Structures of human gastrin-releasing peptide receptors bound to antagonist and agonist for cancer and itch therapy.

Gastrin releasing peptide receptor (GRPR), a member of the bombesin (BBN) G protein-coupled receptors, is aberrantly overexpressed in several malignant tumors, including those of the breast, prostate, pancreas, lung, and central nervous system. Additionally, it also mediates non-histaminergic itch and pathological itch conditions in mice. Thus, GRPR could be an attractive target for cancer and itch therapy. Here, we report the inactive state crystal structure of human GRPR in complex with the non-peptide antagonist PD176252, as well as two active state cryo-electron microscopy (cryo-EM) structures of GRPR bound to the endogenous peptide agonist gastrin-releasing peptide and the synthetic BBN analog [D-Phe6, ß-Ala11, Phe13, Nle14] Bn (6-14), in complex with Gq heterotrimers. These structures revealed the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins signaling of GRPR, which are expected to accelerate the structure-based design of GRPR antagonists and agonists for the treatments of cancer and pruritus.

Synthesis, preclinical, and initial clinical evaluation of integrin αVß3 and gastrin-releasing peptide receptor (GRPR) dual-targeting radiotracer [68Ga]Ga-RGD-RM26-03.

Integrin receptor αvß3 and gastrin-releasing peptide receptor (GRPR) expression of tumors could be detected using PET imaging with radiolabeled Arg-Gly-Asp (RGD) and the antagonistic bombesin analog RM26, respectively. The purpose of this study was to investigate the dual receptor-targeting property of the heterodimer RGD-RM26-03 (denoted as LNC1015), demonstrate the tumor diagnostic value of [68Ga]Ga-LNC1015 in preclinical experiments, and evaluate its preliminary clinical feasibility. METHODS: LNC1015 was designed and synthesized by linking cyclic RGD and the RM26 peptide. Preclinical pharmacokinetics were detected in a PC3 xenograft model using microPET and biodistribution studies. The clinical feasibility of [68Ga]Ga-LNC1015 PET/CT was performed in patients with breast cancer, and the results were compared with those of 18F-fluorodeoxyglucose (FDG). RESULTS: [68Ga]Ga-LNC1015 had good stability in saline for at least 2 h, and favorable binding affinity and specificity were demonstrated in vitro and in vivo. The tumor uptake and retention of [68Ga]Ga-LNC1015 during PET imaging were improved compared with its monomeric counterparts [68Ga]Ga-RGD and [68Ga]Ga-RM26 at all the time points examined. In our initial clinical studies, the tumor uptake and tumor-to-background ratio (TBR) of primary and metastatic lesions in [68Ga]Ga-LNC1015 PET/CT were significantly higher than those in [18F]FDG PET/CT, resulting in high lesion detection rate and tumor delineation. CONCLUSION: The dual targeting radiotracer [68Ga]Ga-LNC1015 showed significantly improved tumor uptake and retention, as well as lower liver uptake than [68Ga]Ga-RGD and [68Ga]Ga-RM26 monomer. The first-in-human study showed high TBRs in patients, suggesting favorable pharmacokinetics and high clinical feasibility for PET/CT imaging of cancer.

Genetic and pharmacological inhibition of GRPR protects against acute kidney injury via attenuating renal inflammation and necroptosis.

Gastrin-releasing peptide (GRP) binds to its receptor (GRP receptor [GRPR]) to regulate multiple biological processes, but the function of GRP/GRPR axis in acute kidney injury (AKI) remains unknown. In the present study, GRPR is highly expressed by tubular epithelial cells (TECs) in patients or mice with AKI, while histone deacetylase 8 may lead to the transcriptional activation of GRPR. Functionally, we uncovered that GRPR was pathogenic in AKI, as genetic deletion of GRPR was able to protect mice from cisplatin- and ischemia-induced AKI. This was further confirmed by specifically deleting the GRPR gene from TECs in GRPRFlox/Flox//KspCre mice. Mechanistically, we uncovered that GRPR was able to interact with Toll-like receptor 4 to activate STAT1 that bound the promoter of MLKL and CCL2 to induce TEC necroptosis, necroinflammation, and macrophages recruitment. This was further confirmed by overexpressing STAT1 to restore renal injury in GRPRFlox/Flox/KspCre mice. Concurrently, STAT1 induced GRP synthesis to enforce the GRP/GRPR/STAT1 positive feedback loop. Importantly, targeting GRPR by lentivirus-packaged small hairpin RNA or by treatment with a novel GRPR antagonist RH-1402 was able to inhibit cisplatin-induced AKI. In conclusion, GRPR is pathogenic in AKI and mediates AKI via the STAT1-dependent mechanism. Thus, targeting GRPR may be a novel therapeutic strategy for AKI.

Side-by-side comparison of the two widely studied GRPR radiotracers, radiolabeled NeoB and RM2, in a preclinical setting.

INTRODUCTION: NeoB and RM2 are the most investigated gastrin-releasing peptide receptor (GRPR)-targeting radiotracers in preclinical and clinical studies. Therefore, an extensive side-by-side comparison of the two radiotracers is valuable to demonstrate whether one has advantages over the other. Accordingly, this study aims to compare the in vitro and in vivo characteristics of radiolabeled NeoB and RM2 to guide future clinical studies. METHOD: The stability of the radiolabeled GRPR analogs was determined in phosphate buffered saline (PBS), and commercially available mouse and human serum. Target affinity was determined by incubating human prostate cancer PC-3 cells with [177Lu]Lu-NeoB or [177Lu]Lu-RM2, + / - increasing concentrations of unlabeled NeoB, RM2, or Tyr4-bombesin (BBN). To determine uptake and specificity cells were incubated with [177Lu]Lu-NeoB or [177Lu]Lu-RM2 + / - Tyr4-BBN. Moreover, in vivo studies were performed to determine biodistribution and pharmacokinetics. Finally, radiotracer binding to various GRPR-expressing human cancer tissues was investigated. RESULTS: Both radiotracers demonstrated high stability in PBS and human serum, but stability in mouse serum decreased substantially over time. Moreover, both radiotracers demonstrated high GRPR affinity and specificity, but a higher uptake of [177Lu]Lu-NeoB was observed in in vitro studies. In vivo, no difference in tumor uptake was seen. The most prominent difference in uptake in physiological organs was observed in the GRPR-expressing pancreas; [177Lu]Lu-RM2 had less pancreatic uptake and a shorter pancreatic half-life than [177Lu]Lu-NeoB. Furthermore, [177Lu]Lu-RM2 presented with a lower tumor-to-kidney ratio, while the tumor-to-blood ratio was lower for [177Lu]Lu-NeoB. The autoradiography studies revealed higher binding of radiolabeled NeoB to all human tumor tissues. CONCLUSION: Based on these findings, we conclude that the in vivo tumor-targeting capability of radiolabeled NeoB and RM2 is similar. Additional studies are needed to determine whether the differences observed in physiological organ uptakes, i.e., the pancreas, kidneys, and blood, result in relevant differences in organ absorbed doses when the radiotracers are applied for therapeutic purposes.

Modified PROMISE criteria for standardized interpretation of gastrin-releasing peptide receptor (GRPR)-targeted PET.

PURPOSE: There are image interpretation criteria to standardize reporting prostate-specific membrane antigen (PSMA)-targeted positron emission tomography (PET). As up to 10% of prostate cancer (PC) do not express PSMA, other targets such as gastrin-releasing peptide receptor (GRPR) are evaluated. Research on GRPR-targeted imaging has been slowly increasing in usage at staging and biochemical recurrence (BCR) of PC. We therefore propose a modification of the Prostate Cancer Molecular Imaging Standardized Evaluation (PROMISE) criteria (mPROMISE) for GRPR-targeted PET. METHODS: [68 Ga]Ga-RM2 PET data from initially prospective studies performed at our institution were retrospectively reviewed: 44 patients were imaged for staging and 100 patients for BCR PC. Two nuclear medicine physicians independently evaluated PET according to the mPROMISE criteria. A third expert reader served as standard reference. Interreader reliability was computed for GRPR expression, prostate bed (T), lymph node (N), skeleton (Mb), organ (Mc) metastases, and final judgment of the scan. RESULTS: The interrater reliability for GRPR PET at staging was moderate for GRPR expression (0.59; 95% confidence interval [CI] 0.40, 0.78), substantial for T-stage (0.78; 95% CI 0.63, 0.94), and almost perfect for N-stage (0.97; 95% CI 0.92, 1.00) and final judgment (0.92; 95% CI 0.82, 1.00). The interreader agreement at BCR showed substantial agreement for GRPR expression (0.70; 95% CI 0.59, 0.81) and final judgment (0.65; 95% CI 0.53, 0.78), while almost perfect agreement was seen across the major categories (T, N, Mb, Mc). Acceptable performance of the mPROMISE criteria was found for all subsets when compared to the standard reference. CONCLUSION: Interpreting GRPR-targeted PET using the mPROMISE criteria showed its reliability with substantial or almost perfect interrater agreement across all major categories. The proposed modification of the PROMISE criteria will aid clinicians in decreasing the level of uncertainty, and clinical trials to achieve uniform evaluation, reporting, and comparability of GRPR-targeted PET. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT03113617 and NCT02624518.

Nanostrategies for Therapeutic and Diagnostic Targeting of Gastrin-Releasing Peptide Receptor.

Advances in nanomedicine bring the attention of researchers to the molecular targets that can play a major role in the development of novel therapeutic and diagnostic modalities for cancer management. The choice of a proper molecular target can decide the efficacy of the treatment and endorse the personalized medicine approach. Gastrin-releasing peptide receptor (GRPR) is a G-protein-coupled membrane receptor, well known to be overexpressed in numerous malignancies including pancreatic, prostate, breast, lung, colon, cervical, and gastrointestinal cancers. Therefore, many research groups express a deep interest in targeting GRPR with their nanoformulations. A broad spectrum of the GRPR ligands has been described in the literature, which allows tuning of the properties of the final formulation, particularly in the field of the ligand affinity to the receptor and internalization possibilities. Hereby, the recent advances in the field of applications of various nanoplatforms that are able to reach the GRPR-expressing cells are reviewed.

Scandium-44: Diagnostic Feasibility in Tumor-Related Angiogenesis.

Angiogenesis-related cell-surface molecules, including integrins, aminopeptidase N, vascular endothelial growth factor, and gastrin-releasing peptide receptor (GRPR), play a crucial role in tumour formation. Radiolabelled imaging probes targeting angiogenic biomarkers serve as valuable vectors in tumour identification. Nowadays, there is a growing interest in novel radionuclides other than gallium-68 (68Ga) or copper-64 (64Cu) to establish selective radiotracers for the imaging of tumour-associated neo-angiogenesis. Given its ideal decay characteristics (Eß+average: 632 KeV) and a half-life (T1/2 = 3.97 h) that is well matched to the pharmacokinetic profile of small molecules targeting angiogenesis, scandium-44 (44Sc) has gained meaningful attention as a promising radiometal for positron emission tomography (PET) imaging. More recently, intensive research has been centered around the investigation of 44Sc-labelled angiogenesis-directed radiopharmaceuticals. Previous studies dealt with the evaluation of 44Sc-appended avb3 integrin-affine Arg-Gly-Asp (RGD) tripeptides, GRPR-selective aminobenzoyl-bombesin analogue (AMBA), and hypoxia-associated nitroimidazole derivatives in the identification of various cancers using experimental tumour models. Given the tumour-related hypoxia- and angiogenesis-targeting capability of these PET probes, 44Sc seems to be a strong competitor of the currently used positron emitters in radiotracer development. In this review, we summarize the preliminary preclinical achievements with 44Sc-labelled angiogenesis-specific molecular probes.

Dual-tracer PET/CT-targeted, mpMRI-targeted, systematic biopsy, and combined biopsy for the diagnosis of prostate cancer: a pilot study.

PURPOSE: Growing evidence proved the efficacy of multi-parametric MRI (mpMRI) and prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT)-guided targeted biopsy (TB) in prostate cancer (PCa) diagnosis, but there is no direct comparison between mpMRI-TB and PSMA PET/CT-TB. Gastrin-releasing peptide receptor (GRPR) is highly expressed in PCa, which can compensate for the unstable expression of PSMA in PCa. Therefore, we designed a study to compare the efficiency of mpMRI-TB, dual-tracer (GRPR and PSMA) PET/CT-TB, systematic biopsy, and combined biopsy for the diagnosis of prostate cancer. METHODS: One hundred twelve suspicious PCa patients were enrolled from September 2020 to June 2021. Patients with anyone of positive dual-tracer PET/CT or mpMRI underwent TB, and all enrolled patients underwent systematic biopsy (SB) after TB. The primary outcome was the detection rates of PCa in different biopsy strategies. Secondary outcomes were the performance of three imaging methods, omission diagnostic rates, and upgrading and downgrading of biopsy samples relative to those of prostatectomy specimens in different biopsy strategies. McNemar's tests and Bonferroni correction in multiple comparisons were used to compare the primary and secondary outcomes. RESULTS: In 112 men, clinically significant PCa (grade group[GG] ≥ 2) accounted for 34.82% (39/112), and nonclinically significant PCa (GG = 1) accounted for 4.46% (5/112). 68 Ga-PSMA PET/CT-TB achieved higher PCa detection rate (69.77%) and positive ratio of biopsy cores (0.44) compared with SB (39.29% and 0.12) and mpMRI-TB (36.14% and 0.23), respectively (P < 0.005). Dual-tracer PET/CT screen out patients for avoiding 52.67% (59/112) unnecessary biopsy, whereas dual-tracer PET/CT-TB plus SB achieved high detection rate (77.36%) without misdiagnosis of csPCa. CONCLUSION: Dual-tracer PET/CT might screen patients for avoiding unnecessary biopsy. Dual-tracer PET/CT-TB plus SB might be a more effective and promising strategy for the definite diagnosis of clinically significant PCa than mpMRI-TB.

Safety of [177Lu]Lu-NeoB treatment: a preclinical study characterizing absorbed dose and acute, early, and late organ toxicity.

PURPOSE: The radiolabeled gastrin-releasing peptide receptor (GRPR)-targeting antagonist NeoB is a promising radioligand for imaging and therapy of GRPR-expressing malignancies. In the current study, we aimed to discover the target organs of toxicity and the radiotoxic effects to these organs, when repeated dosages of [177Lu]Lu-NeoB are administered to healthy female and male mice. METHODS: Animals received either 3 injections, with a 7-day interval, of vehicle (control group 1), 1200 pmol [175Lu]Lu-NeoB (control group 2) or 40 MBq/400 pmol, 80 MBq/800 pmol, and 120 MBq/1200 pmol [177Lu]Lu-NeoB (treatment groups 1, 2, and 3, respectively). At week 5, 19, and 43 after the first injection acute, early, and late organ toxicity, respectively, was determined. For this, histopathological and blood analyses were performed. To correlate the observed toxicity to absorbed dose, we also performed extensive biodistribution and dosimetry studies. RESULTS: The biodistribution study showed the highest absorbed doses in GRPR-expressing pancreas, the liver, and the kidneys (the main organs of excretion). Both control groups and almost all animals of treatment group 1 did not show any treatment-related toxicological effects. Despite the high absorbed doses, no clear microscopic signs of toxicity were found in the pancreas and the liver. Histological analysis indicated kidney damage in the form of hydronephrosis and nephropathy in treatment groups 2 and 3 that were sacrificed at the early and late time point. In the same groups, increased blood urea nitrogen levels were found. CONCLUSION: In general, repeated administration of [177Lu]Lu-NeoB was tolerated. The most significant radiotoxic effects were found in the kidneys, similar to other clinically applied radioligands. The results of this study underline the potential of [177Lu]Lu-NeoB as a promising option for clinical therapy.

Investigation of Tumor Cells and Receptor-Ligand Simulation Models for the Development of PET Imaging Probes Targeting PSMA and GRPR and a Possible Crosstalk between the Two Receptors.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) have both been used in nuclear medicine as targets for molecular imaging and therapy of prostate (PCa) and breast cancer (BCa). Three bioconjugate probes, the PSMA specific: [68Ga]Ga-1, ((HBED-CC)-Ahx-Lys-NH-CO-NH Glu or PSMA-11), the GRPR specific: [68Ga]Ga-2, ((HBED-CC)-4-amino-1-carboxymethyl piperidine-[D-Phe6, Sta13]BN(6-14), a bombesin (BN) analogue), and 3 (the BN analogue: 4-amino-1-carboxymethyl piperidine-[(R)-Phe6, Sta13]BN(6-14) connected with the fluorescent dye, BDP-FL), were synthesized and tested in vitro with PCa and BCa cell lines, more specifically, with PCa cells, PC-3 and LNCaP, with BCa cells, T47D, MDA-MB-231, and with the in-house created PSMA-overexpressing PC-3(PSMA), T47D(PSMA), and MDA-MB-231(PSMA). In addition, biomolecular simulations were conducted on the association of 1 and 2 with PSMA and GRPR. The PSMA overexpression resulted in an increase of cell-bound radioligand [68Ga]Ga-1 (PSMA) for PCa and BCa cells and also of [68Ga]Ga-2 (GRPR), especially in those cell lines already expressing GRPR. The results were confirmed by fluorescence-activated cell sorting with a PE-labeled PSMA-specific antibody and the fluorescence tracer 3. The docking calculations and molecular dynamics simulations showed how 1 enters the PSMA funnel region and how pharmacophore Glu-urea-Lys interacts with the arginine patch, the S1', and S1 subpockets by forming hydrogen and van der Waals bonds. The chelating moiety of 1, that is, HBED-CC, forms additional stabilizing hydrogen bonding and van der Waals interactions in the arene-binding site. Ligand 2 is diving into the GRPR transmembrane (TM) helical cavity, thereby forming hydrogen bonds through its amidated end, water-mediated hydrogen bonds, and π-π interactions. Our results provide valuable information regarding the molecular mechanisms involved in the interactions of 1 and 2 with PSMA and GRPR, which might be useful for the diagnostic imaging and therapy of PCa and BCa.

Discovery of a novel GRPR antagonist for protection against cisplatin-induced acute kidney injury.

The side effects of acute Kidney Injury (AKI) and nephrotoxicity limit the application of cisplatin in cancer treatment. Inflammation and oxidative stress paly important role in the pathogenesis of cisplatin-induced AKI. Gastrin-releasing peptide receptor (GRPR) plays an important role in inflammatory response. In this study, we designed 34 new Pd176252 analogs, most synthesized compounds could reduce cisplatin-induced HK2 cell death. Of these compounds, 9b had strong binding affinity with GRPR, and significantly increased HK2 cell viability. Compound 9b significantly downregulated the level of creatinine, blood urea nitrogen (BUN), and malondialdehyde (MDA), and recovered the glutathione (GSH) level in cisplatin-induced AKI model. It also decreased the level of kidney injury molecule-1(KIM-1) in vitro and vivo. In the further pathogenesis studies, 9b downregulated level of inflammatory factors (TNF-α, IL-1ß, IL-6 and MCP-1), suppressed the nuclear factor-kappa B (NF-kB) phosphorylation, and decreased GRPR level. The results suggested that ameliorating cisplatin-induced AKI actions of 9b was involved in downregulation of TNF-α, IL-1ß, IL-6, and MCP-1, inhibition of NF-kB activation, and reduction of GRPR and oxidative stress level.

Synaptic control of spinal GRPR+ neurons by local and long-range inhibitory inputs.

Spinal gastrin-releasing peptide receptor-expressing (GRPR+) neurons play an essential role in itch signal processing. However, the circuit mechanisms underlying the modulation of spinal GRPR+ neurons by direct local and long-range inhibitory inputs remain elusive. Using viral tracing and electrophysiological approaches, we dissected the neural circuits underlying the inhibitory control of spinal GRPR+ neurons. We found that spinal galanin+ GABAergic neurons form inhibitory synapses with GRPR+ neurons in the spinal cord and play an important role in gating the GRPR+ neuron-dependent itch signaling pathway. Spinal GRPR+ neurons also receive inhibitory inputs from local neurons expressing neuronal nitric oxide synthase (nNOS). Moreover, spinal GRPR+ neurons are gated by strong inhibitory inputs from the rostral ventromedial medulla. Thus, both local and long-range inhibitory inputs could play important roles in gating itch processing in the spinal cord by directly modulating the activity of spinal GRPR+ neurons.

PET Probes for Preclinical Imaging of GRPR-Positive Prostate Cancer: Comparative Preclinical Study of [68Ga]Ga-NODAGA-AMBA and [44Sc]Sc-NODAGA-AMBA.

Gastrin-releasing peptide receptors (GRPR) are overexpressed in prostate cancer (PCa). Since bombesin analogue aminobenzoic-acid (AMBA) binds to GRPR with high affinity, scandium-44 conjugated AMBA is a promising radiotracer in the PET diagnostics of GRPR positive tumors. Herein, the GRPR specificity of the newly synthetized [44Sc]Sc-NODAGA-AMBA was investigated in vitro and in vivo applying PCa PC-3 xenograft. After the in-vitro assessment of receptor binding, PC-3 tumor-bearing mice were injected with [44Sc]Sc/[68Ga]Ga-NODAGA-AMBA (in blocking studies with bombesin) and in-vivo PET examinations were performed to determine the radiotracer uptake in standardized uptake values (SUV). 44Sc/68Ga-labelled NODAGA-AMBA was produced with high molar activity (approx. 20 GBq/µmoL) and excellent radiochemical purity. The in-vitro accumulation of [44Sc]Sc-NODAGA-AMBA in PC-3 cells was approximately 25-fold higher than that of the control HaCaT cells. Relatively higher uptake was found in vitro, ex vivo, and in vivo in the same tumor with the 44Sc-labelled probe compared to [68Ga]Ga-NODAGA-AMBA. The GRPR specificity of [44Sc]Sc-NODAGA-AMBA was confirmed by significantly (p ≤ 0.01) decreased %ID and SUV values in PC-3 tumors after bombesin pretreatment. The outstanding binding properties of the novel [44Sc]Sc-NODAGA-AMBA to GRPR outlines its potential to be a valuable radiotracer in the imaging of GRPR-positive PCa.

The Neurokinin-1 Receptor is Expressed with Gastrin-Releasing Peptide Receptor in Spinal Interneurons and Modulates Itch.

The neurokinin-1 receptor (NK1R; encoded by Tacr1) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch in rodents. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. To address this limitation, we leveraged a newly developed Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization (FISH) to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn (DDH), as well as the lateral spinal nucleus (LSN), of mouse and human spinal cord. Retrograde labeling studies in mice from the parabrachial nucleus (PBN) show that less than 20% of superficial Tacr1CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn (SDH) neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry.SIGNIFICANCE STATEMENT The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons process itch input and how itch signals are encoded within the spinal cord is not fully understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These NK1R neurons comprise a subset of gastrin-releasing peptide receptor (GRPR) interneurons and are thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in human spinal cord, underscoring the translational relevance of our findings in mice. This work is the first to suggest a role for NK1R interneurons in itch and extends our understanding of the complexities of spinal itch circuitry.

Are heterobivalent GRPR- and VPAC1R-bispecific radiopeptides suitable for efficient in vivo tumor imaging of prostate carcinomas?

Receptor-specific peptides labeled with positron emitters play an important role in the clinical imaging of several malignancies by positron emission tomography (PET). Radiolabeled heterobivalent bispecific peptidic ligands (HBPLs) can target more than one receptor type and by this - besides exhibiting other advantages - increase tumor imaging sensitivity. In the present study, we show the initial in vivo evaluation of the most potent heterobivalent gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-bispecific radiotracer and determined its tumor visualization potential via PET/CT imaging. For this purpose, the most potent described HBPL was synthesized together with its partly scrambled heterobivalent monospecific homologs and its monovalent counterparts. The agents were efficiently labeled with 68Ga3+ and evaluated in an initial PET/CT tumor imaging study in a human prostate carcinoma (PCa) xenograft rat tumor model established for this purpose. None of the three 68Ga-HBPLs enabled a clear tumor visualization and a considerably higher involvement in receptor-mediated uptake was found for the GRPR-binding part of the molecule than for the VPAC1R-binding one. Of the monovalent radiotracers, only [68Ga]Ga-NODA-GA-PESIN could efficiently delineate the tumor, confirming the results. Thus, this work sets the direction for future developments in the field of GRPR- and VPAC1R-bispecific radioligands, which should be based on other VPAC1R-specific peptides than PACAP-27.

Targeting GRP receptor: Design, synthesis and preliminary biological characterization of new non-peptide antagonists of bombesin.

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues' side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.

New insights into the mechanisms behind mechanical itch.

Gentle tactile stimuli, such as insects crawling on the skin, can cause itching sensation called mechanical itch. Recent studies have begun to shed light on the neural mechanisms of mechanical itch. Interestingly, the neural pathway for mechanical itch is apparently different from that for chemical itch triggered by the activation of pruriceptors with various mediators. Mechanical itch dysesthesia is frequently seen in patients with chronic itch. Mechanisms of this dysesthesia are plausibly involved in central sensitization. In this review, we summarize the current knowledge of mechanical itch under normal and pathological conditions.

In Vitro Evaluation and Biodistribution Studies of HPMA Copolymers Targeting the Gastrin Releasing Peptide Receptor in Prostate Cancer.

PURPOSE: The development of diagnostic and therapeutic agents utilizing small peptides (e.g., bombesin (BBN)) to target the overexpression of the gastrin-releasing peptide receptor (GRPR) in cancers has been widely investigated. Herein, we examine the capabilities of BBN-modified HPMA copolymers to target the GRPR. METHODS: Four positive, four negative, and two zwitterionic BBN HPMA copolymer conjugates of varying peptide content and charge were synthesized. In vitro and in vivo studies were conducted in a GRPR-overexpressing prostate cancer cell line (PC-3) and a normal CF-1 mouse model, respectively. RESULTS: Cellular uptake of the conjugates were found to be charge and BBN density dependent. The positively-charged conjugates illustrated a direct relationship between the extent of cellular internalization, ranging from 0.7 to 20%, and BBN-incorporation density. The negative and zwitterionic conjugates showed low PC-3 uptake values. Blocking studies confirmed the GRPR-targeting effect of the positively-charged constructs. In vivo studies of the positively-charged copolymers resulted in rapid blood clearance by the mononuclear phagocyte system (MPS)-associated tissues (e.g., liver and spleen). CONCLUSION: Positively-charged BBN-HPMA copolymer conjugates demonstrated good GRPR-targeting and internalization in vitro. However, the impact of peptide density and charge on in vivo MPS recognition are parameters that must be optimized in future agent development.

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.

Peptide-Conjugated Long-Lived Theranostic Imaging for Targeting GRPr in Cancer and Immune Cells.

Gastrin-releasing peptide receptor (GRPr) plays proliferative and inflammatory roles in living systems. Here, we report a highly selective GRPr antagonist (JMV594)-tethered iridium(III) complex for probing GRPr in living cancer cells and immune cells. This probe exhibited desirable photophysical properties and also displayed negligible cytotoxicity, overcoming the inherent toxicity of the iridium(III) complex. Its long emission lifetime enabled its luminescence signal to be readily distinguished from the interfering fluorescence of organic dyes by using a time-resolved technique. This probe selectively visualized living cancer cells via specific binding to GRPr, while it also modulated the function of GRPr on TNF-α secretion in immune cells. To our knowledge, this is the first peptide-conjugated iridium(III) complex developed as a GRPr bioimaging probe and modulator of GRPr activity. This theranostic agent shows great potential at unmasking the diverse roles of GRPr in living systems.