RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Astato/química , Bombesina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Compostos Radiofarmacêuticos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Bombesina/análogos & derivados , Bombesina/síntese química , Bombesina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Células PC-3 , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Bombesina/química , Lutécio , Radioisótopos , Receptores da Bombesina/metabolismo , Animais , Bombesina/síntese química , Bombesina/metabolismo , Humanos , Masculino , Camundongos , Oligopeptídeos/química , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , RadioquímicaRESUMO
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.
Assuntos
Bombesina/química , Quitosana/química , Radioisótopos de Gálio/química , Nanopartículas de Magnetita/química , Imagem Molecular/métodos , Animais , Ligação Competitiva , Bombesina/sangue , Bombesina/síntese química , Morte Celular , Linhagem Celular Tumoral , Quitosana/síntese química , Feminino , Humanos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/ultraestrutura , Camundongos Nus , Tamanho da Partícula , Tomografia por Emissão de Pósitrons , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Distribuição Tecidual , Difração de Raios XRESUMO
A new fluorine-containing azadibenzocyclooctyne (ADIBO-F) was designed using a synthetically accessible pathway. The fluorine-18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21-35 % radiochemical yield in 30â minutes of synthetic time. ADIBO-F has been incorporated into azide-functionalized, cancer-targeting peptides through a strain-promoted alkyne-azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide-based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptorâ 1a (GHSR-1a) and gastrin-releasing peptide receptor (GRPR), with IC50 values of 9.7 and 0.50â nm, respectively. This is a new and rapid labelling option for the incorporation of fluorine-18 into biomolecules for PET imaging.
Assuntos
Bombesina/análogos & derivados , Ciclo-Octanos/química , Grelina/análogos & derivados , Compostos Heterocíclicos com 3 Anéis/química , Compostos Radiofarmacêuticos/química , Alcinos/síntese química , Alcinos/química , Bombesina/síntese química , Química Click , Reação de Cicloadição , Ciclo-Octanos/síntese química , Radioisótopos de Flúor , Grelina/síntese química , Compostos Heterocíclicos com 3 Anéis/síntese química , Marcação por Isótopo/métodos , Estrutura Molecular , Tomografia por Emissão de Pósitrons/métodos , Estudo de Prova de Conceito , Compostos Radiofarmacêuticos/síntese químicaRESUMO
We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [99mTc]1, ([99mTc]demobesin 1, 99mTc-[N4'-diglycolate-dPhe6,Leu-NHEt13]BBN(6-13)). [99mTc]1 has shown superior biological profile compared to analogous agonist-based 99mTc-radioligands. We herein present a small library of [99mTc]1 mimics generated after structural modifications in (a) the linker ([99mTc]2, [99mTc]3, [99mTc]4), (b) the peptide chain ([99mTc]5, [99mTc]6), and (c) the C-terminus ([99mTc]7 or [99mTc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptor-affine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [99mTc]1-[99mTc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [99mTc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [99mTc]1 (5.4 ± 0.7%ID/g at 24 h pi).
Assuntos
Bombesina/análogos & derivados , Neoplasias Experimentais/diagnóstico por imagem , Compostos de Organotecnécio/síntese química , Compostos Radiofarmacêuticos/síntese química , Receptores da Bombesina/antagonistas & inibidores , Animais , Ligação Competitiva/efeitos dos fármacos , Bombesina/síntese química , Bombesina/farmacocinética , Feminino , Humanos , Marcação por Isótopo/métodos , Camundongos , Camundongos SCID , Compostos de Organotecnécio/farmacocinética , Células PC-3 , Radioisótopos , Compostos Radiofarmacêuticos/farmacocinética , Rênio , Tecnécio/química , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Increasing evidence of peptide receptor overexpression in various cancer cells, warrant the development of receptor specific radiolabeled peptides for molecular imaging and therapy in nuclear medicine. Gastrin-releasing-peptide (GRP) receptor, are overexpressed in a variety of human cancer cells. The present study report the synthesis and biological evaluation of new bombesin (BBN) analogs, HYNIC-Asp-[Phe13]BBN(7-13)-NH-CH2-CH2-CH3:BA1, HYNIC-Pro-[Tyr13Met14]BBN(7-14)NH2:BA2 as prospective tumor imaging agent with compare to BBN(7-14)NH2:BS as standard. The pharmacophores were radiolabeled in high yields with 99mTc, characterized for their stability in serum and saline, cysteine/histidine and were found to be substantially stable. Internalization/externalization and receptor binding studies were assessed using MDA-MB-231 cells and showed high receptor binding-affinity and favourable internalization. Fluorescence studies revealed that BA1 changed the morphology of the cells and could localize in the nucleus more effectively than BA2/BS. Cell-viability studies displayed substantial antagonistic and nuclear-internalization effect of BA1. BA1 also exhibited antiproliferative effect on MDA-MB-231 cell by inducing apoptosis. In vivo behaviour of the radiopeptides was evaluated in GRP receptor positive tumor bearing mice. The 99mTc-BA1/99mTc-BA2 demonstrated rapid blood/urinary clearance through the renal pathway and comparatively more significant tumor uptake image and favourable tumor-to-non-target ratios provided by 99mTc-BA1. The specificity of the in vivo uptake was confirmed by co-injection with BS. Moreover, 99mTc-BA1 provided a much clearer tumor image in scintigraphic studies than others. Thus the combination of favourable in vitro and in vivo properties renders BA1 as more potential antagonist bombesin-peptide for targeting GRP-receptor positive tumor. These properties are encouraging to carry out further experiments for non-invasive receptor targeting potential diagnostinc and therapeutic agent for tumors.
Assuntos
Bombesina/genética , Neoplasias da Mama/tratamento farmacológico , Peptídeos/genética , Receptores da Bombesina/genética , Animais , Apoptose/efeitos dos fármacos , Bombesina/administração & dosagem , Bombesina/síntese química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Peptídeos/administração & dosagem , Peptídeos/síntese química , Cintilografia , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/síntese química , Receptores da Bombesina/administração & dosagem , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Attaching polar pharmacological modifiers to molecular imaging probes is a common strategy to modulate their pharmacokinetic profiles to improve such parameters as the clearance rate of radiotracers and/or metabolites, and to enhance signal-to-background ratios. We combined the tumor-targeting peptide sequence of bombesin (BBN) with glucuronic acid and the single-photon emission computed tomography (SPECT) radionuclide 99m Tc by the "click-to-chelate" methodology. The 99m Tc-tricarbonyl-labeled glucuronated BBN conjugate was compared with a reference compound lacking the carbohydrate. The radiolabeled conjugates displayed similar characteristics inâ vitro (cell internalization, receptor affinity), but the hydrophilicity of the glycated version was significantly increased. While the tumor uptake of the two radioconjugates in xenografted mice was similar, the glycated peptide exhibited unexpected higher uptake in organs of the hepatobiliary excretion pathway than the more lipophilic reference compound. Control experiments suggest that this may be the result of unspecific accumulation of metabolites in which the glucuronic acid moiety does not act as an innocent pharmacological modifier.
Assuntos
Bombesina/farmacocinética , Quelantes/farmacocinética , Química Click , Imagem Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Neoplasias Experimentais/metabolismo , Compostos de Organotecnécio/farmacocinética , Peptídeos/farmacocinética , Animais , Bombesina/síntese química , Bombesina/química , Quelantes/síntese química , Quelantes/química , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Camundongos , Camundongos Nus , Sondas Moleculares/síntese química , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/patologia , Compostos de Organotecnécio/química , Peptídeos/química , Relação Estrutura-Atividade , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Células Tumorais CultivadasRESUMO
Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6-14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6-14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6-14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics.
Assuntos
Bombesina/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Bombesina/síntese química , Bombesina/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Receptores da Bombesina/biossíntese , Receptores da Bombesina/metabolismo , Relação Estrutura-AtividadeRESUMO
The aim of this work was to synthesize Lys(1)(α,γ-Folate)-Lys(3)((177)Lu-DOTA)-Bombesin (1-14) ((177)Lu-Folate-BN), as well as to assess its potential for molecular imaging and targeted radiotherapy of breast tumors expressing folate receptors (FR) and gastrin-releasing peptide receptors (GRPR). Radiation absorbed doses of (177)Lu-Folate-BN (74 MBq, i.v.) estimated in athymic mice with T47D-induced breast tumors (positive to FR and GRPR), showed tumor doses of 23.9±2.1 Gy. T47D-tumors were clearly visible (Micro-SPECT/CT images). (177)Lu-Folate-BN demonstrated properties suitable as a theranostic radiopharmaceutical.
Assuntos
Bombesina/análogos & derivados , Neoplasias da Mama/radioterapia , Complexos de Coordenação/síntese química , Complexos de Coordenação/uso terapêutico , Lutécio/uso terapêutico , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Bombesina/síntese química , Bombesina/química , Bombesina/uso terapêutico , Neoplasias da Mama/diagnóstico por imagem , Linhagem Celular Tumoral , Complexos de Coordenação/química , Feminino , Humanos , Camundongos , Camundongos Nus , Imagem Multimodal , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Radioisótopos/uso terapêutico , Compostos Radiofarmacêuticos/química , Dosagem Radioterapêutica , Nanomedicina Teranóstica/métodos , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for (68) Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually.
Assuntos
Acetatos/química , Bombesina/análogos & derivados , Radioisótopos de Gálio/química , Compostos Heterocíclicos com 1 Anel/química , Fragmentos de Peptídeos/síntese química , Compostos Radiofarmacêuticos/síntese química , Bombesina/síntese química , Bombesina/química , Humanos , Fragmentos de Peptídeos/química , Estabilidade Proteica , Compostos Radiofarmacêuticos/química , Soro , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
UNLABELLED: Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH2 = bombesin, a GRPr-specific peptide targeting probe. METHODS: The PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH2], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with (64)CuCl2 and (nat)CuCl2. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.). RESULTS: Competitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-((nat)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18h p.i. with collateral, background radiation also being observed in non-target tissue. CONCLUSIONS: DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2 in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues.
Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Bombesina/metabolismo , Radioisótopos de Cobre , Glutamato Carboxipeptidase II/metabolismo , Receptores da Bombesina/metabolismo , Acetatos/química , Aminoácidos Neutros/química , Ácido Aminocaproico/química , Animais , Transporte Biológico , Bombesina/síntese química , Bombesina/química , Linhagem Celular Tumoral , Técnicas de Química Sintética , Feminino , Glutaratos/química , Compostos Heterocíclicos com 1 Anel/química , Humanos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Radioquímica , Ureia/análogos & derivados , Ureia/químicaRESUMO
UNLABELLED: Gastrin-releasing peptide receptors (GRPRs) expressed on human tumors can serve as molecular targets for radiolabeled peptide analogs based on the frog tetradecapeptide bombesin (BBN). We have recently expanded this approach toward human GRP(18-27) sequences and introduced (99m)Tc-demomedin C, our first radiotracer based on GRP(18-27), showing favorable biologic characteristics during preclinical evaluation in rodents. We now present a series of (99m)Tc-demomedin C analogs, generated by single-Gly(24) or double-Gly(24)/Met(27) substitutions in the peptide chain, and compare their performance in GRPR-positive in vitro and in vivo models. METHODS: The SARNC ([(N4)Gly(18)]GRP(18-27)) analogs (SARNC2 dAla(24), SARNC3 dAla(24)/Nle(27), SARNC4 dAla(24)/Leu(27), SARNC5 ßAla(24), and SARNC6 Sar(24)) were synthesized on the solid support and purified by high-performance liquid chromatography (HPLC). Competition binding experiments against [(125)I-Tyr(4)]BBN were conducted in GRPR-positive PC-3 cell membranes. Internalization of (99m)Tc radioligands was compared in PC-3 cells at 37°C. Metabolic stability was studied by HPLC analysis of blood samples collected 5 min after injection of radiopeptides in mice. Biodistribution was performed by injecting a (99m)Tc-SARNC bolus (185 kBq [5 µCi], 100 µL, 10 pmol of peptide ± 40 nmol of Tyr(4)-BBN: in vivo GRPR blockade) in severe combined immune deficient mice bearing PC-3 xenografts. RESULTS: SARNCs bound to GRPR with high affinity (range of 50% inhibitory concentration [IC50] values, 0.3 nM [SARNC5] to 9.3 nM [SARNC4]). (99m)Tc-SARNCs specifically internalized in PC-3 cells, with (99m)Tc-SARNC5 displaying the fastest internalization rate. (99m)Tc-SARNCs showed distinct degradation rates (17% [(99m)Tc-SARNC3] to >50% [(99m)Tc-SARNC4] remaining intact). All (99m)Tc-SARNCs efficiently and specifically localized in GRPR-positive PC-3 xenografts in mice (4.4 percentage injected dose per gram [%ID/g] [(99m)Tc-SARNC4] to 12.0 %ID/g [(99m)Tc-SARNC2] at 4 h after injection). (99m)Tc-SARNC6 displayed the highest tumor-to-nontumor ratios followed by (99m)Tc-SARNC2. CONCLUSION: This structure-activity relationship study has shown the impact of single-Gly(24) or double-Gly(24)/Met(27) substitutions in the (99m)Tc-SARNC1 motif on key biologic parameters, including GRPR affinity, internalization efficiency, and in vivo stability, which eventually determine the pharmacokinetic profile of resulting radiopeptides. By revealing improved analogs, this study has strengthened the applicability perspectives of radioligands based on human GRP sequences in the detection and therapy of GRPR-expressing tumors in humans.
Assuntos
Bombesina/síntese química , Fragmentos de Peptídeos/síntese química , Traçadores Radioativos , Tecnécio , Animais , Bombesina/metabolismo , Bombesina/farmacocinética , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Técnicas de Química Sintética , Humanos , Ligantes , Masculino , Camundongos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinéticaRESUMO
Acetylene-bearing 2-[(18)F]fluoropyridines [(18)F]FPy5yne and PEG-[(18)F]FPyKYNE were prepared via efficient nucleophilic heteroaromatic [(18)F]fluorination of their corresponding 2-trimethylammoniumpyrdinyl precursors. The prosthetic groups were conjugated to azide- and PEG3-modified bombesin(6-14) analogues via copper-catalyzed azide-alkyne cycloaddition couplings to yield mono- and di-mini-PEGylated ligands for PET imaging of the gastrin- releasing peptide receptor. The PEG3- and PEG2/PEG3-bearing (18)F peptides showed decreased lipophilicity relative to an analogous non-mini-PEGylated (18)F peptide. Assessment of water-soluble peptide pharmacokinetics and tumour-targeting capabilities in a mouse model of prostate cancer is currently underway.
Assuntos
Bombesina , Radioisótopos de Flúor , Neoplasias Experimentais/diagnóstico , Neoplasias da Próstata/diagnóstico , Piridinas , Receptores da Bombesina/análise , Animais , Bombesina/síntese química , Bombesina/química , Modelos Animais de Doenças , Radioisótopos de Flúor/química , Ligantes , Masculino , Camundongos , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Piridinas/químicaRESUMO
Novel bombesin (BBN) antagonists were synthesized by coupling the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to H-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (JMV594) through linkers of increasing number of (ßAla)x residues (x = 1-3). Labeling with (111)In afforded the respective radiotracers in high purity and high specific activity. Bioconjugate affinity for the gastrin releasing peptide receptor (GRPR) as determined against [(125)I-Tyr(4)]BBN was high (IC50 values in the lower nanomolar range). Radioligands poorly internalized in PC-3 cells at 37 °C. Radiopeptides remained >60% intact 5 min after entering the bloodstream of healthy mice. After injection in SCID mice bearing human PC-3 xenografts all analogues showed high tumor uptake and rapid background clearance via the kidneys into urine. Interestingly, pancreatic uptake, albeit GRPR-specific, declined rapidly with time. (111)In-DOTA-(ßAla)2-JMV594 achieved the highest tumor values among the group (17.0 ± 2.8%ID/g vs. 8-10%ID/g, respectively, at 4 h pi) indicating that the (ßAla)2-linker favors in vivo interaction of radiopeptides with the GRPR.
Assuntos
Bombesina/síntese química , Bombesina/farmacologia , Radioisótopos de Índio , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/metabolismo , Sequência de Aminoácidos , Animais , Bombesina/análogos & derivados , Linhagem Celular Tumoral , Técnicas de Química Sintética , Feminino , Compostos Heterocíclicos com 1 Anel/química , Humanos , Marcação por Isótopo , Ligantes , Camundongos , Oligopeptídeos/químicaRESUMO
The synthesis of a series of new zinc phthalocyanine-peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine-bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor.
Assuntos
Complexos de Coordenação/síntese química , Indóis/síntese química , Oligopeptídeos/síntese química , Fármacos Fotossensibilizantes/síntese química , Zinco , Alcinos/síntese química , Alcinos/metabolismo , Alcinos/farmacologia , Animais , Bombesina/análogos & derivados , Bombesina/síntese química , Bombesina/metabolismo , Bombesina/farmacologia , Linhagem Celular Tumoral , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Peptídeo Liberador de Gastrina/metabolismo , Humanos , Indóis/metabolismo , Indóis/farmacologia , Integrinas/metabolismo , Isoindóis , Camundongos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologiaRESUMO
1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [(99m)Tc(CO)(3)](+) core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys(Dap) in which the ε-NH(2) group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH(2) group of the α-amino acid portion is Fmoc- and the -NH(2) of Dap are Boc-protected. Fmoc-l-Lys(Dap(Boc)) was either conjugated to the N- and C-terminus of bombesin BBN(7-14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced the native lysine in a cyclic RGD peptide with l-Lys(Dap). For all peptides, quantitative labeling with the [(99m)Tc(CO)(3)](+) core at a 10 µM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from [Re(OH(2))(3)(CO)(3)](+) and Lys(Dap)-BBN(7-14) or cyclo-(RGDyK(Dap)), respectively. Determination of integrin receptor binding showed low to medium nanomolar affinities for various receptor subtypes. The IC(50) of cyclo-(RGDyK(Dap[Re(CO)(3)])) for α(v)ß(3) is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude mice showed reasonable α(v)ß(3)-integrin specific tumor uptake. Altogether, orthogonally protected l-Lys(Dap) represents a highly versatile building block for integration in any peptide sequence. Lys(Dap)-precursors allow high-yield (99m)Tc-labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+), forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics.
Assuntos
Bombesina/análogos & derivados , Lisina/análogos & derivados , Melanoma/diagnóstico , Oligopeptídeos/química , Compostos de Organotecnécio/química , Fragmentos de Peptídeos/química , Propionatos/química , Técnicas de Síntese em Fase Sólida/métodos , Animais , Bombesina/síntese química , Bombesina/química , Linhagem Celular Tumoral , Quelantes/síntese química , Quelantes/química , Humanos , Integrina alfaVbeta3/metabolismo , Lisina/síntese química , Melanoma/metabolismo , Camundongos , Camundongos Nus , Oligopeptídeos/síntese química , Compostos de Organotecnécio/síntese química , Fragmentos de Peptídeos/síntese química , Propionatos/síntese químicaRESUMO
The synthesis and preclinical evaluation of [(99m)Tc]Demomedin C in GRPR-expressing models are reported. Demomedin C resulted by coupling a Boc-protected N(4)-chelator to neuromedin C (human GRP(18-27)), which, after (99m)Tc-labeling, afforded [(99m)Tc]Demomedin C. Demomedin C showed high affinity and selectivity for the GRPR during receptor autoradiography on human cancer samples (IC(50) in nM: GRPR, 1.4 ± 0.2; NMBR, 106 ± 18; and BB(3)R, >1000). It triggered GRPR internalization in HEK-GRPR cells and Ca(2+) release in PC-3 cells (EC(50) = 1.3 nM). [(99m)Tc]Demomedin C rapidly and specifically internalized at 37 °C in PC-3 cells and was stable in mouse plasma. [(99m)Tc]Demomedin C efficiently and specifically localized in human PC-3 implants in mice (9.84 ± 0.81%ID/g at 1 h pi; 6.36 ± 0.85%ID/g at 4 h pi, and 0.41 ± 0.07%ID/g at 4 h pi block). Thus, human GRP-based radioligands, such as [(99m)Tc]Demomedin C, can successfully target GRPR-expressing human tumors in vivo while displaying attractive biological features--e.g. higher GRPR-selectivity--vs their frog-homologues.
Assuntos
Bombesina/síntese química , Oligopeptídeos/síntese química , Compostos de Organotecnécio/síntese química , Fragmentos de Peptídeos/síntese química , Compostos Radiofarmacêuticos/síntese química , Receptores da Bombesina/metabolismo , Tecnécio , Sequência de Aminoácidos , Animais , Anuros , Autorradiografia , Ligação Competitiva , Bombesina/química , Bombesina/farmacocinética , Neoplasias Brônquicas/diagnóstico por imagem , Neoplasias Brônquicas/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias do Íleo/diagnóstico por imagem , Neoplasias do Íleo/metabolismo , Ligantes , Masculino , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Transplante de Neoplasias , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Bombesina/agonistas , Especificidade da Espécie , Relação Estrutura-Atividade , Distribuição Tecidual , Transplante HeterólogoRESUMO
Bombesins (BN) containing (99m)Tc '4+1' complexes may be useful to detect tumors expressing the gastrin-releasing peptide receptor (GRPR). Derivatives of the formula [(99m)Tc(NS(3)R)(L2-BN(st))] were synthesized, in which Tc(III) is coordinated by an isocyanide L2-BN(st) bearing the peptide (BN(st)=ßAla-ßAla-Gln-Trp-Ala-Val-Gly-His-Cha-Nle-NH(2)) and a tetradentate chelator NS(3)R. NS(3)R consists of 2,2',2â³-nitrilotriethanethiol (NS(3)) bearing a crown ether (NS(3)crown), an aliphatic amine (NS(3)en) and a tricarboxylic acid (NS(3)(COOH)(3)). Non-radioactive Re compounds were prepared and analysed by electrospray ionization mass spectrometry. The structural similarity to the (99m)Tc conjugates was demonstrated by their identical HPLC elution profiles. The lipophilicity of [(99m)Tc(NS(3)R)(L2-BN(st))] decreased depending on the coligands NS(3)crown (log D(O/W), pH=7.4, 0.98 ± 0.11), NS(3)en (-0.49 ± 0.07) and NS(3)(COOH)(3) (-2.01 ± 0.09). Biodistribution in normal rats was characterized by an increasing kidney uptake and a decreasing uptake into the liver corresponding to the reduced lipophilicity of the conjugates. The pancreatic uptake expressed by the organ/blood ratio of standardized uptake values at 60 min p.i. in rats was 8.6 ± 1.2 for [(99m)Tc(NS(3)en)(L2-BN(st))] and higher compared to the other conjugates. The pancreas/liver ratio of the SUV at 60 min p.i. in rats was highest for [(99m)Tc(NS(3)(COOH)(3))(L2-BN(st))] at 8.4 ± 1.3. [(99m)Tc(NS(3)en)(L2-BN(st))] was further studied in tumor-bearing mice and its pancreas/blood and pancreas/liver ratios were lower, however the pancreas/kidney ratios were higher in mice compared to rats. The activity uptake of [(99m)Tc(NS(3)en)(L2-BN(st))] into the PC-3 tumor xenografts was low (%ID/g: 0.83 ± 0.18 at 60 min; SUV: 0.21 ± 0.05 at 60 min) but specific.
Assuntos
Bombesina/análogos & derivados , Bombesina/farmacocinética , Complexos de Coordenação/farmacocinética , Cianetos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio/química , Animais , Bombesina/síntese química , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Cianetos/síntese química , Estabilidade de Medicamentos , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Camundongos , Camundongos Nus , Transplante de Neoplasias/diagnóstico por imagem , Pâncreas/diagnóstico por imagem , Pâncreas/metabolismo , Fragmentos de Peptídeos , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Ratos , Receptores da Bombesina/metabolismo , Rênio/química , Distribuição TecidualAssuntos
Bombesina/análogos & derivados , Química Click/métodos , Compostos Radiofarmacêuticos/síntese química , Azidas/química , Ligação Competitiva , Bombesina/síntese química , Bombesina/química , Linhagem Celular Tumoral , Cobre/química , Radioisótopos de Flúor , Humanos , Marcação por Isótopo , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Ligação Proteica , Ensaio Radioligante , Compostos Radiofarmacêuticos/química , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/metabolismoRESUMO
Magnetic resonance imaging (MRI) has become the leading imaging tool for providing fine anatomical and physiology details. Optical imaging is offering a sensitive and specific method for in vivo molecular imaging of targeting molecules. The goal of this study is to design, synthesize, and characterize a new target-specific dual contrast agent for MR and optical imaging. Hence, [Gd(TTDA-NP)(H(2)O)](2-) was prepared and characterized. In addition, an 8-amino acid Bombesin analogue (BN) peptide substrate, which can target prostate, breast, and colon cancer, was synthesized by solid-phase peptide synthesis and subsequently conjugated with [Gd(TTDA-NP)(H(2)O)](2-) to form BN conjugated Gd-TTDA-NP-BN. The water-exchange rate (k(ex)(298)) for [Gd(TTDA-NP)(H(2)O)](2-) (110×10(6)s(-1)) is significantly higher than that of [Gd(DTPA)(H(2)O)](2-) complex and the rotational correlation time (τ(R)) for [Gd(TTDA-NP)(H(2)O)](2-) (145ps) is also higher than those of [Gd(TTDA)(H(2)O)](2-) (104ps) and [Gd(DTPA)(H(2)O)](2-) (103ps). The Gd-TTDA-NP-BN shows remarkable high relaxivity (7.12mM(-1)s(-1)) comparing to that of [Gd(TTDA-NP)(H(2)O)](2-). The fluorescence studies showed that the Gd-TTDA-NP-BN could efficiently enter PC-3 cells. Additionally, the human cancer cells xenografts using Gd-TTDA-NP-BN-Cy5.5 as an optical imaging probe clearly visualized subcutaneous PC-3 tumor and demonstrated its targeting ability to the gastrin releasing peptide (GRP) receptor overexpression. Furthermore, the biodistribution studies demonstrated significantly high tumor uptake (25.97±1.07% ID/g) and high tumor-to-normal tissue ratios at one hour post-injection of Gd-TTDA-NP-BN-Cy5.5 in the animal model. These results suggest that the Gd-TTDA-NP-BN-Cy5.5 is a superior probe for in vivo optical imaging. Importantly, the MR imaging studies showed notable signal enhancement (44.9±4.2%) on the tumor, indicating a high level accumulation of the contrast agent within the PC-3 tumor sites. Hence, targeting of prostate cancer cells was observed under in vitro and in vivo MR imaging studies using Gd-TTDA-NP-BN contrast agent. We conclude that Gd-TTDA-NP-BN and Gd-TTDA-NP-BN-Cy5.5 can be potentially used as the contrast agents for targeting GRP receptor overexpressing cells and tumors.