Bombesin Antagonist Research Articles

Preclinical assessment of IRDye800CW-labeled gastrin-releasing peptide receptor-targeting peptide for near infrared-II imaging of brain malignancies.

We aimed to develop a new biocompatible gastrin-releasing peptide receptor (GRPR) targeted optical probe, IRDye800-RM26, for fluorescence image-guided surgery (FGS) of brain malignancies in near-infrared window II (NIR-II) imaging. We developed a novel GRPR targeting probe using a nine-amino-acid bombesin antagonist analog RM26 combined with IRDye800CW, and explored the fluorescent probe according to optical properties. Fluorescence imaging characterization in NIR-I/II region was performed in vitro and in vivo. Following simulated NIR-II image-guided surgery, we obtained time-fluorescent intensity curves and time-signal and background ratio curves. Further, we used histological sections of brain from tumor-beating mice model to compare imaging specificity between 5-aminolevulinic acid (5-ALA) and IRDye800-RM26, and evaluated biodistribution and biocompatibility. IRDye800-RM26 had broad emission ranging from 800 to 1200 nm, showing considerable fluorescent intensity in NIR-II region. High-resolution NIR-II imaging of IRDye800-RM26 can enhance the advantages of NIR-I imaging. Dynamic and real time fluorescence imaging in NIR-II region showed that the probe can be used to treat brain malignancies in mice between 12 and 24 h post injection. Its specificity in targeting glioblastoma was superior to 5-ALA. Biodistribution analysis indicated IRDye800-RM26 excretion in the kidney and liver. Histological and blood test analyses did not reveal acute severe toxicities in mice treated with effective dose (40 μg) of the probe for NIR-II imaging. Because of the considerable fluorescent intensity in NIR-II region and high spatial resolution, biocompatible and excretable IRDye800-RM26 holds great potentials for FGS, and is essential for translation into human use.

Bioorthogonal Chemistry Approach for the Theranostics of GRPR-Expressing Cancers.

Several gastrin-releasing peptide receptor (GRPR) antagonists with improved in vivo behavior have been recently developed and tested in the clinic. However, despite the generally mild side effects of peptide receptor radionuclide therapy (PRRT), toxicity has been observed due to high doses delivered to nontarget tissues, especially in the kidneys and pancreas. Previous experiences with radiolabeled peptides opened a unique opportunity to explore GRPR pretargeting using clickable bombesin antagonists. Toward this goal, we used clickable DOTA-like radiocomplexes which have been previously evaluated by our group. We functionalized a potent GRPR antagonist with a clickable TCO moiety using two different linkers. These precursors were then studied to select the compound with the highest GRPR binding affinity and the best pharmacokinetics to finally explore the advantages of the devised pretargeting approach. Our results provided an important proof of concept toward the development of bioorthogonal approaches to GRPR-expressing cancers, which are worth investigating further to improve the in vivo results. Moreover, the use of clickable GRPR antagonists and DOTA/DOTAGA derivatives allows for fine-tuning of their pharmacokinetics and metabolic stability, leading to a versatile synthesis of new libraries of (radio)conjugates useful for the development of theranostic tools toward GRPR-expressing tumors.

PH-responsive graphene oxide loaded with targeted peptide and anticancer drug for OSCC therapy.

Oral squamous cell carcinoma (OSCC) is the most common type of cancer occurring in the oral and maxillofacial regions. Despite of the advances in the diagnosis and treatment, the overall 5-year survival rate has remained about 40%–50% in the past decades. Various nanotechnology-based carrier systems have been investigated for their potentials in the OSCC treatment. However, because of the lack of active targeting of tumors, their application is limited. Studies have shown that gastrin-releasing peptide receptors (GRPRs) are overexpressed on many human cancers, including head and neck squamous cell carcinoma. Herein, we aimed to develop a GRPR-targeted nano-graphene oxide (NGO) nanoprobe drug delivery system for OSCC therapy. DOX@NGO-BBN-AF750 was synthesized by the non-covalent bonding method to couple carboxylated NGO with BBN-AF750 (bombesin antagonist peptides conjugated to Alexa Fluor 750) and DOX (doxorubicin) through π-π and hydrogen bonding. Internalization and antitumor activities were carried out in human HSC-3 cancer cells. The tumor pH microenvironment was simulated to study the release of antitumor drug DOX from the DOX@NGO-ant BBN-AF750 complex under different pH conditions. DOX@NGO-BBN-AF750 showed internalization into HSC-3 cells. The IC50 (50% inhibitory concentration) was 5 µg/ml for DOX@NGO-BBN-AF750 in HSC-3 cells. Furthermore, DOX@NGO-BBN-AF750 showed a pH-sensitive drug release rate, and a dose-dependent and pH-responsive cytotoxicity in HSC-3 cells. DOX@NGO-BBN-AF750 presents the characteristics ensuring a slow release of DOX from the nanoprobe, thereby protecting the drug from degradation and prolonging the half-life of the drug. This report provides a versatile strategy to achieving targeted and imaging-guided therapy of OSCC.

Value of 68Ga-labeled bombesin antagonist (RM2) in the detection of primary prostate cancer comparing with [18F]fluoromethylcholine PET-CT and multiparametric MRI—a phase I/II study

ObjectivesThe bombesin derivative RM2 is a GRPr antagonist with strong binding affinity to prostate cancer (PCa). In this study, the impact of [68Ga]Ga-RM2 positron emission tomography-computed tomography (PET-CT) for the detection of primary PCa was compared with that of [18F]FCH PET-CT and multiparametric magnetic resonance imaging (mpMRI).MethodsThis phase I/II study was conducted in 30 biopsy-positive PCa subjects. The patients were stratified into high (10 patients), intermediate (10 patients), and low risk (10 patients) for extraglandular metastases as defined by National Comprehensive Cancer Network (NCCN) criteria (NCCN Clinical Practice Guidelines in Oncology, 2016). The prostate gland was classified in 12 anatomic segments for data analysis of the imaging modalities as well as histopathologic findings. The segment with the highest radiotracer uptake was defined as the “index lesion.” All cases were scheduled to undergo prostatectomy with pelvic lymph node (LN) dissection in intermediate- and high-risk patients. Intraprostatic and pelvic nodal [68Ga]Ga-RM2 and [18F]FCH PET-CT findings were correlated with mpMRI and histopathologic results.ResultsOf the 312 analyzed regions, 120 regions (4 to 8 lesions per patient) showed abnormal findings in the prostate gland. In a region-based analysis, overall sensitivity and specificity of [68Ga]Ga-RM2 PET-CT in the detection of primary tumor were 74% and 90%, respectively, while it was 60% and 80% for [18F]FCH PET-CT and 72% and 89% for mpMRI. Although the overall sensitivity of [68Ga]Ga-RM2 PET-CT was higher compared to that of [18F]FCH PET-CT and mpMRI, the statistical analysis showed only significant difference between [68Ga]Ga-RM2 PET-CT and [18F]FCH PET-CT in the intermediate-risk group (p = 0.01) and [68Ga]Ga-RM2 PET-CT and mpMRT in the high-risk group (p = 0.03). In the lesion-based analysis, there was no significant difference between SUVmax of [68Ga]Ga-RM2 and [18F]FCH PET-CT in the intraprostatic malignant lesions ([68Ga]Ga-RM2: mean SUVmax: 5.98 ± 4.13, median: 4.75; [18F]FCH: mean SUVmax: 6.08 ± 2.74, median: 5.5; p = 0.13).Conclusions[68Ga]Ga-RM2 showed promising PET tracer for the detection of intraprostatic PCa in a cohort of patients with different risk stratifications. However, significant differences were only found between [68Ga]Ga-RM2 PET-CT and [18F]FCH PET-CT in the intermediate-risk group and [68Ga]Ga-RM2 PET-CT and mpMRT in the high-risk group. In addition, GRP-R-based imaging seems to play a complementary role to choline-based imaging for full characterization of PCa extent and biopsy guidance in low- and intermediate-metastatic-risk PCa patients and has the potential to discriminate them from those at higher risks.Key Points• [68Ga]Ga-RM2 is a promising PET tracer with a high detection rate for intraprostatic PCa especially in intermediate-risk prostate cancer patients.• GRPr-based imaging seems to play a complementary role to choline-based or PSMA-based PET/CT imaging in selected low- and intermediate-risk PCa patients for better characterization and eventually biopsy guidance of prostate cancer disease.

Clickable Radiocomplexes With Trivalent Radiometals for Cancer Theranostics: In vitro and in vivo Studies.

Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [90Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages.

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.

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models

IntroductionRadiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. MethodsDOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. ResultsRadiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70–80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). ConclusionsComparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. Advances in knowledge and implications for patient careThe favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

Comparison of biological properties of [177 Lu]Lu-ProBOMB1 and [177 Lu]Lu-NeoBOMB1 for GRPR targeting.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer and other solid malignancies. Following up on our work on [68 Ga]Ga-ProBOMB1 that had better imaging characteristics than [68 Ga]Ga-NeoBOMB1, we investigated the effects of substituting 68 Ga for 177 Lu to determine if the resulting radiopharmaceuticals could be used with a therapeutic aim. We radiolabeled the bombesin antagonist ProBOMB1 (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH2 ) with lutetium-177 and compared it with [177 Lu]Lu-NeoBOMB1 (obtained in 54.2 ± 16.5% isolated radiochemical yield with >96% radiochemical purity and 440.8 ± 165.1 GBq/μmol molar activity) for GRPR targeting. Lu-NeoBOMB1 had better binding affinity for GRPR than Lu-ProBOMB1 (Ki values: 2.26 ± 0.24 and 30.2 ± 3.23nM). [177 Lu]Lu-ProBOMB1 was obtained in 53.7 ± 5.4% decay-corrected radiochemical yield with 444.2 ± 193.2 GBq/μmol molar activity and >95% radiochemical purity. In PC-3 prostate cancer xenograft mice, tumor uptake of [177 Lu]Lu-ProBOMB1 was 3.38 ± 1.00, 1.32 ± 0.24, and 0.31 ± 0.04%ID/g at 1, 4, and 24 hours pi. However, the uptake in tumor was lower than [177 Lu]Lu-NeoBOMB1 at all time points. [177 Lu]Lu-ProBOMB1 was inferior to [177 Lu]Lu-NeoBOMB1, which had better therapeutic index for the organs receiving the highest doses.

Methylthiazolyl Tacn Ligands for Copper Complexation and Their Bifunctional Chelating Agent Derivatives for Bioconjugation and Copper-64 Radiolabeling: An Example with Bombesin.

We present here the synthesis of two new bifunctionalized azachelators, no2th-EtBzNCS and Hno2th1tha, as bioconjugable analogues of two previously described di- and trimethylthiazolyl 1,4,7-triazacyclononane (tacn) ligands, no2th and no3th, for potential uses in copper-64 (64Cu) positron emission tomography imaging. The first one bears an isothiocyanate group on the remaining free nitrogen atom of the tacn framework, while the second one presents an additional carboxylic function on one of the three heterocyclic pendants. Their syntheses required regiospecific N-functionalization of the macrocycles. In order to investigate their suitability for in vivo applications, a complete study of their copper(II) chelation was performed. The acid-base properties of the ligands and their thermodynamic stability constants with copper(II) and zinc(II) cations were determined using potentiometric techniques. Structural studies were conducted in both solution and the solid state, consolidated by theoretical calculations. The kinetic inertness in an acidic medium of both copper(II) complexes was determined by spectrophotometry, while cyclic voltammetry experiments were performed to evaluate the stability at the copper(I) redox state. UV-vis, NMR (of the zinc complexes), electron paramagnetic resonance spectroscopy, and density functional theory studies showed excellent agreement between the solution structures of the complexes and their crystallographic data. These investigations unambiguously prove that these bifunctional derivatives display similar coordination properties as their no2th and no3th counterparts, opening the door to targeted bioapplications. The no2th-EtBzNCS and Hno2th1tha ligands were then conjugated to a bombesin antagonist peptide for targeting the gastrin-releasing peptide receptor (GRPr). To highlight the potential of the two chelators for radiopharmaceutical development, the 64Cu-radiolabeling properties, in vitro stability, and binding affinity to GRPr of the corresponding bioconjugates were determined. Altogether, the results of this work warrant the further development of 64Cu-based radiopharmaceuticals comprising our novel bifunctional chelators.

Design, Synthesis, and in Vitro and in Vivo Evaluation of High Affinity and Specificity Near-Infrared Fluorescent Bombesin Antagonists for Tumor Imaging.

The bombesin (BBN) antagonist binds with high affinity to the gastrin releasing peptide receptor (GRPr), a receptor overexpressed on many human cancers. We present an investigation employing BBN antagonist for highly specific near-infrared fluorescence (NIRF) imaging of GRPr-positive tumors. Nine NIRF-dye labeled BBN antagonists with differing linkers and dyes were synthesized and characterized to screen for the optimal agent. Three novel agents, AF750-G-pip-Sta-BBN (1), AF750-GSG-Sta-BBN (2), and AF750-6Ahx-Sta-BBN (3), exhibited an excellent binding-specificity and affinity to human PC-3 prostate cancer cells in vitro, and a remarkable in vivo tumor-selectivity and NIRF imaging sensitivity in PC-3 tumor-bearing mice. Compound 1 showed the fastest, and 3, the slowest, pharmacokinetics on the tumor sites. Despite of high tumor uptake, 2 had a low pancreas uptake distinct from 1 and 3 at 0.44 nmol dose. This difference was attributed to the inherent linker properties such as the hydrophilicity, polarity, and charge.

Synthesis and biological activity of an Anderson polyoxometalate bis‐functionalized with a Bombesin‐analog peptide

AbstractPolyoxometalates (POMs) are multi metallic and polyanionic oxides whose functionalization with organic moieties can generate new properties. Among possible strategies to generate molecular diversity and find applications in different fields, the postfunctionalization of a POM with peptides is particularly interesting and easily achievable. In this article, we present the functionalization of the Anderson‐Evans polyoxomolybdate ([MnMo6O24]3–) with a Bombesin antagonist peptide, to highlight the interplay between these 2 domains, in terms of structural changes and assembly. Moreover, since Bombesin analogs show a marked binding affinity and specificity for some subtypes of the Gastrin Releasing Peptide Receptor (GRP‐R), the impact of the peptide on the antitumor activity of the Anderson‐Evans polyoxomolybdate POM has been explored.

Bombesin antagonist based radiotherapy of prostate cancer combined with WST-11 vascular targeted photodynamic therapy

Kim et al . (1) from Memorial Sloan-Kettering Cancer Center investigated the possibility to enhance the effect of radionuclide therapy by vascular-targeted photodynamic (VTP) therapy in an experimental prostate cancer animal model. They used bombesin-antagonist peptide as a targetor which is binding gastrin releasing peptide receptor (GRPr) and it was labeled with high energy beta-emitting radionuclide 90 Y and the radionuclide was linked with DOTA-chelate. This peptide receptor radionuclide therapy (PRRT) compound, 90 Y-DOTA-AR (bombesin antagonist) radiopeptide was followed by therapy with WST-11 (Weizmann STeba-11 drug; a negatively charged water-soluble palladium-bacteriochlorophyll derivative, Tookad ® Soluble) application. This VTP approach generates reactive oxygen/nitrogen species within tumor blood vessels, resulting in their instantaneous destruction and causes rapid tumor necrosis. Radionuclide emitting charged particle forms Cherenkov luminescence imaging (CLI) which can be used to monitor the retention of the accumulated 90 Y-PRRT. In this work, they demonstrated that the instantaneous arrest of tumor vasculature trapped radiopharmaceuticals in the vicinity of the tumor and thus improved the efficacy of targeted radiotherapy (1).

Structural Determinants in the Binding of BB2 Receptor Ligands: In Silico, X-Ray and NMR Studies in PD176252 Analogues.

The mammalian bombesin receptor family comprises three G proteincoupled receptors: the neuromedin B receptor, the gastrin-releasing peptide receptor (BB2), and the bombesin receptor subtype 3. BB2 receptor plays a role in gastrointestinal functions; however, at present the role of this subtype in physiological and pathological conditions is unknown due to the lack of specific binders for all subclasses of bombesin receptors. Here, we present a study focused on the properties of the peptoid bombesin antagonist called PD176252, and other structural analogues with the aim to elucidate causes of their different affinity towards the BB2 receptor. By means of computational techniques, based on QSAR, docking and homology building, supported by experimental data (X-ray diffraction and NMR spectroscopy) fresh insights on binding modes of this class of biological targets were achieved.

La TEP au 68Ga-PSMA et autres traceurs TEP

In a close future, PSMA-PET will be a key tool in the management at all stages of patients with prostatic adenocarcinoma. It will be complementary to MRI for the locoregional extension and for the delineation of tumor volumes for external beam radiotherapy. It will supplant choline-PET and standard imaging by CT and bone scintigraphy for both initial staging and restaging. For metastatic diseases, it will select patients for radionuclide therapy with PSMA ligands radiolabeled with electron emitters isotopes and will be able to evaluate the effectiveness. A flare-up effect of hormone therapy seems to exist and evaluation of the androgenic blockade by PET-PSMA should be done with caution. Bombesin antagonists are currently in clinical development.

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26.

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors.

Selection of optimal chelator improves the contrast of GRPR imaging using bombesin analogue RM26.

Bombesin (BN) analogs bind with high affinity to gastrin-releasing peptide receptors (GRPRs) that are up-regulated in prostate cancer and can be used for the visualization of prostate cancer. The aim of this study was to investigate the influence of radionuclide-chelator complexes on the biodistribution pattern of the 111In-labeled bombesin antagonist PEG2-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (PEG2-RM26) and to identify an optimal construct for SPECT imaging. A series of RM26 analogs N-terminally conjugated with NOTA, NODAGA, DOTA and DOTAGA via a PEG2 spacer were radiolabeled with 111In and evaluated both in vitro and in vivo. The conjugates were successfully labeled with 111In with 100% purity and retained binding specificity to GRPR and high stability. The cellular processing of all compounds was characterized by slow internalization. The IC50 values were in the low nanomolar range, with lower IC50 values for positively charged natIn-NOTA-PEG2-RM26 (2.6 ± 0.1 nM) and higher values for negatively charged natIn-DOTAGA-PEG2-RM26 (4.8 ± 0.5 nM). The kinetic binding studies showed KD values in the picomolar range that followed the same pattern as the IC50 data. The biodistribution of all compounds was studied in BALB/c nu/nu mice bearing PC-3 prostate cancer xenografts. Tumor targeting and biodistribution studies displayed rapid clearance of radioactivity from the blood and normal organs via kidney excretion. All conjugates showed similar uptake in tumors at 4 h p.i. The radioactivity accumulation in GRPR-expressing organs was significantly lower for DOTA- and DOTAGA-containing constructs compared to those containing NOTA and NODAGA. 111In-NOTA-PEG2-RM26 with a positively charged complex showed the highest initial uptake and the slowest clearance of radioactivity from the liver. At 4 h p.i., DOTA- and DOTAGA-coupled analogs showed significantly higher tumor-to-organ ratios compared to NOTA- and NODAGA-containing variants. The NODAGA conjugate demonstrated the best retention of radioactivity in tumors, and, at 24 h p.i., had the highest contrast to blood, muscle and bones.

Detection of prostate cancer with the [68Ga]-labeled bombesin antagonist RM2 in patients undergoing radical prostatectomy.

80 Background: Gastrin-releasing-peptide receptor (GRPr) is overexpressed in prostate carcinoma (PCa) and offer new means to detect prostate cancer. The bombesin derivative RM2 (DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) is a GRPr antagonist with high binding affinity (Kd 3 nM). A first-in-man study was performed to assess tumor targeting potential of [68Ga]RM2 in patients with local PCa using PET/CT. Based on promising results a Phase I/II study in local PCa was initiated. Methods: In the initial study eleven men with biopsy-proven PCa scheduled for radical prostatectomy were enrolled. The patients (pt) received an i.v. dose of 140 MBq of [68Ga]RM2 followed by dynamic imaging for at least 60 min p.i. The PET assessment included visual evaluation of [68Ga]RM2 intra-prostatic and nodal uptake as well as quantification. An ongoing Phase I/II study is designed to enroll 30+50 biopsy-positive PCa patients. The initial 30 pt have been enrolled, stratified into high, intermediate and low risk of prostate Ca recurrence as defined by NCCN criteria. Results: High uptake of [68Ga]RM2 is seen in pancreas and the urinary system with low background in the rest of the body. Despite of high bladder activity focal or multifocal intraprostatic uptake was detectable. In the first study 10 of 11 pt had positive scans. Subsector analysis of PET positivity vs. pathology yielded a sensitivity, specificity and accuracy of 89, 81, and 83%, respectively. SUVmax was measured in 14 lesions from 9 pts, classified in two groups according to postoperative Gleason score (GS). The higher GS group showed a trend toward higher SUV-values, 10.0 ± 6.7 vs. 6.2 ± 2.5 (t-test: t = -1.21, p-value = 0.28). Both of which were elevated compared to normal tissue 1.2 ± 0.8 The ongoing study is designed with a power of 95% to discriminate between the two GS groups with statistical significance (p = 0.05). Conclusions: [68Ga]RM2 is a promising new tracer with a high detection rate for PCa. Preliminary data indicates higher uptake in more aggressive cancers. The current study is assessing the diagnostic accuracy and impact of presurgical risk assessment on tumor detection in newly diagnosed PCa patients. Clinical trial information: NCT02483884.

Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development.

Retinoic acid (RA) and ghrelin levels are altered in human hypoplastic lungs when compared to healthy lungs. Although considerable data have been obtained about RA, ghrelin and bombesin in the congenital diaphragmatic hernia (CDH) rat model, neuroendocrine factors have never been associated with the RA signalling pathway in this animal model. In this study, the interaction between neuroendocrine factors and RA was explored in the CDH rat model. The authors found that normal fetal lung explants treated with RA, bombesin and ghrelin showed an increase in lung growth. Hypoplastic lungs presented higher expression levels of the RA receptors α and γ. Moreover bombesin and ghrelin supplementation, in vitro, to normal lungs increased RA receptor α/γ expression whereas administration of bombesin and ghrelin antagonists to normal and hypoplastic lungs decreased it. These data reveal for the first time that there is a link between neuroendocrine factors and RA, and that neuroendocrine factors sensitise the lung to the RA action through RA receptor modulation. Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, β and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ.

PO-0754 Neuroendocrine Regulation Of Retinoic Acid Receptors During Lung Branching In Normal And Hypoplastic Lungs

The pathogenesis of pulmonary hypoplasia and congenital diaphragmatic hernia (CDH) is unknown. CDH represents a spectrum of lung hypoplasia and consequent pulmonary hypertension (PH) that leads to high morbidity and mortality of patients. We studied neuroendocrine factors and retinoic acid in order to achieve the relation underlying them. At 13.5 days post-conception normal and CDH lungs were cultured in vitro during four days with DMSO, retinoic acid, bombesin, ghrelin, bombesin antagonist and ghrelin antagonist. Morphometric analysis were done after the culture and Western Blot (WB) was performed to quantify the protein levels of retinoic acid receptors (RAR). Immunohistochemestry (IHC) was performed as well on normal, nitrofen and CDH E17.5 lungs. When compared with controls, CDH lungs presented higher expression of RAR in IHC and WB. Moreover in normal lungs after the administration of bombesin and ghrelin the expression of RAR also increases and in case of retinoic acid administration it decreases. Regarding bombesin and ghrelin antagonists administration, RAR expression decreases as it was expected. In terms of morphometry, treated groups showed an increase in branching, perimeter and area. This study, shows for the first time that retinoic acid deficit on CDH lungs is associated with neuroendocrine factors overexpression. Furthermore, neuroendocrine factors such as ghrelin and bombesin sensitise for RAR expression.

PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists.

The gastrin-releasing peptide receptor (GRPR) was shown to be expressed with high density on several types of cancers. Radiolabeled peptides for imaging and targeted radionuclide therapy have been developed. In this study, we evaluated the potential of statine-based bombesin antagonists, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) through oligoethyleneglycol spacers, labeled with (177)Lu and we determined the effect of polyethyleneglycol (PEG) spacer length on in vitro and in vivo properties. The bombesin antagonists were synthesized on solid phase using Fmoc chemistry; the spacers Fmoc-dPEGx-OH (x=2, 4, 6 and 12) and the DOTA(tBu)3 were coupled using a standard procedure. The peptides were labeled with (177)Lu and evaluated in vitro (lipophilicity, serum stability, internalization and binding affinity assays). Biodistribution studies were performed in PC-3 tumor-bearing nude mice. The solid-phase synthesis was straightforward with an overall yield ranging from 30% to 35% based on the first Fmoc cleavage. The hydrophilicity increased with spacer length (logD: -1.95 vs -2.22 of PEG2 and PEG12 analogs, respectively). There is a tendency of increased serum stability by increasing the spacer length (T1/2=246±4 and 584±20 for PEG2 and PEG6 analogs, respectively) which seems to reverse with the PEG12 analog. The IC50 values are similar with the only significant difference of the PEG12 analog. The (177)Lu-labeled PEG4 and PEG6 conjugates showed similar pharmacokinetic with high tumor uptake and excellent tumor-to-kidney ratios (7.8 and 9.7 at 4h for the PEG4 and PEG6 derivatives, respectively). The pancreas uptake was relatively high at 1h but it shows fast washout (0.46%±0.02% IA/g and 0.29%±0.08% IA/g already at 4h). Among all the studied analogs the PEG4 and PEG6 showed significantly better properties. The very high tumor-to-non-target organ ratios, in particular tumor-to-kidney ratios, already at early time point will be important in regard to safety concerning kidney toxicity.