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.

68Ga-Labeled [Thz14]Bombesin(7-14) Analogs: Promising GRPR-Targeting Agonist PET Tracers with Low Pancreas Uptake.

With overexpression in various cancers, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer imaging and therapy. However, the high pancreas uptake of reported GRPR-targeting radioligands limits their clinical application. Our goal was to develop 68Ga-labeled agonist tracers for detecting GRPR-expressing tumors with positron emission tomography (PET), and compare them with the clinically validated agonist PET tracer, [68Ga]Ga-AMBA. Ga-TacBOMB2, TacBOMB3, and TacBOMB4, derived from [Thz14]Bombesin(7-14), were confirmed to be GRPR agonists by a calcium mobilization study, and their binding affinities (Ki(GRPR)) were determined to be 7.62 ± 0.19, 6.02 ± 0.59, and 590 ± 36.5 nM, respectively, via in vitro competition binding assays. [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA clearly visualized PC-3 tumor xenografts in a PET imaging study. [68Ga]Ga-TacBOMB2 showed comparable tumor uptake but superior tumor-to-background contrast ratios when compared to [68Ga]Ga-AMBA. Moreover, [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 showed a much lower rate of uptake in the pancreas (1.30 ± 0.14 and 2.41 ± 0.72%ID/g, respectively) than [68Ga]Ga-AMBA (62.4 ± 4.26%ID/g). In conclusion, replacing Met14 in the GRPR-targeting sequence with Thz14 retains high GRPR-binding affinity and agonist properties. With good tumor uptake and tumor-to-background uptake ratios, [68Ga]Ga-TacBOMB2 is promising for detecting GRPR-expressing tumors. The much lower pancreas uptake of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 suggests that [Thz14]Bombesin(7-14) is a promising targeting vector for the design of GRPR-targeting radiopharmaceuticals, especially for radioligand therapy application.

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.

Synthesis and Biological Evaluation of 99mTc(I) Tricarbonyl Complexes Dual-Targeted at Tumoral Mitochondria.

For effective Auger therapy of cancer, the Auger-electron emitters must be delivered to the tumor cells in close proximity to a radiosensitive cellular target. Nuclear DNA is considered the most relevant target of Auger electrons to have augmented radiotoxic effects and significant cell death. However, there is a growing body of evidence that other targets, such as the mitochondria, could be relevant subcellular targets in Auger therapy. Thus, we developed dual-targeted 99mTc(I) tricarbonyl complexes containing a triphenylphosphonium (TPP) moiety to promote accumulation of 99mTc in the mitochondria, and a bombesin peptide to provide specificity towards the gastrin releasing peptide receptor (GRPr) overexpressed in prostate cancer cells. The designed dual-targeted complex, 99mTc-TPP-BBN, is efficiently internalized by human prostate cancer PC3 cells through a specific GRPr-mediated mechanism of uptake. Moreover, the radioconjugate provided an augmented accumulation of 99mTc in the mitochondria of the target tumor cells, most probably following its intracellular cleavage by cathepsin B. In addition, 99mTc-TPP-BBN showed an enhanced ability to reduce the survival of PC3 cells, in a dose-dependent manner.

Preclinical Evaluation of NHS-Activated Gold Nanoparticles Functionalized with Bombesin or Neurotensin-Like Peptides for Targeting Colon and Prostate Tumours.

Recent advances and large-scale use of hybrid imaging modalities like PET-CT have led to the necessity of improving nano-drug carriers that can facilitate both functional and metabolic screening in nuclear medicine applications. In this study, we focused on the evaluation of four potential imaging nanoparticle structures labelled with the 68Ga positron emitter. For this purpose, we functionalized NHS-activated PEG-gold nanoparticles with 68Ga-DOTA-Neuromedin B, 68Ga-DOTA-PEG(4)-BBN(7-14), 68Ga-DOTA-NT and 68Ga-DOTA-Neuromedin N. In vitro binding kinetics and specific binding to human HT-29 colon carcinoma cells and DU-145 prostate carcinoma cells respectively were assessed, over 75% retention being obtained in the case of 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP in prostate tumour cells and over 50% in colon carcinoma cells. Biodistribution in NU/J mice highlighted a three-fold uptake increase in tumours at 30 min post-injection of 68Ga-DOTA-NT-AuNP and 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP compared to 68Ga-DOTA-NT and 68Ga-DOTA-PEG(4)-BBN(7-14) respectively, therewith fast distribution in prostate and colon tumours and minimum accumulation in non-targeted tissues.

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

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

Preliminary Evaluation of Astatine-211-Labeled Bombesin Derivatives for Targeted Alpha Therapy.

There are various diagnostic and therapeutic agents for prostate cancer using bombesin (BBN) derivatives, but astatine-211 (211At)-labeled BBN derivatives have yet to be studied. This study presented a preliminary evaluation of 211At-labeled BBN derivative. Several nonradioactive iodine-introduced BBN derivatives (IB-BBNs) with different linkers were synthesized and their binding affinities measured. Because IB-3 exhibited a comparable affinity to native BBN, [211At]AB-3 was synthesized and the radiochemical yields of [211At]AB-3 was 28.2 ± 2.4%, with a radiochemical purity of >90%. The stability studies and cell internalization/externalization experiments were performed. [211At]AB-3 was taken up by cells and internalized; however, radioactivity effluxed from cells over time. In addition, the biodistribution of [211At]AB-3, with and without excess amounts of BBN, were evaluated in PC-3 tumor-bearing mice. Despite poor stability in murine plasma, [211At]AB-3 accumulated in tumor tissue (4.05 ± 0.73%ID/g) in PC-3 tumor-bearing mice, which was inhibited by excess native BBN (2.56 ± 0.24%ID/g). Accumulated radioactivity in various organs is probably due to free 211At. Peptide degradation in murine plasma and radioactivity efflux from cells are areas of improvement. The development of 211At-labeled BBN derivatives requires modifying the BBN sequence and preventing deastatination.

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

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

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

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

The Influence of Different Spacers on Biological Profile of Peptide Radiopharmaceuticals for Diagnosis and Therapy of Human Cancers.

BACKGROUND: Cancer is the leading cause of death worldwide. Early detection can reduce the disadvantageous effects of diseases and the mortality in cancer. Nuclear medicine is a powerful tool that has the ability to diagnose malignancy without harming normal tissues. In recent years, radiolabeled peptides have been investigated as potent agents for cancer detection. Therefore, it is necessary to modify radiopeptides in order to achieve more effective agents. OBJECTIVE: This review describes modifications in the structure of radioconjugates with spacers who have improved the specificity and sensitivity of the peptides that are used in oncologic diagnosis and therapy. METHODS: To improve the biological activity, researchers have conjugated these peptide analogs to different spacers and bifunctional chelators. Many spacers of different kinds, such as hydrocarbon chain, amino acid sequence, and poly (ethyleneglycol) were introduced in order to modify the pharmacokinetic properties of these biomolecules. RESULTS: Different spacers have been applied to develop radiolabeled peptides as potential tracers in nuclear medicine. Spacers with different charge and hydrophilicity affect the characteristics of peptide conjugate. For example, the complex with uncharged and hydrophobic spacers leads to increased liver uptake, while the composition with positively charged spacers results in high kidney retention. Therefore, the pharmacokinetics of radio complexes correlates to the structure and total charge of the conjugates. CONCLUSION: Radio imaging technology has been successfully applied to detect a tumor in the earliest stage. For this purpose, the assessment of useful agents to diagnose the lesion is necessary. Developing peptide radiopharmaceuticals using spacers can improve in vitro and in vivo behavior of radiotracers leading to better noninvasive detection and monitoring of tumor growth.

Probing two PESIN-indocyanine-dye-conjugates: significance of the used fluorophore.

Peptide-dye-conjugates hold a great promise in application for biological and medical imaging of cellular processes and in delineation and characterization of human tumors. In particular, indocyanine dyes are of great interest due to their reported superior properties such as absorption and emission in the near-infrared (NIR) spectral range, favorable Stokes shifts and their well-studied safety profile in humans. In this study, we investigated and describe the influence of indocyanine dyes on different properties of the final peptide-dye-conjugates. As a target peptide, PESIN, a bombesin derivative, was used as a model peptide which addresses GRP receptors overexpressed on different malignancies. Here, we map similarities and differences of the fluorescent conjugates and by this elucidate the influence of the dyes on different properties of the formed conjugates. We performed the dye syntheses, subsequent bioconjugation reactions and in the following investigated the optical properties, water/octanol distribution coefficients and target receptor affinities by in vitro competitive binding studies on PC-3 cells. The obtained results give a handrail to medical and biological researchers planning studies involving indocyanine dye biomolecule conjugates.

Preliminary studies of 68Ga-NODA-USPION-BBN as a dual-modality contrast agent for use in positron emission tomography/magnetic resonance imaging.

The aim of this study was to propose a new dual-modality nanoprobe for positron emission tomography/magnetic resonance imaging (PET/MRI) for the early diagnosis of breast cancer. For synthesis of the nanoprobe, polyethylene glycol-coated ultra-small superparamagnetic iron-oxide nanoparticles (USPION) armed with NODA-GA chelate and grafted with bombesin (BBN) were radiolabeled with 68Ga. After characterization, in vitro studies to evaluate the cell binding affinity of the nanoprobe were done by performing Perl's Prussian blue cell staining and MRI imaging. Finally, for in vivo studies, magnetic resonance images were taken in SCID mice bearing breast cancer tumor pre- and post-injection, and a multimodal nanoScan PET/computed tomography was used to perform preclinical imaging of the radiolabeled nanoparticles. Afterwards, a biodistribution study was done on sacrificed mice. The results showed that the highest r1 and r2 values were measured for USPIONs at 20 and 60 MHz, respectively. From the in vitro studies, the optical density of the cells after incubation increased with the increase of the iron concentration and the duration of incubation. However, the T2 values decreased when the iron concentration increased. Furthermore, from in vivo studies, the T2 and signal intensity decreased during the elapsed time post-injection in the tumor area. In this study, the in vitro studies showed that the affinity of cancer cells to nanoprobe increases meaningfully after conjugation with BBN, and also by increasing the duration of incubation and the iron concentration. Meanwhile, the in vivo results confirmed that the blood clearance of the nanoprobe happened during the first 120 min post-injection of the radiolabeled nanoprobe and also confirmed the targeting ability of that to a gastrin-releasing peptide receptor positive tumor.

Novel Gastrin-Releasing Peptide Receptor Targeted Near-Infrared Fluorescence Dye for Image-Guided Surgery of Prostate Cancer.

PURPOSE: Prostate cancer (PCa), the most widespread male cancer in western countries, is generally eradicated by surgery, especially if localized. However, during surgical procedures, it is not always possible to identify malignant tissues by visual inspection. Among the possible consequences, there is the formation of positive surgical margins, often associated with recurrence. In this work, the gastrin-releasing peptide receptor (GRPR), overexpressed in the prostatic carcinoma and not in healthy tissues or in benign hyperplasia (BPH), is proposed as target molecule to design a novel near-infrared fluorescent (NIRF) probe for image-guided prostatectomy. PROCEDURES: The NIRF dye Sulfo-Cy5.5 was conjugated to a Bombesin-like peptide (BBN), targeting GRPR. The final product, called BBN-Cy5.5, was characterized and tested in vitro on PC-3, DU145, and LnCAP cell lines, using unconjugated Sulfo-Cy5.5 as control. In vivo biodistribution studies were performed by optical imaging in PC-3 tumor-bearing and healthy mice. Finally, simulation of the surgical protocol was carried out. RESULTS: BBN-Cy5.5 showed high water solubility and a good relative quantum yield. The ability of the probe to recognize the GRPR, highly expressed in PC-3 cells, was tested both in vitro and in vivo, where a significant tumor accumulation was achieved 24 h post-injection. Furthermore, a distinguishable fluorescent signal was visible in mice bearing PCa, when the surgery was simulated. By contrast, low signal was found in healthy or BPH-affected mice. CONCLUSIONS: This work proposes a new NIRF probe ideal to target GRPR, biomarker of PCa. The promising data obtained suggest that the dye could allow the real-time intraoperative visualization of prostate cancer.

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.

Bombesin-functionalized superparamagnetic iron oxide nanoparticles for dual-modality MR/NIRFI in mouse models of breast cancer.

BACKGROUND: The early and accurate detection afforded by imaging techniques significantly reduces mortality in cancer patients. However, it is still a great challenge to achieve satisfactory performance in tumor diagnosis using any single-modality imaging method. Magnetic resonance imaging (MRI) has excellent soft tissue contrast and high spatial resolution, but it suffers from low sensitivity. Fluorescence imaging has high sensitivity, but it is limited by penetration depth. Thus, the combination of the two modes could result in synergistic benefits. Here, we design and characterize a novel dual-modality MR/near-infrared fluorescence imaging (MR/NIRFI) nanomicelle and test its imaging properties in mouse models of breast cancer. METHODS: The nanomicelles were prepared by incorporating superparamagnetic iron oxide (SPIO) nanoparticles into 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] micelles to which an NIRF dye and a tumor-targeted peptide (N3-Lys-bombesin, Bom) were conjugated. The nanomicelles were characterized for particle size, zeta potential and morphology. The transverse relaxivity, targeting specificity and imaging ability of the nanomicelles for MR/NIRFI were also examined. RESULTS: The fabricated nanomicelles displayed a well-defined spherical morphology with a mean diameter of 145±56 nm and a high transverse relaxivity (493.9 mM-1·s-1, 3.0T). In MRI, the T2 signal reduction of tumors in the Bom-targeted group was 24.1±5.7% at 4 hrs postinjection, whereas only a 0.1±3.4% (P=0.003) decrease was observed in the nontargeted group. In NIRFI, the contrast increased gradually in the targeted group, and the tumor/muscle ratio increased from 3.7±0.3 at 1 hr to 4.7±0.1 at 2 hrs and to 6.4±0.2 at 4 hrs. No significant changes were observed in the nontargeted group at any time points. CONCLUSION: Considering all our results, we conclude that these novel MR/NIRFI dual-modality nanomicelles could be promising contrast agents for cancer diagnosis.

177Lu-Bombesin-PLGA (paclitaxel): A targeted controlled-release nanomedicine for bimodal therapy of breast cancer.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in >75% of breast cancers. 177Lu-Bombesin (177Lu-BN) has demonstrated the ability to target GRPr and facilitate efficient delivery of therapeutic radiation doses to malignant cells. Poly(d,l­lactide­co­glycolide) acid (PLGA) nanoparticles can work as smart drug controlled-release systems activated through pH changes. Considering that paclitaxel (PTX) is a first-line drug for cancer treatment, this work aimed to synthesize and chemically characterize a novel polymeric PTX-loaded nanosystem with grafted 177Lu-BN and to evaluate its performance as a targeted controlled-release nanomedicine for concomitant radiotherapy and chemotherapy of breast cancer. PLGA(PTX) nanoparticles were synthesized using the single emulsification-solvent evaporation method with PVA as a stabilizer in the presence of PTX. Thereafter, the activation of PLGA carboxylic groups for BN attachment through the Lys1-amine group was performed. Results of the chemical characterization by FT-IR, DLS, HPLC and SEM/TEM demonstrated the successful synthesis of BN-PLGA(PTX) with a hydrodynamic diameter of 163.54 ±â€¯33.25 nm. The entrapment efficiency of paclitaxel was 92.8 ±â€¯3.6%. The nanosystem showed an adequate controlled release of the anticancer drug, which increased significantly due to the pH change from neutral (pH = 7.4) to acidic conditions (pH = 5.3). After labeling with 177Lu and purification by ultrafiltration, 177Lu-BN-PLGA(PTX) was obtained with a radiochemical purity of 99 ±â€¯1%. In vitro and in vivo studies using MDA-MB-231 breast cancer cells (GRPr-positive) demonstrated a 177Lu-BN-PLGA(PTX) specific uptake and a significantly higher cytotoxic effect for the radiolabeled nanosystem than the unlabeled BN-PLGA(PTX) nanoparticles. Using a pulmonary micrometastasis MDA-MB-231 model, the added value of 177Lu-BN-PLGA(PTX) for tumor imaging was confirmed. The 177Lu-BN-PLGA(PTX) nanomedicine is suitable as a targeted paclitaxel delivery system with concomitant radiotherapeutic effect for the treatment of GRPr-positive breast cancer.

68Ga-NOTA-Aca-BBN(7-14) PET imaging of GRPR in children with optic pathway glioma.

PURPOSE: Optic pathway glioma (OPG) is a rare neoplasm that arises predominantly during childhood. Its location in a sensitive region involving the optic pathways, onset in young patients and controversial therapy choice make the management of OPG a challenge in paediatric neuro-oncology. In this study we assessed gastrin-releasing peptide receptor (GRPR)-targeted positron emission tomography (PET) imaging in children with OPG, and the application of a PET/MRI imaging-guided surgery navigation platform. METHODS: Eight children (five boys, mean age 8.81 years, range 5-14 years) with suspicion of optic pathway glioma on MRI were recruited. Written informed consent was obtained from all patients and legal guardians. Brain PET/CT or PET/MRI acquisitions were performed 30 min after intravenous injection of 1.85 MBq/kg body weight of 68Ga-NOTA-Aca-BBN(7-14). Four patients also underwent 18F-FDG brain PET/CT for comparison. All patients underwent surgical resection within 1 week. RESULTS: All 11 lesions (100%) in the eight patients showed prominent 68Ga-NOTA-Aca-BBN(7-14) uptake with excellent contrast in relation to surrounding normal brain tissue. Tumour-to-background ratios (SUVmax and SUVmean) were significantly higher for 68Ga-NOTA-Aca-BBN(7-14) than for 18F-FDG (28.4 ± 5.59 vs. 0.47 ± 0.11 and 18.3 ± 4.99 vs. 0.35 ± 0.07, respectively). Fusion images for tumour delineation were obtained in all patients using the PET/MRI navigation platform. All lesions were pathologically confirmed as OPGs with positive GRPR expression, and 75% were pilocytic astrocytoma WHO grade I and 25% were diffuse astrocytoma WHO grade II. There was a positive correlation between the SUV of 68Ga-NOTA-Aca-BBN(7-14) and the expression level of GRPR (r2 = 0.56, P < 0.01, for SUVmax; r2 = 0.47, P < 0.05, for SUVmean). CONCLUSION: This prospective study showed the feasibility of 68Ga-NOTA-Aca-BBN(7-14) PET in children with OPG for tumour detection and localization. 68Ga-NOTA-Aca-BBN(7-14) PET/MRI may be helpful for assisting surgery planning in OPG patients with severe symptoms, GRPR-targeted PET has the potential to provide imaging guidance for further GRPR-targeted therapy in patients with OPG.

68Ga-radiolabeled bombesin-conjugated to trimethyl chitosan-coated superparamagnetic nanoparticles for molecular imaging: preparation, characterization and biological evaluation.

INTRODUCTION: Nowadays, nanoparticles (NPs) have attracted much attention in biomedical imaging due to their unique magnetic and optical characteristics. Superparamagnetic iron oxide nanoparticles (SPIONs) are the prosperous group of NPs with the capability to apply as magnetic resonance imaging (MRI) contrast agents. Radiolabeling of targeted SPIONs with positron emitters can develop dual positron emission tomography (PET)/MRI agents to achieve better diagnosis of clinical conditions. METHODS: In this work, N,N,N-trimethyl chitosan (TMC)-coated magnetic nanoparticles (MNPs) conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) as a radioisotope chelator and bombesin (BN) as a targeting peptide (DOTA-BN-TMC-MNPs) were prepared and validated using fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), thermogravimetric analysis (TGA), vibrating sample magnetometer (VSM), and powder X-ray diffraction (PXRD) tests. Final NPs were radiolabeled with gallium-68 (68Ga) and evaluated in vitro and in vivo as a potential PET/MRI probe for breast cancer (BC) detection. RESULTS: The DOTA-BN-TMC-MNPs with a particle size between 20 and 30 nm were efficiently labeled with 68Ga (radiochemical purity higher than 98% using thin layer chromatography (TLC)). The radiolabeled NPs showed insignificant toxicity (>74% cell viability) and high affinity (IC50=8.79 µg/mL) for the gastrin-releasing peptide (GRP)-avid BC T-47D cells using competitive binding assay against 99mTc-hydrazinonicotinamide (HYNIC)-gamma-aminobutyric acid (GABA)-BN (7-14). PET and MRI showed visible uptake of NPs by T-47D tumors in xenograft mouse models. CONCLUSION: 68Ga-DOTA-BN-TMC-MNPs could be a potential diagnostic probe to detect BC using PET/MRI technique.

Bombesin conjugated solid lipid nanoparticles for improved delivery of epigallocatechin gallate for breast cancer treatment.

Epigallocatechin-gallate (EGCG) is a potent anti-cancer therapeutic which effectively controls the growth of cancerous cells through a variety of different pathways. However, its molecular structure is susceptible to modifications due to cellular enzymes affecting its stability, bioavailability and hence, overall efficiency. In this study, we have initially encapsulated EGCG in the matrix of solid lipid nanoparticles to provide a stable drug carrier. To confer additional specificity towards gastrin releasing peptide receptors (GRPR) overexpressed in breast cancer, EGCG loaded nanoparticles were conjugated with a GRPR-specific peptide. In-vitro cytotoxicity studies showed that the peptide-conjugated formulations possessed greater cytotoxicity to cancer cell lines compared to the non-conjugated formulations. Further, in-vivo studies performed on C57/BL6 mice showed greater survivability and reduction in tumour volume in mice treated with peptide-conjugated formulation as compared to the mice treated with non-conjugated formulation or with plain EGCG. These results warrant the potential of the system designed in this study as a novel and effective drug delivery system in breast cancer therapy.

Synthesis and preclinical evaluation of the 177Lu-DOTA-PSMA(inhibitor)-Lys3-bombesin heterodimer designed as a radiotheranostic probe for prostate cancer.

BACKROUND: Human tumors show intrinsic heterogeneity and changes in phenotype during disease progression, which implies different expression levels of cell surface receptors. The research on new heterodimeric lutetium-177 (Lu)-radiopharmaceuticals interacting with two different targets on tumor cells is a strategy for improvement of radiotheranostic performance. This study aimed to synthesize and characterize the Lu-DOTA-PSMA(inhibitor)-Lys-bombesin (Lu-DOTA-iPSMA-Lys-BN) heterodimer and to evaluate its potential to target prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPr) overexpressed in prostate cancer. METHODS: The heterodimeric conjugate was synthesized and characterized by infrarred, mass, and H-NMR spectroscopies. The ligand was labeled with Lu and the radiochemical purity was assessed by radio-high-performance liquid chromatography. PSMA/GRPr affinity and the heterobivalent effect on cell viability were evaluated in LNCaP and PC3 prostate cancer cell lines. The biodistribution profile (3 and 96 h) was assessed in athymic mice with induced prostate tumors. Using pulmonary LNCaP (PSMA-positive) and PC3 (GRPr-negative) micrometastasis models, the influence of heterobivalency and affinity on tumor uptake was quantified (micro-SPECT/CT). RESULTS: Lu-iPSMA-BN (radiochemical purity>98%) showed specific recognition for PSMA and GRPr (IC50=5.62 and 3.49 nmol/l, respectively) with a significant decrease in cell viability (10.15% of cell viability in LNCaP and 40.10% in PC3 at 48 h), as well as high LNCaP and PC3 tumor uptake (5.21 and 3.21% ID/g at 96 h, respectively). Micro-SPECT/CT imaging showed the heterodimer ability to target the tumors (SUVmax of 1.93±0.30 and 1.76±0.10 in LNCaP and PC3, respectively), possibly influenced by the heterobivalent effect. Lu-DOTA-iPSMA-Lys-BN showed suitable affinity for PSMA and GRPr. CONCLUSION: The results warrant further preclinical studies to establish the Lu-radiotracer theranostic efficacy.