSCC diagnosis; however, some drawbacks need to be overcome. Therefore, it is urgently needed to find a non-invasive targeted technology for OSCC early diagnosis. Fluorescent optical imaging using near infrared (NIR) dyes tagged to tumor specific target will benefit such developments. Gastrin releasing peptide receptor (GRPR) is an attractive target for OSCC imaging and therapy. In this study, we synthesized nano-graphene oxide (NGO) nanoparticles with GRPR-specific peptides AF750-6Ahx-Sta-BBN via hydrogen bond and π–π bonds (NGO-BBN-AF750), and investigated their receptor binding, cell uptake and internalization in HSC-3 cells. NGO-BBN-AF750 and AF750-6Ahx-Sta-BBN showed a similar binding affinity to GRPR on HSC-3 cells. In contrast to AF750-6Ahx-Sta-BBN antagonist peptide, NGO-BBN-AF750 showed cellular internalization property. Overall, this study proposes a NGO nanoclusters-based nanoprobe for GRPR targeted near-infrared fluorescence imaging for OSCC. Nanoparticle-based delivery systems have shown highly significant potential in the delivery of a wide range of therapeutic agents.

Heterodimeric Radiotracer Targeting PSMA and GRPR for Imaging of Prostate Cancer-Optimization of the Affinity towards PSMA by Linker Modification in Murine Model.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are promising targets for molecular imaging of prostate cancer (PCa) lesions. Due to the heterogenic overexpression of PSMA and GRPR in PCa, a heterodimeric radiotracer with the ability to bind to both targets could be beneficial. Recently, our group reported the novel heterodimer BQ7800 consisting of a urea-based PSMA inhibitor, the peptide-based GRPR antagonist RM26 and NOTA chelator. The study reported herein, aimed to improve the affinity of BQ7800 towards PSMA by changing the composition of the two linkers connecting the PSMA- and GRPR-targeting motifs. Three novel heterodimeric analogues were synthesized by incorporation of phenylalanine in the functional linker of the PSMA-binding motif and/or shortening the PEG-linker coupled to RM26. The heterodimers were labeled with indium-111 and evaluated in vitro. In the competitive binding assay, BQ7812, featuring phenylalanine and shorter PEG-linker, demonstrated a nine-fold improved affinity towards PSMA. In the in vivo biodistribution study of [111In]In-BQ7812 in PC3-pip tumor-bearing mice (PSMA and GRPR positive), the activity uptake was two-fold higher in the tumor and three-fold higher in kidneys than for [111In]In-BQ7800. Herein, we showed that the affinity of a bispecific PSMA/GRPR heterodimer towards PSMA could be improved by linker modification.

Critical role of GRP receptor-expressing neurons in the spinal transmission of imiquimod-induced psoriatic itch.

AimAmple evidence indicates that gastrin‐releasing peptide receptor (GRPR)–expressing neurons play a critical role in the transmission of acute itch. However, the pathophysiology of spinal mechanisms underlying intractable itch such as psoriasis remains unclear. In this study, we aimed to determine whether itch‐responsive GRPR+ neurons contribute to the spinal transmission of imiquimod (IMQ)‐induced psoriatic itch.MethodsTo generate a psoriasis model, C57BL/6J mice received a daily topical application of 5% IMQ cream on their shaved back skin for 7‐10 consecutive days. GRP+ neurons were inhibited using Cre‐dependent expression of Gi‐designer receptors exclusively activated by designer drugs (DREADDs), while GRPR+ neurons were ablated by intrathecal administration of bombesin‐saporin.ResultsRepeated topical application of IMQ elicited psoriasis‐like dermatitis and scratching behaviors. The mRNA expression levels of GRP and GRPR were upregulated in the cervical spinal dorsal horn (SDH) on days 7 and 10 after IMQ application. Either chemogenetic silencing of GRP+ neurons by Gi‐DREADD or ablation of GRPR+ neurons significantly attenuated IMQ‐induced scratching behaviors.ConclusionThe GRP‐GRPR system might be enhanced in the SDH, and itch‐responsive GRPR+ neurons largely contribute to intractable itch in a mouse model of psoriasis.

Voxel-based comparison of [68Ga]Ga-RM2-PET/CT and [68Ga]Ga-PSMA-11-PET/CT with histopathology for diagnosis of primary prostate cancer

BackgroundFocal therapies or focally escalated therapies of primary prostate cancer are becoming more and more important. This increases the need to identify the exact extension of the intraprostatic tumor and possible dominant intraprostatic lesions by imaging techniques. While the prostate-specific membrane antigen (PSMA) is already a well-established target for imaging of prostate cancer cells, the gastrin-releasing peptide receptor (GRPR) seems to provide interesting additional information. Histopathology was used to examine the extent to which the single and combined image information of PET scans targeting GRPR and PSMA might lead to better tumor delineation.MethodsEight patients with histologically proven primary prostate cancer underwent two positron emission tomography with computer tomography scans, [68Ga]Ga-RM2-PET/CT (RM2-PET) and [68Ga]Ga-PSMA-11-PET/CT (PSMA-PET), prior to radical prostatectomy. RM2-PET data were correlated voxel-wise to a voxel-based model of the histopathologic tumor volume information. The results were compared to, correlated to, and combined with the correlation of PSMA-PET data analyzed analogously.ResultsIn 4/8 patients, RM2-PET showed a higher signal in histologically proven tumor regions compared to PSMA. There were also tumor regions where PSMA-PET showed a higher signal than GRPR in 4/8 patients. A voxel-wise correlation of RM2-PET against histopathology yielded similar results compared to the correlation of PSMA-PET against histopathology, while PSMA-PET is the slightly better performing imaging technique. The combined information of both tracers yielded the best overall result, although this effect was not statistically significant compared to RM2-PET alone.ConclusionsQualitative and quantitative findings in this preliminary study with 8 patients indicate that RM2-PET and PSMA-PET partially show not only the same, but also distinct regions of prostate cancer. Patients with pPCa might profit from information given by tracers targeting GRPR and PSMA simultaneously, in terms of a better delineation of the gross tumor volume.

Sorbitol as a Polar Pharmacological Modifier to Enhance the Hydrophilicity of 99mTc-Tricarbonyl-Based Radiopharmaceuticals.

The organometallic technetium-99m tricarbonyl core, [99mTc][Tc(CO)3(H2O)3]+, is a versatile precursor for the development of radiotracers for single photon emission computed tomography (SPECT). A drawback of the 99mTc-tricarbonyl core is its lipophilicity, which can influence the pharmacokinetic properties of the SPECT imaging probe. Addition of polar pharmacological modifiers to 99mTc-tricarbonyl conjugates holds the promise to counteract this effect and provide tumor-targeting radiopharmaceuticals with improved hydrophilicities, e.g., resulting in a favorable fast renal excretion in vivo. We applied the “Click-to-Chelate” strategy for the assembly of a novel 99mTc-tricarbonyl labeled conjugate made of the tumor-targeting, modified bombesin binding sequence [Nle14]BBN(7–14) and the carbohydrate sorbitol as a polar modifier. The 99mTc-radiopeptide was evaluated in vitro with PC-3 cells and in Fox-1nu mice bearing PC-3 xenografts including a direct comparison with a reference conjugate lacking the sorbitol moiety. The glycated 99mTc-tricarbonyl peptide conjugate exhibited an increased hydrophilicity as well as a retained affinity toward the Gastrin releasing peptide receptor and cell internalization properties. However, there was no significant difference in vivo in terms of pharmacokinetic properties. In particular, the rate and route of excretion was unaltered in comparison to the more lipophilic reference compound. This could be attributed to the intrinsic properties of the peptide and/or its metabolites. We report a novel glycated (sorbitol-containing) alkyne substrate for the “Click-to-Chelate” methodology, which is potentially of general applicability for the development of 99mTc-tricarbonyl based radiotracers displaying an enhanced hydrophilicity.

Supercritical fluid assembly of albendazole liposomes targeting gastrin-releasing peptide receptor overexpressing tumors.

Aim: To develop albendazole (ABZ)-loaded bombesin(6-14) (BBN(6-14)) functionalized liposomes for targeting GRPR to enhance delivery to cancer cells. Materials & methods: ABZ-loaded liposomes were formulated using supercritical CO2 technology; functionalized with a GRPR-targeted lipid-anchored BBN(6-14) peptide; and evaluated for effects on cell viability, particle size and targeted cell uptake. Results: BBN(6-14)-coated ABZ liposomes decreased cell viability compared with nonfunctionalized ABZ liposomes. The level of GRPR expression positively correlated with intracellular uptake and decreased cell viability. The reduced cell viability, higher cell uptake and GRPR expression were observed in the order PC-3 > Caco-2 > HepG2 cells. Conclusion: BBN(6-14)-functionalized ABZ liposomes showed enhanced reduction in cell viability compared with nonfunctionalized ABZ liposomes.

A prospective study of 68Ga-RM2 PET/MRI in patients with biochemically recurrent prostate cancer and negative conventional imaging.

e17536 Background: 68Ga-RM2 is a synthetic bombesin receptor antagonist targeting gastrin-releasing peptide receptors (GRPr) that are overexpressed in several human tumors, including prostate cancer (PC). Methods: We enrolled 114 men with BCR PC, 45-83-year-old (mean±SD: 68.2±7.0). Imaging started at 40-89 minutes (mean±SD: 51.3±9.2 after injection of 113.8-152.6 MBq (mean±SD: 140.7±6.4) of 68Ga-RM2 using a time-of-flight (TOF)-enabled simultaneous positron emission tomography (PET) / magnetic resonance imaging (MRI) scanner. Twenty-four and 23 patients also underwent 68Ga-PSMA11 and 18F-DCFPyL PET/CT, respectively. Results: All patients had rising PSA and negative conventional imaging prior to enrollment. 68Ga-RM2 PET identified recurrent PC in 78 of the 114 participants, while the simultaneous MRI was positive for PC in 45 of the 103 patients. Positivity rate of 68Ga-RM2 PET was: 31.8% for PSA < 0.5 ng/dl ( n= 22), 60% for PSA 0.5 – 1.0 ng/dl ( n= 15), 64.7% for PSA 1.0 – 2.0 ng/dl ( n= 17), 81.8% for PSA 2.0 – 5.0 ng/dl ( n= 22) and 87.2% for PSA > 5.0 ng/dl ( n= 38). PSA velocity values were 1.9±2.7 ng/ml/year (range: 0-9.1) in patients with negative PET scans and 5.8±9 ng/ml/year (range: 0.2-45.4) in patients with positive PET scans ( P: 0.01). Twenty-eight and 34 lesions were detected by 68Ga-RM2 PET and 68Ga-PSMA11 PET, respectively, while 25 lesions in 13 patients were identified by both radio-pharmaceuticals. The mean SUVmax ranged 1.6-51.2 (mean±SD:14.7±12.5) for PSMA and ranged 2.5-52.5 (mean±SD: 10.7±11.6) for RM2 ( P= 0.096). Three lesions in 2 patients were RM2-avid only (all lymph nodes) and 9 lesions in 7 patients were PSMA avid only (7 lymph nodes, 1 skeletal and 1 lung nodule). 32 and 48 lesions were detected by 68Ga-RM2 PET and 18F-DCFPyL PET, respectively. 28 lesions in 12 patients were identified by both radio-pharmaceuticals. The mean SUVmax ranged 1.7-79.3 (mean±SD: 22.2±23) for DCFPyL and ranged 1.7-46.8 (mean±SD: 7±9.2) for RM2 (P < 0.01). Four lesions in 2 patients were RM2 avid only (1 adrenal and 3 skeletal); 17 lesions in 6 patients were DCFPyL avid only (7 lymph nodes, 9 skeletal and 1 prostate). Conclusions: 68Ga-RM2 may identify higher risk patients given the highly statistically significant difference PSA velocity values between patients with negative and positive scans and may be a complementary radiopharmaceutical to the PSMA-targeting tracers to ultimately allow for personalized medicine. Clinical trial information: NCT02624518 .

Recent updates and developments in PET imaging of prostate cancer.

A number of positron emission tomography (PET) radiotracers have been developed to improve the sensitivity and specificity of imaging for prostate cancer. These radiotracers include the bone-seeking agent Na18F as well as more tumor-specific compounds such as 11C-choline and 18F-fluciclovine. In this review, we will discuss the advantages and disadvantages of these PET radiotracers for the imaging of men with prostate cancer across a range of clinical contexts. We will also touch upon radiotracers in late clinical development that have not gained regulatory approval, including those targeted against prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR).

Association between gastrin-releasing peptide receptor expression as assessed with [68Ga]Ga-RM2 PET/CT and histopathological tumor regression after neoadjuvant chemotherapy in primary breast cancer

IntroductionThe gastrin-releasing peptide receptor is overexpressed in breast cancer (BC) tissue and can be visualized by positron emission tomography (PET) using the GRPR antagonist [68Ga]Ga-RM2. This study assessed tumor binding of RM2 before and after neoadjuvant chemotherapy (NAC) in primary BC with reference to residual tumor size in the resected specimen. Materials and methodsIn this retrospective study, five female patients with biopsy-confirmed estrogen receptor (ER)-positive primary BC (one with bilateral tumors) underwent [68Ga]Ga-RM2 PET/CT before and after NAC. PET/CT was acquired 1 h after injection of 143–224 MBq [68Ga]Ga-RM2. Time from pre-NAC PET to beginning of NAC was 23 ± 4.9 days, from end of NAC to post-NAC PET 18.7 ± 6.3 days, and from post-NAC PET to surgery 9.5 ± 10.8 days. In vivo tumor uptake of [68Ga]Ga-RM2 was assessed before and after NAC and correlated with histopathological response. ResultsAll tumors (6/6) showed strongly increased [68Ga]Ga-RM2 uptake compared to normal breast tissue on pre-NAC PET (mean SUVmax 13.2 ± 7.3; mean SUVpeak 9.4 ± 4.4). [68Ga]Ga-RM2 uptake was significantly reduced on post-NAC PET in all primary tumors (mean SUVmax 2.3 ± 0.8, −79 ± 11%; p = 0.0125; mean SUVpeak 1.6 ± 0.4, −79 ± 10%; p = 0.0096). Residual tumor size in resected specimens correlated well with SUVmax (r = 0.91, p = 0.0057) and SUVpeak (r = 0.88, p = 0.0196) on [68Ga]Ga-RM2 PET/CT after NAC. Conclusion and implications for patient careIn this pilot study, residual uptake of [68Ga]Ga-RM2 in ER-positive primary BC correlated well with residual vital tumor size after NAC. This suggests that [68Ga]Ga-RM2 PET/CT merits further investigation for response assessment to NAC in patients with ER-positive BC.

Synthesis and Biochemical Evaluation of Samarium-153 Oxide Nanoparticles Functionalized with iPSMA-Bombesin Heterodimeric Peptide.

Developments in the design of lanthanide oxide nanoparticles (NPs) have unleashed a wide variety of biomedical applications. Several types of hepatic cancer cells overexpress two proteins: the gastrin-releasing peptide receptor (GRPr), which specifically recognizes the bombesin (BN) peptide, and the prostate-specific membrane antigen (PSMA), which specifically binds to several peptides that inhibit its activity (iPSMA). This research synthesized and physicochemically characterized Sm₂O₃ nanoparticles functionalized with the iPSMA-BN heterodimeric peptide and studied the effects on their structural, biochemical and preclinical properties after activation by neutron irradiation for possible use in molecular dual-targeted radiotherapy of hepatocellular carcinoma. The Sm₂O₃ NPs were synthesized by the precipitation-calcination method and functionalized with iPSMA-BN peptide using the DOTA macrocycle as a linking agent. Analysis of physicochemical characterization via TEM, EDS, XRD, UV-Vis, FT-IR, DSL, and zeta potential results showed the formation of Sm₂O₃-iPSMA-BN NPs (94.23 ± 5.98 nm), and their physicochemical properties were not affected after neutron activation. The nanosystem showed a high affinity with respect to PSMA and GRPr in HepG2 cells ( Kd = 6.6 ± 1.6 nM) and GRPr in PC3 cells ( Kd = 10.6 ± 1.9 nM). 153Sm₂O₃-iPSMA-BN NPs exhibited radioluminescent properties, making possible in vivo optical imaging of their biodistribution in mice. The results obtained from this research support further preclinical studies designed to evaluate the dosimetry and therapeutic efficacy of 153Sm₂O₃-iPSMA-BN nanoparticles for in vivo imaging and molecular dual-targeted radiotherapy of liver tumors overexpressing PSMA and/or GRPr proteins.

MITIGATE-NeoBOMB1, a Phase I/IIa Study to Evaluate Safety, Pharmacokinetics, and Preliminary Imaging of 68Ga-NeoBOMB1, a Gastrin-Releasing Peptide Receptor Antagonist, in GIST Patients

Gastrin-releasing peptide receptors (GRPRs) are potential molecular imaging targets in a variety of tumors. Recently, a 68Ga-labeled antagonist to GRPRs, NeoBOMB1, was developed for PET. We report on the outcome of a phase I/IIa clinical trial (EudraCT 2016-002053-38) within the EU-FP7 project Closed-loop Molecular Environment for Minimally Invasive Treatment of Patients with Metastatic Gastrointestinal Stromal Tumors ('MITIGATE') (grant agreement no. 602306) in patients with oligometastatic gastrointestinal stromal tumors (GIST). Methods: The main objectives were evaluation of safety, biodistribution, dosimetry, and preliminary tumor targeting of 68Ga-NeoBOMB1 in patients with advanced tyrosine-kinase inhibitors-treated GIST using PET/CT. Six patients with histologically confirmed GIST and unresectable primary lesion or metastases undergoing an extended protocol for detailed pharmacokinetic analysis were included. 68Ga-NeoBOMB1 was prepared using a kit procedure with a licensed 68Ge/68Ga generator. 68Ga-NeoBOMB1 (3 MBq/kg of body weight) was injected intravenously, and safety parameters were assessed. PET/CT included dynamic imaging at 5, 11, and 19 min as well as static imaging at 1, 2, and 3-4 h after injection for dosimetry calculations. Venous blood samples and urine were collected for pharmacokinetic analysis. Tumor targeting was assessed on a per-lesion and per-patient basis. Results: 68Ga-NeoBOMB1 (50 μg) was prepared with high radiochemical purity (yield > 97%). Patients received 174 ± 28 MBq of the radiotracer, which was well tolerated in all patients over a follow-up period of 4 wk. Dosimetry calculations revealed a mean effective dose of 0.029 ± 0.06 mSv/MBq, with the highest organ dose to the pancreas (0.274 ± 0.099 mSv/MBq). Mean plasma half-life was 27.3 min with primarily renal clearance (mean 25.7% ± 5.4% of injected dose 4 h after injection). Plasma metabolite analyses revealed high stability; metabolites were detected only in the urine. In 3 patients, a significant uptake with increasing maximum SUVs (SUVmax at 2 h after injection: 4.3-25.9) over time was found in tumor lesions. Conclusion: This phase I/IIa study provides safety data for 68Ga-NeoBOMB1, a promising radiopharmaceutical for targeting GRPR-expressing tumors. Safety profiles and pharmacokinetics are suitable for PET imaging, and absorbed dose estimates are comparable to those of other 68Ga-labeled radiopharmaceuticals used in clinical routine.

On-cell saturation transfer difference NMR study of Bombesin binding to GRP receptor.

We report the NMR characterization of the molecular interaction between Gastrin Releasing Peptide Receptor (GRP-R) and its natural ligand bombesin (BN). GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation; in addition, being overexpressed on the surface of different human cancer cell lines, it is ideal for the development of new strategies for the selective targeted delivery of anticancer drugs and diagnostic devices to tumor cells. However, the design of new GRP-R binders requires structural information on receptor interaction with its natural ligands. The experimental protocol presented herein, based on on-cell STD NMR techniques, is a powerful tool for the screening and the epitope mapping of GRP-R ligands aimed at the development of new anticancer and diagnostic tools. Notably, the study can be carried out in a physiological environment, at the surface of tumoral cells overespressing GRP-R. Moreover, to the best of our knowledge, this is the first example of an NMR experiment able to detect and investigate the structural determinants of BN/GRP-R interaction.

NFAT5 is involved in GRP‐enhanced secretion of GLP‐1 by high sodium

Organ to organ communication is important in the maintenance of normal fluid and electrolyte balance and blood pressure (BP). Neural mechanisms and gastrointestinal (GIT) hormones mediate the natriuresis of an oral sodium load. The GIT is the first organ exposed to ingested nutrients and most likely to react initially to a sodium load. Sensing the amount of ingested sodium by the GIT may be an important mechanism by which sodium balance is regulated. We have reported that gastrin secreted by G‐cells in the stomach and duodenum is involved in the regulation of sodium balance and BP. Besides gastrin, glucagon‐like peptide‐1 (GLP‐1) is another gut hormone, which may also participate in regulation of sodium balance and BP. It is well known that glucose‐induced GLP‐1 secretion is sodium‐dependent. The increase in intracellular calcium needed for GLP‐1 secretion is also due to sodium‐dependent cell excitability. However, the specific effect of varying sodium concentration on GLP‐1 secretion and its potential mechanism remain to be elucidated. This study aimed to determine the effect of varying sodium concentrations on GLP‐1 secretion and its potential mechanism. Here, we show that sodium can stimulate GLP‐1 secretion and that gastrin‐releasing peptide (GRP) enhances the high sodium‐induced increase in GLP‐1 secretion in a concentration and time‐dependent manner in L‐cells. The exposure of L‐cells to high (170 mM) Na+ increases GLP‐1 protein (produced by degradation of glucagon) or proglucagon mRNA expression, while Low (90 mM) and high (170 mM) Na+ increase NFAT5 and GRP receptor (GRPR) mRNA and protein expression. Silencing NFAT5 abrogates the GRP‐mediated enhancement of high sodium‐induced increase in GLP‐1 secretion and blocks the sodium‐induced increase in GRPR mRNA and protein expressions in L‐cells. Furthermore, NFAT5 silencing reduces the GLP‐1 protein or proglucagon expression in L‐cells exposed to high sodium concentration. NFAT5 silencing also reduces GRPR expression, using the promoter reporter assay; n=3/group for all studies. These results suggest that the high sodium‐induced NFAT5 expression increases expression of GRPR; GRP acting on the increased GRPR expression increases GLP‐1 secretion from L‐cells. These data give a new perspective on the mechanisms of GLP‐1 secretion, and the direct relationship between sodium and GLP‐1. These may contribute to better utilization of existing GLP‐1 based drugs and further GLP‐1‐based drug development.Effect of NaCl or GRP on GLP‐1 secretion in CCL‐251 L cells (human colorectal adenocarcinoma cells=GLP‐1 secreting cells).The same amount of cells were exposed to 90 mM, 145 mM, or 170 mM NaCl medium, and A, treated with varying concentrations of GRP (10, 100, 500, or 1000 nmol) for 4 h, or B, treated with 1000 nmol GRP at different times (1 h, 2 h, 3 h, or 4 h). GLP‐1 in the cell culture supernatant was measured by ELISA. The results are expressed as ng/ml. *P<0.05 vs. 90 mM with or without GRP. #P<0.05 vs. others, one‐way ANOVA, Holm‐Sidak test, n=3/treatment.Figure 1

Functional roles of neuromedin B and gastrin‐releasing peptide in regulating itch and pain in the spinal cord of primates

Neuromedin B (NMB) has been demonstrated to elicit itch and pain in rodents. However, the functional role of NMB and its cognate receptor (NMBR) in regulating itch and pain in the primate spinal cord is unknown. The aim of this study was to determine the functional profile of spinal NMB and compare its behavioral effects with those elicited by spinal gastrin‐releasing peptide (GRP) in rhesus monkeys (Macaca mulatta). Following intrathecal administration, GRP (3–10 nmol) dose‐dependently elicited robust scratching responses. In contrast, intrathecal NMB (30–200 nmol) only elicited mild scratching responses. Intrathecal GRP‐ and NMB‐elicited scratching activities were attenuated by a GRP receptor (GRPR) antagonist (RC‐3095) and a NMBR antagonist (PD168368), respectively. Unlike intrathecal substance P (100 nmol) producing thermal allodynia manifested as a reduced tail‐withdrawal latency in 46C water, intrathecal administration of GRP or NMB (1–100 nmol) did not change the thermal nociceptive threshold of primates. In addition, intrathecal administration of a mu opioid receptor agonist, DAMGO (3 nmol), inhibited capsaicin‐induced thermal allodynia. Unlike DAMGO, both RC‐3095 and PD16838 (100–300 nmol) intrathecally did not produce antiallodynic effects. Taken together, these pharmacological findings indicate that spinal NMB‐NMBR and GRP‐GRPR systems have a minimum role in the neurotransmission of pain and both spinal GRPR and NMBR function independently to elicit itch/scratching responses in primates.Support or Funding InformationThe US‐PHS grants AR064456 and AR069861

LBA02-09 INTERIM ANALYSIS RESULTS OF A PROSPECTIVE STUDY OF 68 GA-RM2 PET/MRI IN PATIENTS WITH BIOCHEMICALLY RECURRENT PROSTATE CANCER AND NEGATIVE CONVENTIONAL IMAGING

Introduction:68Ga-RM2 is a synthetic bombesin receptor antagonist targeting gastrin-releasing peptide receptors (GRPr) that are overexpressed in several human tumors, including prostate cancer (PC)...

A comparative study of peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 in preclinical prostate tumour models

IntroductionPeptide-based imaging agents targeting prostate-specific membrane antigen (PSMA) have revolutionized the evaluation of biochemical recurrence of prostate cancer (PCa) but lacks sensitivity at very low serum prostate specific antigen (PSA) levels. Once recurrence is suspected, other positron emission tomography (PET) radiotracers could be of interest to discriminate between local and distant relapse. We studied [18F]fluorodeoxyglucose ([18F]FDG) targeting glucose metabolism, [18F]fluorocholine ([18F]FCH) targeting membrane metabolism and peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 targeting PSMA, gastrin releasing peptide receptor (GRPr), αvβ3 integrin and neurotensin type 1 receptor (NTSR1) respectively, in different PCa tumour models. MethodsMice were xenografted with 22Rv1, an androgen-receptor (AR)-positive, PCa cell line that expresses PSMA and PC3, an AR-negative one that does not express PSMA. PET imaging using the different radiotracers was performed sequentially and the uptake characteristics compared to one other. NTSR1 and PSMA expression levels were analysed in tumours by immunohistochemistry. Results[18F]FDG displayed low but sufficient uptake to visualize PC3 and 22Rv1 derived tumours. We also observed a low efficacy of [18F]FCH PET imaging and a low [68Ga]Ga-NODAGA-RGD tumour uptake in those tumours. As expected, an elevated tumour uptake was obtained for [68Ga]Ga-PSMA-11 in 22Rv1 derived tumour although no uptake was measured in the androgen independent cell line PC3, derived from a bone metastasis of a high-grade PCa. Moreover, in PC3 cell line, we obtained good tumour uptake, high tumour-to-background contrast using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3.Immunohistochemistry analysis confirmed high NTSR1 expression in PC3 derived tumours and conversely high PSMA expression in 22Rv1 derived tumours. ConclusionPET imaging using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3 demonstrates that GRPr and NTSR1 could represent viable alternative targets for diagnostic or therapeutic applications in PCa with limited PSMA expression levels. More preclinical and clinical studies will follow to explore this potential. Advances in knowledge and implications for patientPeptide-based imaging agents targeting PSMA represent a major progress in the evaluation of biochemical recurrence of PCa but sometimes yield false negative results in some lesions. Continuing efforts have thus been made to evaluate other radiotracers. Our preclinical results suggest that [68Ga]labelled bombesin and neurotensin analogues could serve as alternative PET radiopharmaceuticals for diagnostic or therapy in cases of PSMA-negative PCa.

Inhibition of Gastrin-Releasing Peptide Attenuates Phosphate-Induced Vascular Calcification

Vascular calcification is the pathological deposition of calcium/phosphate in the vascular system and is closely associated with cardiovascular morbidity and mortality. Here, we investigated the role of gastrin-releasing peptide (GRP) in phosphate-induced vascular calcification and its potential regulatory mechanism. We found that the silencing of GRP gene and treatment with the GRP receptor antagonist, RC-3095, attenuated the inorganic phosphate-induced calcification of vascular smooth muscle cells (VSMCs). This attenuation was caused by inhibiting phenotype change, apoptosis and matrix vesicle release in VSMCs. Moreover, the treatment with RC-3095 effectively ameliorated phosphate-induced calcium deposition in rat aortas ex vivo and aortas of chronic kidney disease in mice in vivo. Therefore, the regulation of the GRP-GRP receptor axis may be a potential strategy for treatment of diseases associated with excessive vascular calcification.

Radiolabeled GRPR Antagonists for Imaging of Disseminated Prostate Cancer - Influence of Labeling Chemistry on Targeting Properties.

Radionuclide molecular imaging of Gastrin-Releasing Peptide Receptor (GRPR) expression promises unparalleled opportunities for visualizing subtle prostate tumors, which due to small size, adjacent benign tissue, or a challenging location would otherwise remain undetected by conventional imaging. Achieving high imaging contrast is essential for this purpose and the molecular design of any probe for molecular imaging of prostate cancer should be aimed at obtaining as high tumor-to-organ ratios as possible. This short review summarizes the key imaging modalities currently used in prostate cancer, with a special focus on radionuclide molecular imaging. Emphasis is laid mainly on the issue of radiometals labeling chemistry and its influence on the targeting properties and biodistribution of radiolabeled GRPR antagonists for imaging of disseminated prostate cancer. A comprehensive literature search of the PubMed/MEDLINE, and Scopus library databases was conducted to find relevant articles. The combination of radionuclide, chelator and required labeling chemistry was shown to have a significant influence on the stability, binding affinity and internalization rate, off-target interaction with normal tissues and blood proteins, interaction with enzymes, activity uptake and retention in excretory organs and activity uptake in tumors of radiolabeled bombesin antagonistic analogues. Labeling chemistry has a very strong impact on the biodistribution profile of GRPRtargeting peptide based imaging probes and needs to be considered when designing a targeting probe for high contrast molecular imaging. Taking into account the complexity of in vivo interactions, it is not currently possible to accurately predict the optimal labeling approach. Therefore, a detailed in vivo characterization and optimization is essential for the rational design of imaging agents.