RESUMO
Gastrin-releasing peptide receptor (GRPR), overexpressed in many solid tumors, is a promising imaging marker and therapeutic target. Most reported GRPR-targeted radioligands contain a C-terminal amide. Based on the reported potent antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, we synthesized C-terminal hydroxamate-derived [68Ga]Ga-LW02075 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) and [68Ga]Ga-LW02050 ([68Ga]Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), and compared them with the closely related and clinically validated [68Ga]Ga-SB3 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt). Binding affinities (Ki) of Ga-SB3, Ga-LW02075, and Ga-LW02050 were 1.20 ± 0.31, 1.39 ± 0.54, and 8.53 ± 1.52 nM, respectively. Both Ga-LW02075 and Ga-LW02050 were confirmed to be GRPR antagonists by calcium release assay. Imaging studies showed that PC-3 prostate cancer tumor xenografts were clearly visualized at 1 h post injection by [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 in PET images, but not by [68Ga]Ga-LW02075. Ex vivo biodistribution studies conducted at 1 h post injection showed that the tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 ± 1.00 vs. 6.98 ± 1.36 %ID/g), followed by [68Ga]Ga-LW02075 (3.97 ± 1.71 %ID/g). [68Ga]Ga-SB3 had the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [68Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 ± 0.11 %ID/g. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing cancer lesions.
Assuntos
Radioisótopos de Gálio , Ácidos Hidroxâmicos , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Receptores da Bombesina , Receptores da Bombesina/metabolismo , Receptores da Bombesina/antagonistas & inibidores , Radioisótopos de Gálio/química , Animais , Humanos , Tomografia por Emissão de Pósitrons/métodos , Camundongos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacocinética , Ácidos Hidroxâmicos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Linhagem Celular Tumoral , Distribuição Tecidual , Masculino , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismoRESUMO
Acute kidney injury (AKI) manifests as a clinical syndrome characterised by the rapid accumulation of metabolic wastes, such as blood creatinine and urea nitrogen, leading to a sudden decline in renal function. Currently, there is a lack of specific therapeutic drugs for AKI. Previously, we identified gastrin-releasing peptide receptor (GRPR) as a pathogenic factor in AKI. In this study, we investigated the therapeutic potential of a novel Chinese medicine monomer, aurantiamide (AA), which exhibits structural similarities to our previously reported GRPR antagonist, RH-1402. We compared the therapeutic efficacy of AA with RH-1402 both in vitro and in vivo using various AKI models. Our results demonstrated that, in vitro, AA attenuated injury, necroptosis, and inflammatory responses in human renal tubular epithelial cells subjected to repeated hypoxia/reoxygenation and lipopolysaccharide stimulation. In vivo, AA ameliorated renal tubular injury and inflammation in mouse models of ischemia/reperfusion and cecum ligation puncture-induced AKI, surpassing the efficacy of RH-1402. Furthermore, molecular docking and cellular thermal shift assay confirmed GRPR as a direct target of AA, which was further validated in primary cells. Notably, in GRPR-silenced HK-2 cells and GRPR systemic knockout mice, AA failed to mitigate renal inflammation and injury, underscoring the importance of GRPR in AA's mechanism of action. In conclusion, our study has demonstrated that AA serve as a novel antagonist of GRPR and a promising clinical candidate for AKI treatment.
Assuntos
Injúria Renal Aguda , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose , Receptores da Bombesina , Animais , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/patologia , Humanos , Necroptose/efeitos dos fármacos , Camundongos , Masculino , Linhagem Celular , Receptores da Bombesina/metabolismo , Receptores da Bombesina/antagonistas & inibidores , Inflamação/tratamento farmacológico , Modelos Animais de Doenças , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêuticoRESUMO
Each tumor has its own distinctive molecular identity. Treatment, therefore, should be tailored to this unique cancer phenotype. Theragnostics uses the same compound for targeted imaging and treatment, radiolabeled to an appropriate radionuclide, respectively. Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, and radiolabeled GRPR antagonists have shown high diagnostic performance at staging and biochemical recurrence. Several GRPR-targeting theragnostic compounds have been developed preclinically. Their translation into clinics is underway with 4 clinical trials recruiting participants with GRPR-expressing tumors.
Assuntos
Tomografia por Emissão de Pósitrons , Neoplasias da Próstata , Compostos Radiofarmacêuticos , Receptores da Bombesina , Humanos , Masculino , Receptores da Bombesina/antagonistas & inibidores , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/radioterapia , Compostos Radiofarmacêuticos/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/uso terapêuticoRESUMO
Radiolabeled gastrin-releasing peptide receptor (GRPR) antagonists have shown great promise for the theranostics of prostate cancer; however, their suboptimal metabolic stability leaves room for improvements. It was recently shown that the replacement of Gly11 with Sar11 in the peptidic [D-Phe6,Leu13-NHEt,des-Met14]BBN(6-14) chain stabilized the [99mTc]Tc-DB15 radiotracer against neprilysin (NEP). We herein present DOTAGA-PEG2-(Sar11)RM26 (AU-RM26-M1), after Gly11 to Sar11-replacement. The impact of this replacement on the metabolic stability and overall biological performance of [111In]In-AU-RM26-M1 was studied using a head-to-head comparison with the unmodified reference [111In]In-DOTAGA-PEG2-RM26. In vitro, the cell uptake of [111In]In-AU-RM26-M1 could be significantly reduced in the presence of a high-excess GRPR-blocker that demonstrated its specificity. The cell uptake of both radiolabeled GRPR antagonists increased with time and was superior for [111In]In-AU-RM26-M1. The dissociation constant reflected strong affinities for GRPR (500 pM for [111In]In-AU-RM26-M1). [111In]In-AU-RM26-M1 showed significantly higher stability in peripheral mice blood at 5 min pi (88 ± 8% intact) than unmodified [111In]In-DOTAGA-PEG2-RM26 (69 ± 2% intact; p < 0.0001). The administration of a NEP inhibitor had no significant impact on the Sar11-compound (91 ± 2% intact; p > 0.05). In vivo, [111In]In-AU-RM26-M1 showed high and GRPR-mediated uptake in the PC-3 tumors (7.0 ± 0.7%IA/g vs. 0.9 ± 0.6%IA/g in blocked mice) and pancreas (2.2 ± 0.6%IA/g vs. 0.3 ± 0.2%IA/g in blocked mice) at 1 h pi, with rapid clearance from healthy tissues. The tumor uptake of [111In]In-AU-RM26-M1 was higher than for [111In]In-DOTAGA-PEG2-RM26 (at 4 h pi, 5.7 ± 1.8%IA/g vs. 3 ± 1%IA/g), concordant with its higher stability. The implanted PC-3 tumors were visualized with high contrast in mice using [111In]In-AU-RM26-M1 SPECT/CT. The Gly11 to Sar11-substitution stabilized [111In]In-DOTAGA-PEG2-(Sar11)RM26 against NEP without negatively affecting other important biological features. These results support the further evaluation of AU-RM26-M1 for prostate cancer theranostics after labeling with clinically relevant radionuclides.
Assuntos
Neoplasias da Próstata , Receptores da Bombesina , Animais , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Medicina de Precisão , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/metabolismoRESUMO
INTRODUCTION: Prostate and breast cancer represent a leading cause of cancer-related death worldwide with a dramatic social and demographic impact. Gastrin-releasing peptide receptors (GRPRs), part of the bombesin (BBN) family, have been found overexpressed in both the aforementioned malignancies, and have emerged as a potentially useful target to combine imaging and therapy in a unique, synergistic approach, namely 'theranostics.' AREAS COVERED: The biological characteristics of GRPRs, as well as their aberrant expression in breast and prostate cancer, are covered. Furthermore, the role of the different available GRPR agonists and antagonists, labeled with radionuclides suitable for molecular imaging through single photon computed tomography (SPECT) or positron emission computed (PET/CT), is reviewed, with a particular focus on the potential theranostic implications. EXPERT OPINION: GRPR-targeted molecular imaging of breast and prostate cancer gave promising results in pre-clinical studies. Notably, GRPRs' expression was found to be inversely correlated with disease progression in both prostate and breast cancer. Among the different GRPR agonists and antagonists applied as imaging probes, RM26 presented particularly interesting applications, with meaningful theranostic potential, but its diagnostic performance resulted highly influenced by the choice of the chelator-radionuclide complex, being long-life radionuclides more suitable for obtaining high-contrast imaging.
Assuntos
Neoplasias da Mama , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Imagem Molecular , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Radioisótopos/uso terapêutico , Receptores da Bombesina/agonistas , Receptores da Bombesina/antagonistas & inibidores , FemininoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Bombesina/farmacologia , Desenho de Fármacos , Receptores da Bombesina/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Bombesina/análogos & derivados , Bombesina/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Receptores da Bombesina/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.
Assuntos
Antineoplásicos/farmacologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/tratamento farmacológico , Receptores da Bombesina/antagonistas & inibidores , Animais , Antineoplásicos/química , Radioisótopos de Cobalto , Radioisótopos de Cobre , Humanos , Masculino , Camundongos , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Células PC-3 , Neoplasias da Próstata/metabolismo , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismoRESUMO
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.
Assuntos
Bombesina/química , Bombesina/farmacocinética , Radioisótopos de Gálio/química , Tumores do Estroma Gastrointestinal/diagnóstico por imagem , Tumores do Estroma Gastrointestinal/patologia , Receptores da Bombesina/antagonistas & inibidores , Segurança , Idoso , Idoso de 80 Anos ou mais , Bombesina/efeitos adversos , Bombesina/farmacologia , Feminino , Tumores do Estroma Gastrointestinal/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Radiometria , Distribuição TecidualRESUMO
Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)+ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensation. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR+ neurons in the spinal dorsal horn (SDH). GRPR+ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR+ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP+ neurons in SDH contain glutamate, we investigated the role of GRP+ (GRP+/Glu+) neurons in regulating itch. Chemogenetic inhibition of GRP+ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP+ neurons or ablation of GRPR+ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR+ AMPAR+ neurons in SDH, mediating itch sensation. GRP-GRPR and the glutamate-AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.
Assuntos
Neurônios/metabolismo , Prurido/metabolismo , Receptores de AMPA/metabolismo , Receptores da Bombesina/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Animais , Bombesina/análogos & derivados , Bombesina/farmacologia , Ciclopropanos/toxicidade , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Prurido/induzido quimicamente , Receptores de AMPA/antagonistas & inibidores , Receptores da Bombesina/antagonistas & inibidores , Corno Dorsal da Medula Espinal/efeitos dos fármacosRESUMO
Recurrent and intractable chronic itch is a worldwide problem, but mechanisms, especially the neural mechanisms, underlying chronic itch still remain unclear. In this study, we investigated the peripheral and spinal mechanisms responsible for prolonged itch in a mouse model of allergic contact dermatitis induced by squaric acid dibutylester. We found that repeated exposure of mice to squaric acid dibutylester evoked persistent spontaneous scratching and significantly aberrant cutaneous and systemic immune responses lasting for weeks. Squaric acid dibutylester-induced itch requires both nonhistaminergic and histaminergic pathways, which are likely relayed by GRPR and NPRA in the spinal cord, respectively. Employing genetic, pharmacologic, RNAscope assay, and cell-specific ablation methods, we dissected a neural circuit for prolonged itch formed as Grpr+ neurons act downstream of Npr1+ neurons in the spinal cord. Taken together, our data suggested that targeting GRPR and NPRA may provide effective treatments for allergic contact dermatitis-associated chronic pruritus.
Assuntos
Dermatite Alérgica de Contato/imunologia , Prurido/imunologia , Receptores do Fator Natriurético Atrial/metabolismo , Receptores da Bombesina/metabolismo , Animais , Antipruriginosos/farmacologia , Antipruriginosos/uso terapêutico , Doença Crônica , Ciclobutanos/imunologia , Dermatite Alérgica de Contato/complicações , Dermatite Alérgica de Contato/tratamento farmacológico , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Vias Neurais/efeitos dos fármacos , Vias Neurais/imunologia , Neurônios/metabolismo , Prurido/tratamento farmacológico , Prurido/patologia , Receptores do Fator Natriurético Atrial/antagonistas & inibidores , Receptores da Bombesina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/imunologia , Pele/inervação , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Targeting tumor-expressed receptors using selective molecules for diagnostic, therapeutic, or both diagnostic and therapeutic (theragnostic) purposes is a promising approach in oncologic applications. Such approaches have increased significantly over the past decade. Peptides such as gastrin-releasing peptide receptors targeting radiopharmaceuticals are small molecules with fast blood clearance and urinary excretion. They demonstrate good tissue diffusion, low immunogenicity, and highly selective binding to their target cell-surface receptors. They are also easily produced. Gastrin-releasing peptide receptors, part of the bombesin family, are overexpressed in many tumors, including breast and prostate cancer, and therefore represent an attractive target for future development.
Assuntos
Neoplasias da Mama/química , Neoplasias da Próstata/química , Receptores da Bombesina/análise , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Feminino , Peptídeo Liberador de Gastrina/análise , Peptídeo Liberador de Gastrina/fisiologia , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/terapia , Compostos Radiofarmacêuticos , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/fisiologia , Distribuição TecidualRESUMO
Receptor-targeted radiopharmaceuticals based on low-molecular-weight carriers offer many clinically advantageous attributes relative to macromolecules but have generally been hampered by their rapid clearance from tumors, thus diminishing tumor-to-nontarget tissue ratios. Herein, we present a strategy using irreversible inhibitors (E-64 derivative) of cysteine cathepsins (CCs) as trapping agents to increase the tumor retention of receptor-targeted agents. Methods: We incorporated these CC-trapping agents into agonistic and antagonistic pharmacophores targeting the gastrin-releasing peptide receptor (GRPR). The synthesized radioconjugates with either an incorporated CC inhibitor or a matching control were examined using in vitro and in vivo models of the GRPR-positive, PC-3 human prostate cancer cell line. Results: From the in vitro studies, multiple techniques confirmed that the CC-trapping, GRPR-targeted constructs were able to increase cellular retention by forming intracellular macromolecule adducts. In PC-3 tumor-bearing xenograft mice, the CC-trapping, GRPR-targeted agonistic and antagonistic constructs led to an approximately 2-fold increase in tumor retention with a corresponding improvement in most tumor-to-nontarget tissue ratios over 72 h. Conclusion: CC endolysosomal trapping provides a pathway to increase the efficacy and clinical potential of low-molecular-weight, receptor-targeted agents.
Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacologia , Receptores da Bombesina/agonistas , Receptores da Bombesina/antagonistas & inibidores , Animais , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Células PC-3 , Compostos Radiofarmacêuticos/farmacocinética , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Distribuição TecidualRESUMO
Gastrin-releasing peptide receptors (GRPRs) are promising targets in oligometastatic prostate cancer. We have recently used 55Co (T1/2 = 17.5 h) as a label for next day PET imaging of GRPR expression obtaining high imaging contrast. The radionuclide-chelator combination can significantly influence the biodistribution of radiopeptides. Therefore, in this study, we hypothesized that the properties of 55Co-labeled PEG2-RM26 can be improved by identifying the optimal macrocyclic chelator. All analogues (X-PEG2-RM26, X = NOTA,NODAGA,DOTA,DOTAGA) were successfully labeled with radiocobalt with high yields and demonstrated high stability. The radiopeptides bound specifically and with picomolar affinity to GRPR and their cellular processing was characterized by low internalization. The best binding capacity was found for DOTA-PEG2-RM26. Ex vivo biodistribution in PC-3 xenografted mice was characterized by rapid blood clearance via renal excretion. Tumor uptake was similar for all conjugates at 3 h pi, exceeding the uptake in all other organs. Higher kidney uptake and longer retention were associated with N-terminal negative charge (DOTAGA-containing conjugate). Tumor-to-organ ratios increased over time for all constructs, although significant chelator-dependent differences were observed. Concordant with affinity measurements, DOTA-analog had the best retention of activity in tumors, resulting in the highest tumor-to-blood ratio 24 h pi, which translated into high contrast PET/CT imaging (using 55Co).
Assuntos
Bombesina/farmacocinética , Radioisótopos de Cobalto/farmacocinética , Compostos Macrocíclicos/química , Compostos Organometálicos/química , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/patologia , Receptores da Bombesina/antagonistas & inibidores , Animais , Apoptose , Bombesina/análogos & derivados , Bombesina/farmacologia , Proliferação de Células , Quelantes/química , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neurotransmissores/química , Neurotransmissores/farmacocinética , Neurotransmissores/farmacologia , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The acyclic chelator HBED-CC has attained huge clinical significance owing to high thermodynamic and kinetic stability of 68 Ga-HBED-CC chelate. It provides an excellent platform for quick preparation of 68 Ga-based radiotracers in high yield. Thus, the present study aimed at conjugation of gastrin releasing peptide receptor (GRPr) antagonist, RM26, with HBED-CC chelator for 68 Ga-labeling. In vitro and vivo behavior of the peptide tracer, 68 Ga-HBED-CC-PEG2 -RM26, was assessed and compared with 68 Ga-NODAGA-PEG2 -RM26. The peptide tracers, 68 Ga-HBED-CC-PEG2 -RM26 and 68 Ga-NODAGA-PEG2 -RM26, prepared either by wet chemistry or formulated using freeze-dried kits exhibited excellent radiochemical yield and in vitro stability. The two peptide tracers cleared rapidly from the blood. Biodistribution studies in normal mice demonstrated slightly higher or comparable uptake of 68 Ga-HBED-CC-PEG2 -RM26 in GRPr-expressing organs pancreas, stomach, and intestine. The preliminary studies suggest high potential of 68 Ga-HBED-CC-PEG2 -RM26 for further investigation as a GRPr imaging agent and the wide scope of HBED-CC chelator in development of 68 Ga-based peptide tracers.
Assuntos
Ácido Edético/análogos & derivados , Radioisótopos de Gálio/química , Receptores da Bombesina/antagonistas & inibidores , Técnicas de Química Sintética , Ácido Edético/síntese química , Ácido Edético/química , Ácido Edético/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Células PC-3 , RadioquímicaRESUMO
Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33-39%), reduced cell proliferation at day 3 (-53%) and reduced lipid accumulation at day 21 (-86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/fisiologia , Receptores da Bombesina/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Indóis/farmacologia , Camundongos , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Perilipina-1/genética , Perilipina-1/metabolismo , Piridinas/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/genéticaRESUMO
Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin-derived GRPR-antagonist PEG2 -RM26 for labeling with 177 Lu and further determined the effect of treatment with 177 Lu-labeled peptide alone or in combination with the anti-HER2 antibody trastuzumab in a murine model. The PEG2 -RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide-chelator conjugates were labeled with 177 Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC-3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA-PEG2 -RM26, (C) 177 Lu-DOTAGA-PEG2 -RM26, (D) trastuzumab or (E) 177 Lu-DOTAGA-PEG2 -RM26 in combination with trastuzumab. 177 Lu-DOTAGA-PEG2 -RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/µmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (KD = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with 177 Lu-DOTAGA-PEG2 -RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment-related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four 177 Lu-labeled PEG2 -RM26 analogs, we concluded that 177 Lu-DOTAGA-PEG2 -RM26 was the most promising analog for TRT. Radiotherapy using 177 Lu-DOTAGA-PEG2 -RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti-HER2 therapy additionally improved survival.
Assuntos
Antineoplásicos/farmacologia , Lutécio/química , Polietilenoglicóis/química , Neoplasias da Próstata/tratamento farmacológico , Radioisótopos/química , Receptores da Bombesina/antagonistas & inibidores , Trastuzumab/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Terapia Combinada/métodos , Xenoenxertos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células PC-3 , Próstata/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Spinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itch-like behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output.
Assuntos
Peptídeo Liberador de Gastrina/genética , Prurido/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Comportamento Animal , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neurônios , Optogenética , Bloqueadores dos Canais de Potássio/farmacologia , Prurido/genética , Prurido/psicologia , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/genética , Receptores de Glutamato/fisiologia , Receptores Pré-Sinápticos/metabolismo , Medula Espinal/citologiaRESUMO
Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, representing attractive targets for diagnosis and therapy with bombesin (BBN)-like radioligands. GRPR-antagonists have lately attracted much attention owing to inherent biosafety and favorable pharmacokinetics. We herein present the GRPR-antagonist SB9 structurally resembling the known BBN-based agonist AMBA (SB9 = [Leu13 NHEt-desMet14 ]AMBA). The profiles of 111 In-SB9 and 111 In-AMBA were directly compared in PC-3 cells and tumor-bearing mice. SB9 and AMBA displayed high GRPR affinities. 111 In-AMBA strongly internalized in PC-3 cells, while 111 In-SB9 remained bound on the cell surface showing a typical GRPR-radioantagonist profile. 111 In-SB9 was more stable than 111 In-AMBA, but coinjection of the neprilysin (NEP) inhibitor phosphoramidon (PA) stabilized both in vivo. The radioligands displayed high tumor uptake (20.23 ± 3.41 %ID/g and 18.53 ± 1.54 %ID/g, respectively, at 4 hours pi), but 111 In-SB9 washed faster from background. PA coinjection led to significant increase of tumor uptake, combined with better clearance for 111 In-SB9. In short, this study has revealed superior pharmacokinetics and higher stability for the GRPR-antagonist 111 In-SB9 vs the corresponding agonist 111 In-AMBA consolidating previous evidence that GRPR antagonists are preferable to agonists for tumor imaging and therapy. It has also demonstrated that further pharmacokinetic improvements were feasible by in situ metabolic radioligand stabilization using PA.
Assuntos
Radioisótopos de Índio , Oligopeptídeos/farmacologia , Neoplasias da Próstata/patologia , Receptores da Bombesina/antagonistas & inibidores , Animais , Transporte Biológico , Estabilidade de Medicamentos , Compostos Heterocíclicos com 1 Anel/química , Humanos , Radioisótopos de Índio/uso terapêutico , Masculino , Camundongos , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Oligopeptídeos/uso terapêutico , Células PC-3 , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/radioterapia , Distribuição TecidualRESUMO
Neuromedin B is expressed in nociceptive and itch-sensitive dorsal root ganglia neurons, but its peripheral pruritogenic potential is not well described. The potential of neuromedin B as a pruritogen and pro-inflammatory peptide in the skin was tested in vivo in an acute model in mice and monkeys as well as an allergic dermatitis model in mice. To identify the underlying mechanisms in vitro real time PCR analysis for neuromedin B and its receptor expression in murine mast cells and dorsal root ganglia as well as functional calcium imaging in the ganglia was applied. Neuromedin B induces itch when injected intradermally, and the peripheral signal is likely transmitted through the activation of dorsal root ganglia. Thus, neuromedin B could be an interesting new therapeutic target for peripheral processing of itch at the level of sensory neurons.
Assuntos
Degranulação Celular , Mastócitos/fisiologia , Neurocinina B/análogos & derivados , Prurido/induzido quimicamente , Células Receptoras Sensoriais/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Dermatite Alérgica de Contato/etiologia , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Expressão Gênica , Indóis/farmacologia , Injeções Intradérmicas , Macaca mulatta , Masculino , Mastócitos/metabolismo , Camundongos , Neurocinina B/administração & dosagem , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismo , Análise de Célula Única , Tolueno 2,4-Di-IsocianatoRESUMO
The Gastrin-Releasing Peptide Receptor (GRPR) is over-expressed in estrogen receptor (ER) positive breast tumors and related metastatic lymph nodes offering the opportunity of imaging and therapy of luminal tumors. 68Ga-RM2 binding and 18F-FDG binding in tumoral zones were measured and compared using tissue micro-imaging with a beta imager on 14 breast cancer samples (10 primaries and 4 associated metastatic lymph nodes). Results were then assessed against ER expression, progesterone receptor (PR) expression, HER2 over-expression or not and Ki-67 expression. GRPR immunohistochemistry (IHC) was also performed on all samples. We also retrospectively compared 68Ga-RM2 and 18F-FDG bindings to 18F-FDG SUVmax on the pre-therapeutic PET/CT examination, if available. 68Ga-RM2 binding was significantly higher in tumors expressing GRPR on IHC than in GRPR-negative tumors (P = 0.022). In ER+ tumors, binding of 68Ga-RM2 was significantly higher than 18F-FDG (P = 0.015). In tumors with low Ki-67, 68Ga-RM2 binding was also significantly increased compared to 18F-FDG (P = 0.029). Overall, the binding of 68Ga-RM2 and 18F-FDG displayed an opposite pattern in tumor samples and 68Ga-RM2 binding was significantly higher in tumors that had low 18F-FDG binding (P = 0.021). This inverse correlation was also documented in the few patients in whom a 18F-FDG PET/CT examination before surgery was available. Findings from this in vitro study suggest that GRPR targeting can be an alternative to 18F-FDG imaging in ER+ breast tumors. Moreover, because GRPR antagonists can also be labeled with lutetium-177 this opens new avenues for targeted radionuclide therapy in the subset of patients with progressive metastatic disease following conventional treatments.