Gastrin-releasing peptide receptor as theranostic target in estrogen-receptor positive breast cancer: A preclinical study of the theranostic pair [55Co]Co- and [177Lu]Lu-DOTA-RM26.

BACKGROUND: Patients with advanced metastatic estrogen receptor-positive breast cancer often develop resistance to standard treatments, leading to uncontrolled progression. Thus, innovative therapies are urgently needed. The gastrin-releasing peptide receptor (GRPR) is overexpressed in various cancers, including breast cancer, making it an interesting theranostic target. RM26, a GRPR-targeting antagonist, has demonstrated promising in vivo kinetics in prostate cancer models. This study evaluated the theranostic capabilities of [55Co]Co-/[177Lu]Lu-DOTA-RM26 in vitro in estrogen receptor-positive breast cancer cells and assessed the diagnostic potential of [55Co]Co-DOTA-RM26 in vivo in a breast cancer mouse model. METHODS: We analyzed the binding specificity of [57Co]Co-/[177Lu]Lu-DOTA-RM26 in T47D breast cancer cells, using [57Co]Co-DOTA-RM26 as a surrogate for [55Co]Co-DOTA-RM26. The therapeutic efficacy of increasing [177Lu]Lu-DOTA-RM26 concentrations was determined via viability assay in vitro. Ex vivo biodistribution of [57Co]Co-DOTA-RM26 (17.2 ± 2.7 kBq, 33 ± 5.2 pmol/mouse) was investigated in 12 mice (n= 4/group) with orthotopic breast cancer tumors. The mice were sacrificed at 4 and 24 h post-injection (pi), including a blocking group (20 nmol of unlabeled [Tyr4]-Bombesin) at 4 h pi. For imaging, two tumor-bearing mice underwent [55Co]Co-DOTA-RM26 PET/CT, 4 and 24 h pi (2.8 ± 0.2 MBq, 167.5 ± 0.5 pmol/mouse), with or without GRPR blocking. RESULTS: In vitro studies revealed high, specific binding of [57Co]Co-DOTA-RM26 (43 ± 1 % of total added activity per 106 cells (%IA/106)) and [177Lu]Lu-DOTA-RM26 (37 ± 4 %IA/106). The activity was predominantly localized at the cell surface: 71 ± 3 % and 80 ± 6 % for [57Co]Co-DOTA-RM26 and [177Lu]Lu-DOTA-RM26, respectively. [177Lu]Lu-DOTA-RM26 significantly reduced cell viability at all activity concentrations >0.625 MBq/mL (p < 0.0001), with cell viability below 1 % at concentrations ≥5 MBq/mL. Biodistribution data (n = 12) indicated a high, specific tumor uptake of [57Co]Co-DOTA-RM26, surpassing all other tissues significantly at both time points, 3.7 ± 0.6 % of the injected activity per gram (%IA/g) 4 h pi and 0.98 ± 0.05 %IA/g 24 h pi. The kidneys showed the second-highest uptake (2.0 ± 0.1 %IA/g 4 h pi), followed by the pancreas (1.4 ± 0.4 %IA/g 4 h pi). PET/CT imaging with [55Co]Co-DOTA-RM26 supported the biodistribution data and, distinctly visualized the tumor 24 h pi and showed an improved tumor-to-background compared to the earlier time points. Effective GRPR blocking significantly reduced tumor uptake in the PET images 24 h pi. CONCLUSION: These findings suggest that the theranostic pair [55Co]Co-/[177Lu]Lu-DOTA-RM26 holds significant promise as a theranostic agent for estrogen receptor-positive breast cancer.

Linking GERD and the Peptide Bombesin: A New Therapeutic Strategy to Modulate Inflammatory, Oxidative Stress and Clinical Biochemistry Parameters.

Gastroesophageal reflux disease (GERD) represents one of the most prevalent foregut illnesses, affecting a large portion of individuals worldwide. Recent research has shown that inflammatory mediators such as cytokines, chemokines, and enzymes are crucial for causing esophageal mucosa alterations in GERD patients. It seems likely that the expression of various cytokines in the esophageal mucosa also induces oxidative stress by increasing the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). As humoral agents and peptidergic neurotransmitters that may support the enterogastric axis, bombesin and its related bombesin-like peptide, GRP (gastrin releasing peptide), have not been fully investigated. Therefore, considering all these assumptions, this study aimed to evaluate the influence of bombesin in reestablishing biochemical markers linked with inflammation and oxidative/nitrosative stress in GERD pathological settings. C57BL/6 mice were alternatively overfed and fasted for 56 days to induce GERD and then treated with bombesin (0.1, 0.5, and 1 mg/kg intraperitoneally) once daily for 7 days, and omeprazole was used as the positive control. After 7 days of treatment, gastric pain and inflammatory markers were evaluated. Abdominal pain was significantly reduced following bombesin administration, which was also successful in diminishing inflammatory and oxidative/nitrosative stress markers in a manner overlapping with omeprazole. Moreover, bombesin was also able to appreciably modulate gastric pH as a result of the restoration of gastric homeostasis. Overall, these observations indicated that the upregulation of bombesin and interconnected peptides is a promising alternative approach to treat GERD patients.

In vitro and in vivo evaluation of Bombesin-MMAE conjugates for targeted tumour therapy.

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. The upregulation of the bombesin receptor 2 (BB2) in several malignancies and the advantages offered by peptide drug conjugates over antibody drug conjugates in terms of production and tumour targeting motivated us to synthesise and test bombesin conjugates armed with the tubulin binder monomethyl auristatin E. The widely used Val-Cit-PABC was initially included as cathepsin cleavable self-immolative linker for the release of the free drug. However, the poor stability of the Val-Cit-conjugates in mouse plasma encouraged us to consider the optimised alternatives Glu-Val-Cit-PABC and Glu-Gly-Cit-PABC. Conjugate BN-EVcM1, featuring Glu-Val-Cit-PABC, combined suitable stability (t(½) in mouse and human plasma: 8.4 h and 4.6 h, respectively), antiproliferative activity in vitro (IC50 = 29.6 nM on the human prostate cancer cell line PC-3) and the full release of the free payload within 24 h. Three conjugates, namely BN-EGcM1, BN-EVcM1 and BN-EVcM2, improved the accumulation of MMAE in PC-3 human prostate cancer xenograft mice models, compared to the administration of the free drug. Among them, BN-EVcM1 also stood out for the significantly extended survival of mice in in vivo acute efficacy studies and for the significant inhibition of the growth of a PC-3 tumour in mice in both acute and chronic efficacy studies.

Utility of 64Cu-Sarcophagine-Bombesin PET/CT in Men with Biochemically Recurrent Prostate Cancer and Negative or Equivocal Findings on 68Ga-PSMA-11 PET/CT.

Despite a high detection rate of 68Ga-prostate-specific membrane antigen (PSMA) PET/CT in biochemical recurrence (BCR) of prostate cancer, a significant proportion of men have negative 68Ga-PSMA-11 PET/CT results. Gastrin-releasing peptide receptor, targeted by the copper-chelated bombesin analog 64Cu-sarcophagine-bombesin (SAR-BBN) PET/CT, is also overexpressed in prostate cancer. In this prospective imaging study, we investigate the detection rate of 64Cu-SAR-BBN PET/CT in patients with BCR and negative or equivocal 68Ga-PSMA-11 PET/CT results. Methods: Men with confirmed adenocarcinoma of the prostate, prior definitive therapy, and BCR (defined as a prostate-specific antigen [PSA] level > 0.2 ng/mL) with negative or equivocal 68Ga-PSMA-11 PET/CT results within 3 mo were eligible for enrollment. 64Cu-SAR-BBN PET/CT scans were acquired at 1 and 3 h after administration of 200 MBq of 64Cu-SAR-BBN, with further delayed imaging undertaken optionally at 24 h. PSA (ng/mL) was determined at baseline. All PET (PSMA and bombesin) scans were assessed visually. Images were read with masking of the clinical results by 2 experienced nuclear medicine specialists, with a third reader in cases of discordance. Accuracy was defined using a standard of truth that included biopsy confirmation, confirmatory imaging, or response to targeted treatment. Results: Twenty-five patients were enrolled. Prior definitive therapy was radical prostatectomy (n = 24, 96%) or radiotherapy (n = 1, 4%). The median time since definitive therapy was 7 y (interquartile range [IQR], 4-11 y), and the Gleason score was 7 or less (n = 15, 60%), 8 (n = 3, 12%), or 9 (n = 7, 28%). The median PSA was 0.69 ng/mL (IQR, 0.28-2.45 ng/mL). Baseline PSMA PET scans were negative in 19 patients (76%) and equivocal in 6 (24%). 64Cu-SAR-BBN PET-avid disease was identified in 44% (11/25): 12% (3/25) with local recurrence, 20% (5/25) with pelvic node metastases, and 12% (3/25) with distant metastases. The κ-score between readers was 0.49 (95% CI, 0.16-0.82). Patients were followed up for a median of 10 mo (IQR, 9-12 mo). Bombesin PET/CT results were true-positive in 5 of 25 patients (20%), false-positive in 2 of 25 (8%), false-negative in 7 of 25 (28%), and unverified in 11 of 25 (44%). Conclusion: 64Cu-SAR-BBN PET/CT demonstrated sites of disease recurrence in 44% of BCR cases with negative or equivocal 68Ga-PSMA-11 PET/CT results. Further evaluation to confirm diagnostic benefit is warranted.

Structural insights into ligand recognition, selectivity, and activation of bombesin receptor subtype-3.

Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gq protein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3's selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3's ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.

Bombesin protects myocardium against ischemia/reperfusion injury via activation of the Keap1-Nrf2-HO-1 signaling pathway.

AIMS: It has been reported that some peptides released by the gastro-intestinal tract play important roles in the prevention of myocardial ischemia/reperfusion injury (MIRI). Bombesin (BN) is a biologically active peptide released by non-adrenergic non-cholinergic nerves on the gastric antrum mucosa controlled by the vagus nerve. However, there is a lack of reports on the impact of BN on MIRI. This study aimed to explore the influence of BN on MIRI and its underlying mechanism. MATERIALS AND METHODS: MIRI was induced by either 30 min of global ischemia in Langendorff perfused rat hearts, or by ligation of the descending coronary artery for 30 min in anesthetized Spraque-Dawley rats, and both were followed by 120 min reperfusion. Infarct size and left ventricular function were assessed, and lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) levels were measured spectrophotometrically, while cardiomyocyte apoptosis was detected by TUNEL assay. The content of BN in plasma was measured with enzyme-linked immunosorbent assays (ELISA). The expression of caspase 3, Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were quantified. KEY FINDINGS: BN and vagus nerve stimulation improved cardiac contractile function and reduced myocardial infarct size, attenuated oxidative stress damage and myocardial cell apoptosis, increased the expression of Keap1, Nrf2, and HO-1. and these effects were blocked by using a BN receptor antagonist. SIGNIFICANCE: BN provides protection against MIRI, and its underlying mechanism is through activation of the Keap1/Nrf2/HO-1 pathway. This research provides more reliable evidence for the "gut-heart axis dialogue" and explores potential therapeutic approaches for MIRI.

Bimodal MRI/Fluorescence Nanoparticle Imaging Contrast Agent Targeting Prostate Cancer.

We developed a novel site-specific bimodal MRI/fluorescence nanoparticle contrast agent targeting gastrin-releasing peptide receptors (GRPrs), which are overexpressed in aggressive prostate cancers. Biocompatible ultra-small superparamagnetic iron oxide (USPIO) nanoparticles were synthesized using glucose and casein coatings, followed by conjugation with a Cy7.5-K-8AOC-BBN [7-14] peptide conjugate. The resulting USPIO(Cy7.5)-BBN nanoparticles were purified by 100 kDa membrane dialysis and fully characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, and magnetic resonance imaging (MRI) relaxivity, as well as evaluated for in vitro and in vivo binding specificity and imaging efficacy in PC-3 prostate cancer cells and xenografted tumor-bearing mice. The USPIO(Cy7.5)-BBN nanoparticles had a core diameter of 4.93 ± 0.31 nm and a hydrodynamic diameter of 35.56 ± 0.58 nm. The r2 relaxivity was measured to be 70.2 ± 2.5 s-1 mM-1 at 7T MRI. The Cy7.5-K-8AOC-BBN [7-14] peptide-to-nanoparticle ratio was determined to be 21:1. The in vitro GRPr inhibitory binding (IC50) value was 2.5 ± 0.7 nM, indicating a very high binding affinity of USPIO(Cy7.5)-BBN to the GRPr on PC-3 cells. In vivo MRI showed significant tumor-to-muscle contrast enhancement in the uptake group at 4 h (31.1 ± 3.4%) and 24 h (25.7 ± 2.1%) post-injection compared to the blocking group (4 h: 15.3 ± 2.0% and 24 h: -2.8 ± 6.8%; p < 0.005). In vivo and ex vivo near-infrared fluorescence (NIRF) imaging revealed significantly increased fluorescence in tumors in the uptake group compared to the blocking group. These findings demonstrate the high specificity of bimodal USPIO(Cy7.5)-BBN nanoparticles towards GRPr-expressing PC-3 cells, suggesting their potential for targeted imaging in aggressive prostate cancer.

Radiolabeled peptides and their expanding role in clinical imaging and targeted cancer therapy.

There is an expanding body of evidence showing that synthetic peptides in combination with radioactive isotopes can be utilized for medical purposes. This area is of particular interest in oncology where applications in diagnosis and therapy are at different stages of development. We review the contributions in this area by the group originally founded by Carlo Pedone in Naples many years ago. We highlight the work of this group in the context of other developments in this area, focusing on three biologically relevant receptor systems: somatostatin, gastrin-releasing peptide, and cholecystokinin-2/gastrin receptors. We focus on key milestones, state of the art, and challenges in this area of research as well as the current and future outlook for expanding clinical applications.

Biodistribution and radiation dosimetry of [99mTc]Tc-N4-BTG in patients with biochemical recurrence of prostate cancer.

BACKGROUND: In patients with prostate cancer (PCa), imaging with gastrin-releasing peptide receptor (GRPR) ligands is an alternative to PSMA-targeted tracers, particularly if PSMA expression is low or absent. [99mTc]Tc-N4-BTG is a newly developed GRPR-directed probe for conventional scintigraphy and single photon emission computed tomography (SPECT) imaging. The current study aims to investigate the safety, biodistribution and dosimetry of [99mTc]Tc-N4-BTG in patients with biochemical recurrence (BCR) of PCa. RESULTS: No adverse pharmacologic effects were observed. Injection of [99mTc]Tc-N4-BTG resulted in an effective dose of 0.0027 ± 0.0002 mSv/MBq. The urinary bladder was the critical organ with the highest mean absorbed dose of 0.028 ± 0.001 mGy/MBq, followed by the pancreas with 0.0043 ± 0.0015 mGy/MBq, osteogenic cells with 0.0039 ± 0.0005 mGy/MBq, the kidneys with 0.0034 ± 0.0003 mGy/MBq, and the liver with 0.0019 ± 0.0004 mGy/MBq, respectively. No focal tracer uptake suggestive of PCa recurrence could be revealed for any of the patients. CONCLUSION: [99mTc]Tc-N4-BTG appears to be a safe diagnostic agent. Compared to GRPR-targeted PET tracers, this 99mTc-labelled SPECT agent could contribute to a broader application and better availability of this novel approach. Further research to assess its clinical value is warranted.

Gastrin Releasing Peptide Receptors-targeted PET Diagnostics and Radionuclide Therapy for Prostate Cancer Management: Preclinical and Clinical Developments of the Past 5 Years.

Each tumor has its own distinctive molecular identity. Treatment, therefore, should be tailored to this unique cancer phenotype. Theragnostics uses the same compound for targeted imaging and treatment, radiolabeled to an appropriate radionuclide, respectively. Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, and radiolabeled GRPR antagonists have shown high diagnostic performance at staging and biochemical recurrence. Several GRPR-targeting theragnostic compounds have been developed preclinically. Their translation into clinics is underway with 4 clinical trials recruiting participants with GRPR-expressing tumors.

Stabilizing Scaffold for Short Peptides Based on Knottins.

BACKGROUND: Bombesin (BBN) is a short peptide with a high affinity for receptors that are expressed on the surface of various types of cancer cells. However, a full length BBN molecule has low in vivo stability. OBJECTIVE: In our study, we propose the use of peptide toxins, derived from animal and plant toxins, as scaffold molecules to enhance the bioavailability and stability of bombesin. These peptides possess a unique structure known as an inhibitory cystine knot. METHODS: We synthesized structures in which short bombesin was incorporated into various domains of arthropod and plant toxins using solid-phase peptide synthesis. The stability under different conditions was assessed through high-performance liquid chromatography, and binding to cell cultures expressing the bombesin receptor was analyzed. Additionally, toxicity to cell cultures was evaluated using fluorescence microscopy. RESULTS: The data obtained demonstrated that placing the short peptide between the first and second cysteine residues in arachnid toxins results in increased in vitro stability and bioavailability, as well as low cytotoxicity. CONCLUSION: Arachnid toxins with an inhibitory cystine knot can be considered as a scaffold for increasing the stability of therapeutic peptides.

Assessment of the bound conformation of bombesin to the BB1 and BB2 receptors.

Bombesin is an endogenous peptide involved in a wide spectrum of physiological activities ranging from satiety, control of circadian rhythm and thermoregulation in the central nervous system, to stimulation of gastrointestinal hormone release, activation of macrophages and effects on development in peripheral tissues. Actions of the peptide are mediated through the two high affinity G-protein coupled receptors BB1R and BB2R. Under pathophysiological conditions, these receptors are overexpressed in many different types of tumors, such as prostate cancer, breast cancer, small and non-small cell lung cancer and pancreatic cancer. This observation has been used for designing cell markers, but it has not been yet exploited for therapeutical purposes. Despite the enormous biological interest of the peptide, little is known about the stereochemical features that contribute to their activity. On the one hand, mutagenesis studies identified a few receptor residues important for high bombesin affinity and on the other, a few studies focused on the relevance of diverse residues of the peptide for receptor activation. Models of the peptide bound to BB1R and BB2R can be helpful to improve our understanding of the stereochemical features granting bombesin activity. Accordingly, the present study describes the computational process followed to construct such models by means of Steered Molecular Dynamics, using models of the peptide and its receptors. Present results provide new insights into the structure-activity relationships of bombesin and its receptors, as well as render an explanation for the differential binding affinity observed towards BB1R and BB2R. Finally, these models can be further exploited to help for designing novel small molecule peptidomimetics with improved pharmacokinetics profile.

Towards Cancer Nanoradiopharmaceuticals-Radioisotope Nanocarrier System for Prostate Cancer Theranostics Based on Radiation-Synthesized Polymer Nanogels.

Despite the tremendous development of oncology, prostate cancer remains a debilitating malignancy. One of the most promising approaches to addressing this issue is to exploit the advancements of nanomedicine in combination with well-established nuclear medicine and radiotherapy. Following this idea, we have developed a radioisotope nanocarrier platform of electron-beam-synthesized nanogels based on poly(acrylic acid). We have developed a functionalization protocol, showing the very high (>97%) efficiency of the conjugation in targeting a ligand-bombesin derivative. This engineered peptide can bind gastrin-releasing peptide receptors overexpressed in prostate cancer cells; moreover, it bears a radioisotope-chelating moiety. Our nanoplatform exhibits very promising performance in vitro; the radiolabeled nanocarriers maintained high radiochemical purity of >90% in both the labeling buffer and human serum for up to 14 days. The application of the targeted nanocarrier allowed also effective and specific uptake in PC-3 prostate cancer cells, up to almost 30% after 4 h, which is a statistically significant improvement in comparison to carrier-free radiolabeled peptides. Although our system requires further studies for more promising results in vivo, our study represents a vital advancement in radionanomedicine-one of many steps that will lead to effective therapy for castration-resistant prostate cancer.

Radiometal Complexes as Pharmacokinetic Modifiers: A Potent 68Ga-Labeled Gastrin-Releasing Peptide Receptor Antagonist Based on the Macrocyclic Metal Chelator NODIA-Me.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in various cancer types including prostate and breast carcinomas, making it an attractive target for molecular imaging and therapy. In this work, we designed a novel GRPr antagonistic probe comprising metal chelator NODIA-Me. This 1,4,7-triazacyclononane-based chelator forms positively charged metal complexes due to its neutral methylimidazole arms. Because a positive charge at the N-terminus of GRPr conjugates is responsible for high receptor affinity as exemplified by the current gold standard DOTA-RM2, we investigated if a positively charged radiometal complex can be used as a pharmacokinetic modifier to also produce high-affinity GRPr conjugates. In this respect, the bioconjugate NODIA-Me-Ahx-JMV594 was prepared by a combination of solid-phase peptide synthesis and solution-based reactions in a 94% yield. Radiolabeling provided the 68Ga-labeled conjugate in radiochemical yields of >95% and radiochemical purities of >98% with mean molar activities of Am ∼17 MBq nmol-1. The competitive GRPr affinity of the metal-free and 69/71Ga-labeled conjugate was determined to be IC50 = 0.41 ± 0.06 and 1.45 ± 0.06 nM, respectively. The metal-free GRPr antagonist DOTA-RM2 and its corresponding 69/71Ga complex had IC50 values of 1.42 ± 0.07 and 0.98 ± 0.19 nM, respectively. Small-animal PET imaging of mice bearing GRPr(+) PC-3 tumors revealed high radioactivity accumulation in the tumors and in the pancreas as an organ with high levels of GRPr expression. These findings were corroborated by the corresponding ex vivo biodistribution data, in which the tumors and the pancreas exhibited the highest radioactivity accumulation. By coinjection of an excess of NODIA-Me-Ahx-JMV594, uptake in the tumors and GRPr(+) organs was significantly reduced, confirming specific receptor-mediated uptake. The estrogen receptor-positive tumor of a female breast cancer patient was clearly visualized by PET imaging using 68Ga-labeled NODIA-Me-Ahx-JMV594. To summarize, the positive charge at the N-terminus of the conjugate induced by the Ga(NODIA-Me) complex resulted in high GRPr affinity comparable to that of the potent antagonist DOTA-RM2. The conjugate NODIA-Me-Ahx-JMV594 is a promising probe for imaging of GRPr tumors that warrants further evaluation in larger patient cohorts as well as in combination with other radiometals.

Bombesins: A New Frontier in Hybrid Compound Development.

Recently, bombesin (BN) and its analogs have attracted much attention as excellent anticancer agents because they interact with specific receptors widely distributed on the surface of various cancer cells. However, their biological properties proceed far beyond this, given a broad spectrum of activity. Bombesin receptor ligands are effective drugs for the treatment of rheumatoid arthritis or gastrointestinal diseases. However, most diseases are complex, and the use of polytherapy may lead to pharmacokinetic and pharmacodynamic drug-drug interactions, resulting in side effects. Therefore, there is a need to develop effective compounds that also contain BN or its analogs, which are combined with other structural entities, thus generating a so-called hybrid drug. Hybrid drugs that contain bombesin pharmacophore(s) may be proposed as a solution to the problem of polytherapy or the lack of an effective cure. Such structures have now demonstrated the desired efficacy, though information on these aforementioned compounds is relatively scarce. Therefore, our paper aims to encourage researchers to focus on bombesins. Herein, we indicate that the hybrid approach should also be firmly applied to bombesins and the BN receptor family. This paper's structure is divided into two main sections demonstrating bombesins and their properties, as well as recent data on bombesin-based hybrid compounds and their potential usefulness in medicine. Overall, it refers to the discovery and synthesis of modified bombesin-based hybrid compounds.

GRPR-targeting radiotheranostics for breast cancer management.

Breast Cancer (BC) is the most common cancer worldwide and, despite the advancements made toward early diagnosis and novel treatments, there is an urgent need to reduce its mortality. The Gastrin-Releasing Peptide Receptor (GRPR) is a promising target for the development of theranostic radioligands for luminal BC with positive estrogen receptor (ER) expression, because GRPR is expressed not only in primary lesions but also in lymph nodes and distant metastasis. In the last decades, several GRPR-targeting molecules have been evaluated both at preclinical and clinical level, however, most of the studies have been focused on prostate cancer (PC). Nonetheless, given the relevance of non-invasive diagnosis and potential treatment of BC through Peptide Receptor Radioligand Therapy (PRRT), this review aims at collecting the available preclinical and clinical data on GRPR-targeting radiopeptides for the imaging and therapy of BC, to better understand the current state-of-the-art and identify future perspectives and possible limitations to their clinical translation. In fact, since luminal-like tumors account for approximately 80% of all BC, many BC patients are likely to benefit from the development of GRPR-radiotheranostics.

Knockout of the BRAP homolog in mice leads to abnormal tracheal cilia.

Both bombesin receptor-activated protein (BRAP) and its mouse homolog have been found to be expressed in bronchial epithelia but with unclear functions. Using electron microscopy combined with histological assays, we found that BRAP homolog deficiency in mice led to abnormal tracheal cilia. Rab-3A-interacting protein (Rabin8), a protein that might play a role in cilia development, was screened by yeast two-hybrid and further verified to have interaction with human BRAP by co-immunoprecipitation and pulldown assays. The expression levels of Rabin8, together with acetylated α-tubulin, a marker of cilia, were either downregulated by knockdown of BRAP or upregulated by overexpression of BRAP in cultured immortalized human bronchial epithelial cells. These results reveal a role for BRAP in airway cilia formation.

The effects of novel macrocyclic chelates on the targeting properties of the 68Ga-labeled Gastrin releasing peptide receptor antagonist RM2.

BACKGROUND: The gastrin-releasing peptide receptor (GRPr) is a molecular target for the visualization of prostate cancer. Bombesin (BN) analogs are short peptides with a high affinity for GRPr. RM2 is a bombesin-based antagonist. It has been demonstrated that RM2 have superior in vivo biodistribution and targeting properties than high-affinity receptor agonists. This study developed new RM2-like antagonists by introducing the novel bifunctional chelators AAZTA5 and DATA5m to RM2. RESULTS: The effects of different macrocyclic chelating groups on drug targeting properties and the possibility of preparing 68Ga-radiopharmaceuticals in a kit-based protocol were investigated using 68Ga-labeled entities. Both new RM2 variants were labelled with 68Ga3+ resulting in high yields, stability, and low molarity of the ligand. DATA5m-RM2 and AAZTA5-RM2 incorporated 68Ga3+ nearly quantitatively at room temperature within 3-5 min, and the labelling yield for 68Ga-DOTA-RM2 was approximately 10% under the same conditions. 68Ga-AAZTA5-RM2 showed stronger hydrophilicity according to partition coefficient. Although the maximal cellular uptake values of the three compounds were similar, 68Ga-AAZTA5-RM2 and 68Ga-DATA5m-RM2 peaked more rapidly. Biodistribution studies showed high and specific tumor uptake, with a maximum of 9.12 ± 0.81 percentage injected activity per gram of tissue (%ID/g) for 68Ga-DATA5m-RM2 and 7.82 ± 0.61%ID/g for 68Ga-AAZTA5-RM2 at 30 min after injection. CONCLUSIONS: The conditions for complexation of DATA5m-RM2 and AAZTA5-RM2 with gallium-68 are milder, faster and require less amount of precursors than DOTA-RM2. Chelators had an evident influence on the pharmacokinetics and targeting properties of 68Ga-X-RM2 derivatives. Positively charged 68Ga-DATA5m-RM2 provided a high tumor uptake, high image contrast and good capability of targeting GRPr.

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.

Targeting the Gastrin-Releasing Peptide Receptor (GRP-R) in Cancer Therapy: Development of Bombesin-Based Peptide-Drug Conjugates.

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide-drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.