Synthesis and Radiolabeling of Glu-Urea-Lys with 99mTc-Tricarbonyl-Imidazole-Bathophenanthroline Disulfonate Chelation System and Biological Evaluation as Prostate-Specific Membrane Antigen Inhibitor.

Background: The Glu-Urea-Lys (EUK) pharmacophore as prostate-specific membrane antigen (PSMA)-targeted ligand was synthesized, radiolabeled with 99mTc-tricarbonyl-imidazole-BPS chelation system, and biological activities were evaluated. The strategy [2 + 1] ligand is applied for tricarbonyl labeling. (5-imidazole-1-yl)pentanoic acid as a monodentate ligand and bathophenanthroline disulfonate (BPS) as a bidentate ligand formed a chelate system with 99mTc-tricarbonyl. EUK-pentanoic acid-imidazole and EUK were evaluated for PSMA active site using AutoDock 4 software. Materials and Methods: EUK-pentanoic acid-imidazole was synthesized in two steps. BPS was radiolabeled with 99mTc-tricarbonyl at 100°C for 30 min. The purified 99mTc(CO)3(H2O)BPS was used to radiolabel EUK-pentanoic acid-imidazole at 100°C, 30 min. Radiochemical purity, Log P, and stability studies were carried out within 24 h. Affinity of 99mTc(CO)3BPS-imidazole-EUK was performed in the saturation binding studies using LNCaP cells at 37°C for 1 h with a range of 0.001-1000 nM radiolabeled compound range. Internalization studies were performed in LNCaP cells with 1000 nM radiolabeled compound incubated for (0-2) h at 37°C. Biodistribution was studied in normal male Balb/c mice. The artificial intelligence predicts the uptake of radiolabeled compound in tumor. Results: The structures of synthesized compounds were confirmed by mass spectroscopy. Radiochemical purity, Log P, and protein binding were ≥95%, -0.2%, and 23%, respectively. The radiolabeled compound was stable in saline and human plasma within 24 h with radiochemical purity ≥90%. There was no release of 99mTc within 4 h in competition with histidine. The affinity was 82 ± 26.38 nM, and the activity increased inside the cells over time. Biodistribution studies showed radioactivity accumulation in kidneys less than 99mTc-HYNIC-PSMA. There was a moderate accumulation of radioactivity in the liver and intestine. Conclusion: Based on the results, 99mTc(CO)3BPS-imidazole-EUK can potentially be used as an imaging agent for studies at prostate bed and distal areas. The chelate system can be potentially labeled with rhenium for imaging studies (fluorescent or scintigraphy) and therapy.

Vimentin-targeted radiopeptide 99m Tc-HYNIC-(tricine/EDDA)-VNTANST: a promising drug for pulmonary fibrosis imaging.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by the accumulation of extracellular matrix. Because there is no effective treatment for advanced IPF to date, its early diagnosis can be critical. Vimentin is a cytoplasmic intermediate filament that is significantly up-regulated at the surface of fibrotic foci with a crucial role in fibrotic morphological changes. METHODS: In the present study, VNTANST sequence as a known vimentin-targeting peptide was conjugated to hydrazinonicotinic acid (HYNIC) and labeled with 99m Tc. The stability test in saline and human plasma and log P determination were performed. Next, the biodistribution study and single photon emission computed tomography (SPECT) integrated with computed tomography (CT) scanning were performed in healthy and bleomycin-induced fibrosis mice models. RESULTS: The 99m Tc-HYNIC-(tricine/EDDA)-VNTANST showed a hydrophilic nature (log P  = -2.20 ±â€…0.38) and high radiochemical purity > 97% and specific activity (336 Ci/mmol). The radiopeptide was approximately 93% and 86% intact in saline and human plasma within 6 h, respectively. The radiopeptide was substantially accumulated in the pulmonary fibrotic lesions (test vs. control = 4.08 ±â€…0.08% injected dose per gram (ID/g) vs. 0.36 ±â€…0.01% ID/g at 90 min postinjection). SPECT-CT images in fibrosis-bearing mice also indicated the fibrotic foci and kidneys. CONCLUSION: Because there is no available drug for the treatment of advanced pulmonary fibrosis, early diagnosis is the only chance. The 99m Tc-HYNIC-(tricine/EDDA)-VNTANST could be a potential tracer for SPECT imaging of pulmonary fibrosis.

Preclinical study of a new 177Lu-labeled somatostatin receptor antagonist in HT-29 human colorectal cancer cells.

OBJECTIVES: Somatostatin receptor-positive neuroendocrine tumors have been targeted using various peptide analogs radiolabeled with therapeutic radionuclides for years. The better biomedical properties of radioantagonists as higher tumor uptake make these radioligands more attractive than agonists for somatostatin receptor-targeted radionuclide therapy. In this study, we tried to evaluate the efficiency of Luthetium-177 (177Lu) radiolabeled DOTA-Peptide 2 (177Lu-DOTA-Peptide 2) as a new radioantagonist in HT-29 human colorectal cancer in vitro and in vivo. METHODS: DOTA conjugated antagonistic peptide with the sequence of p-Cl-Phe-Cyclo(D-Cys-L-BzThi-D-Aph-Lys-Thr-Cys)-D-Tyr-NH2 (DOTA-Peptide 2) was labeled with 177Lu. In vitro assays (saturation binding assay and internalization test) and animal biodistribution were performed in human colon adenocarcinoma cells (HT-29) and HT-29 tumor-bearing nude mice. RESULTS: 177Lu-DOTA-Peptide 2 showed high stability in acetate buffer and human plasma (>97%). Antagonistic property of 177Lu-DOTA-Peptide 2 was confirmed by low internalization in HT-29 cells (<5%). The desired dissociation constant (Kd =11.14 nM) and effective tumor uptake (10.89 percentage of injected dose per gram of tumor) showed high binding affinity of 177Lu-DOTA-Peptide 2 to somatostatin receptors. CONCLUSION: 177Lu-DOTA-Peptide 2 demonstrated selective and high binding affinity to somatostatin receptors overexpressed on the surface of HT-29 cancer cells, which could make this radiopeptide suitable for somatostatin receptor-targeted radionuclide therapy.

Design, preparation and biological evaluation of a 177Lu-labeled somatostatin receptor antagonist for targeted therapy of neuroendocrine tumors.

Somatostatin receptor-targeted radionuclide therapy has become an effective treatment in patients with neuroendocrine tumors. Recently, investigations on the development of antagonistic peptides are increasing with possible superior biological properties as opposed to the agonists. Herein, we have reported the development of a new somatostatin receptor peptide ligand labeled with 177Lu to achieve a therapeutic ligand for tumor treatment. The interactions of selected and drown ligands using Avogadro software were docked on somatostatin receptor by Dink algorithm. The best docked peptide-chelator conjugate (DOTA-p-Cl-Phe-Cyclo(d-Cys-l-BzThi-d-Aph-Lys-Thr-Cys)-d-Tyr-NH2) (DOTA-Peptide 2) was synthesized using the Fmoc solid-phase method. DOTA-Peptide 2 was radiolabeled with the 177Lu Trichloride (177LuCl3) solution at 95 °C for 30 min and radiochemical purity (RCP) of 177Lu-DOTA-Peptide 2 solution was monitored by radio-HPLC and radio-TLC procedures. The new radiolabeled peptide was evaluated for stability, receptor binding, internalization, biodistribution and single-photon emission computed tomography (SPECT) imaging using C6 glioma cells and C6 tumor-bearing rats. DOTA-Peptide 2 was obtained with 98% purity and efficiently labeled with 177Lu (RCP > 99%). 177Lu-DOTA-Peptide 2 showed a high value of stability in acetate buffer (91.4% at 312 h) and human plasma (>97% at 24 h). Radioconjugate exhibited low internalization (<5%) and high affinity for somatostatin receptors (Kd = 12.06 nM, Bmax = 0.20 pmol/106 cells) using saturation binding assay. Effective tumor uptake of 7.3% ID/g (percentage of injected dose per gram of tumor) at 4 h post-injection and fast clearance of radiopeptide from blood and other organs led to a high tumor-to-normal organ ratios. SPECT/CT imaging clearly showed the activity localization in tumor. The favorable antagonistic properties of 177Lu-DOTA-Peptide 2 on the somatostatin receptors can make it a suitable candidate for peptide receptor radionuclide therapy (PRRT). In the future study, the therapeutic application of this radiopeptide will be evaluated.

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.

Synthesis and Biological Evaluation of Cyclic [99mTc]-HYNIC-CGPRPPC as a Fibrin-Binding Peptide for Molecular Imaging of Thrombosis and Its Comparison with [99mTc]-HYNIC-GPRPP.

PURPOSE: Many patients worldwide suffer from cardiovascular diseases for which an underlying factor is thrombosis. Devising a molecular imaging technique for early detection of thrombosis in a clinical setting is highly recommended. Because fibrin is a major constituent of clots and is present in all types of thrombi but absent in circulation, it is a highly specific and sensitive target for molecular imaging of thrombi. It is assumed that cyclization of peptides will improve the receptor binding affinity and stability of the peptide. In the present study, we have developed linear and cyclic fibrin-binding peptides for thrombus imaging and compared their biological properties. PROCEDURES: Linear HYNIC-GPRPP and cyclic HYNIC-CGPRPPC peptides were synthesized using a standard Fmoc strategy and radiolabeled with Tc-99m. The stability of the radiolabeled peptides in human plasma and their affinity for fibrin and blood clots were determined. Blood clearance and biodistribution were evaluated in rats and mice, respectively. The peptide with the highest affinity was injected to a live rabbit femoral thrombosis model, and scintigraphic images were obtained. RESULTS: In vitro studies show that peptides are stable in human plasma and have a high affinity for human fibrin. They also demonstrated fast blood clearance in rats and high thrombus uptake in the Balb/c mice femoral thrombosis model. Femoral thrombosis was visualized 30 min postinjection of cyclic peptide in a live rabbit model using single photon emission computed tomography (SPECT)/X-ray computed tomography. CONCLUSIONS: The results indicate that the cyclic peptide is a promising agent for molecular imaging of fibrin using SPECT.

Preclinical evaluation of holmium-166 labeled anti-VEGF-A(Bevacizumab).

Radiolabeled antiangiogenic monoclonal antibodies are potential agents for targeted therapy in specific types of malignancies. In this study, (166)Ho-DOTA-Bevacizumab was used in biodistribution studies using single-photon emission computed tomography (SPECT) to acquire dosimetric aspects of the radiolabeled antibody in mice. The liver toxicity of the radiolabeled antibody was also determined using serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase and alkaline phosphatase assay 2-7 days post-injection. The SPECT biodistribution demonstrated a similar pattern as the other radiolabeled anti-vascular endothelial growth factor A (VEGF-A) immunoconjugates. (166)Ho-DOTA-Bevacizumab was revealed as a potential compound for therapy/imaging of VEGF-A expression in oncology.