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OBJECTIVES: Somatostatin receptor positron emission tomography/computed tomography (SSTR-PET/CT) using [68Ga]-labeled tracers is a widely used imaging modality for neuroendocrine tumors (NET). Recently, [18F]SiTATE, a SiFAlin tagged [Tyr3]-octreotate (TATE) PET tracer, has shown great potential due to favorable clinical characteristics. We aimed to evaluate the reproducibility of Somatostatin Receptor-Reporting and Data System 1.0 (SSTR-RADS 1.0) for structured interpretation and treatment planning of NET using [18F]SiTATE. METHODS: Four readers assessed [18F]SiTATE-PET/CT of 95 patients according to the SSTR-RADS 1.0 criteria at two different time points. Each reader evaluated up to five target lesions per scan. The overall scan score and the decision on peptide receptor radionuclide therapy (PRRT) were considered. Inter- and intra-reader agreement was determined using the intraclass correlation coefficient (ICC). RESULTS: The ICC analysis on the inter-reader agreement using SSTR-RADS 1.0 for identical target lesions (ICC ≥ 85%), overall scan score (ICC ≥ 90%), and the decision to recommend PRRT (ICC ≥ 85%) showed excellent agreement. However, significant differences were observed in recommending PRRT among experienced readers (ER) (p = 0.020) and inexperienced readers (IR) (p = 0.004). Compartment-based analysis demonstrated good to excellent inter-reader agreement for most organs (ICC ≥ 74%), except for lymph nodes (ICC ≥ 53%). CONCLUSION: SSTR-RADS 1.0 represents a highly reproducible and consistent framework system for stratifying SSTR-targeted PET/CT scans, even using the novel SSTR-ligand [18F]SiTATE. Some inter-reader variability was observed regarding the evaluation of uptake intensity prior to PRRT as well as compartment scoring of lymph nodes, indicating that those categories require special attention during further clinical validation and might be refined in a future SSTR-RADS version 1.1. CLINICAL RELEVANCE STATEMENT: SSTR-RADS 1.0 is a consistent framework for categorizing somatostatin receptor-targeted PET/CT scans when using [18F]SiTATE. The framework serves as a valuable tool for facilitating and improving the management of patients with NET. KEY POINTS: SSTR-RADS 1.0 is a valuable tool for managing patients with NET. SSTR-RADS 1.0 categorizes patients with showing strong agreement across diverse reader expertise. As an alternative to [68Ga]-labeled PET/CT in neuroendocrine tumor imaging, SSTR-RADS 1.0 reliably classifies [18F]SiTATE-PET/CT.
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INTRODUCTION: PET/CT using 68Ga-labeled somatostatin analogs (SSA) targeting somatostatin receptors (SSR) on the cell surface of well-differentiated neuroendocrine tumors (NET) represents the clinical reference standard for imaging. However, economic and logistic challenges of the 68Ge/68Ga generator-based approach have disadvantages over 18F-labeled compounds. Here, we present the first in-human data of 18F-SiFAlin-TATE, a novel 18F-labeled, SSR-targeting peptide. The aim was to compare the intra-individual biodistribution, tumor uptake, and image quality of 18F-SiFAlin-TATE to the clinical reference standard 68Ga-DOTA-TOC. METHODS: Thirteen patients with NET staged with both 68Ga-DOTA-TOC and 18F-SiFAlin-TATE PET/CT have been included in this retrospective analysis. We compared the biodistribution in normal organs and tumor uptake of NET lesions by SUVmean and SUVmax measurement for tracers. Additionally mean and max tumor-to-liver (TLR) and tumor-to-spleen ratios (TSR) have been calculated by division of SUVmean and SUVmax of tumor lesions by the SUVmean of the liver and spleen, respectively. Additionally, image quality was visually rated by 5 blinded readers and an intra-class correlation (ICC) analysis on inter-observer agreement has been performed. RESULTS: Compared with 68Ga-DOTA-TOC, the biodistribution of 18F-SiFAlin-TATE showed somewhat higher, however, statistically not significant higher uptake in the liver, spleen, and adrenal glands. Significantly higher uptake was observed in the kidneys. Tumor uptake was higher in most tumor lesions with significantly higher uptake in common metastatic sites of NET including the liver (SUVmax 18.8 ± 8.4 vs. 12.8 ± 5.6; p < 0.001), lymph nodes (SUVmax 23.8 ± 20.7 vs. 17.4 ± 16.1; p < 0.001) and bone (SUVmax 16.0 ± 10.1 vs. 10.3 ± 5.7; p < 0.01) for 18F-SiFAlin-TATE. The high tumor uptake resulted in favorable TLR and TSR, comparable with that of 68Ga-DOTA-TOC. The ICC analysis on the inter-observer agreement on image quality was substantial and almost perfect. Image quality was rated as excellent in most cases in both 68Ga-DOTA-TOC and 18F-SiFAlin-TATE PET. CONCLUSION: The favorable characteristics of 18F-SiFAlin-TATE with a high image quality, the kit-like labeling procedure, and the promising clinical performance enable improved logistics and diagnostic possibilities for PET imaging of NET. Our first clinical results warrant further systematic studies investigating the clinical use of 18F-SiFAlin-TATE in NET patients.
Assuntos
Tumores Neuroendócrinos , Compostos Organometálicos , Humanos , Tumores Neuroendócrinos/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Receptores de Somatostatina/metabolismo , Estudos Retrospectivos , Somatostatina , Distribuição TecidualRESUMO
As the gastrin releasing peptide receptor (GRPR) is overexpressed on several tumor types, it represents a promising target for the specific in vivo imaging of these tumors using positron emission tomography (PET). We were able to show that PESIN-based peptide multimers can result in substantially higher GRPR avidities, highly advantageous in vivo pharmacokinetics and tumor imaging properties compared to the respective monomers. However, the minimal distance between the peptidic binders, resulting in the lowest possible system entropy while enabling a concomitant GRPR binding and thus optimized receptor avidities, has not been determined so far. Thus, we aimed here to identify the minimal distance between two GRPR-binding peptides in order to provide the basis for the development of highly avid GRPR-specific PET imaging agents. We therefore synthesized dimers of the GRPR-binding bombesin analogue BBN(7-14) on a dendritic scaffold, exhibiting different distances between both peptide binders. The homodimers were further modified with the chelator NODAGA, radiolabeled with (68)Ga, and evaluated in vitro regarding their GRPR avidity. We found that the most potent of the newly developed radioligands exhibits GRPR avidity twice as high as the most potent reference compound known so far, and that a minimal distance of 62 bond lengths between both peptidic binders within the homodimer can result in concomitant peptide binding and optimal GRPR avidities. These findings answer the question as to what molecular design should be chosen when aiming at the development of highly avid homobivalent peptidic ligands addressing the GRPR.
Assuntos
Bombesina/análogos & derivados , Fragmentos de Peptídeos/química , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/química , Receptores da Bombesina/química , Bombesina/química , Bombesina/metabolismo , Dimerização , Radioisótopos de Gálio/metabolismo , Humanos , Marcação por Isótopo , Masculino , Fragmentos de Peptídeos/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/metabolismo , Receptores da Bombesina/metabolismo , Células Tumorais CultivadasRESUMO
The Silicon-Fluoride-Acceptor (SiFA)-(18)F-labeling strategy has been shown before to enable the straightforward and efficient (18)F-labeling of complex biologically active substances such as proteins and peptides. Especially in the case of peptides, the radiolabeling proceeds kit-like in short reaction times and without the need of complex product workup. SiFA-derivatized, (18)F-labeled Tyr(3)-octreotate (TATE) derivatives demonstrated, besides strong somatostatin receptor (SSTR) binding, favorable in vivo pharmacokinetics as well as excellent tumor visualization by PET imaging. In this study, we intended to determine the influence of the underlying molecular design and used molecular scaffolds of SiFAlin-TATE derivatives on SSTR binding as well as on the in vivo pharmacokinetics of the resulting (18)F-labeled peptides. For this purpose, new SiFAlin-(Asp)n-PEG1-TATE analogs (where n = 1-4) were synthesized, efficiently radiolabeled with (18)F in a kit-like manner and obtained in radiochemical yields of 70-80%, radiochemical purities of ≥97%, and nonoptimized specific activities of 20.1-45.2 GBq/µmol within 20-25 min starting from 0.7-1.5 GBq of (18)F. In the following, the radiotracer's lipophilicities and stabilities in human serum were determined. Furthermore, the SSTR-specific binding affinities were evaluated by a competitive displacement assay on SSTR-positive AR42J cells. The obtained in vitro results support the assumption that aspartic acids are able to considerably increase the radiotracer's hydrophilicity and that their number does not affect the SSTR binding potential of the TATE derivatives. The most promising tracer (18)F-SiFAlin-Asp3-PEG1-TATE [(18)F]6 (LogD = -1.23 ± 0.03, IC50 = 20.7 ± 2.5 nM) was further evaluated in vivo in AR42J tumor-bearing nude mice via PET/CT imaging against the clinical gold standard (68)Ga-DOTATATE as well as the previously developed SiFAlin-TATE derivative [(18)F]3. The results of these evaluations showed that [(18)F]6-although showing very similar chemical and in vitro properties to [(18)F]3-exhibits not only a slowed renal clearance compared to [(18)F]3, but also a higher absolute tumor uptake compared to (68)Ga-DOTATATE, and furthermore enables excellent tumor visualization with high image resolution. These results emphasize the importance of systematic study of the influence of molecular design and applied structure elements of peptidic radiotracers, as these may considerably influence in vivo pharmacokinetics while not affecting other parameters such as radiochemistry, lipophilicity, serum stability, or receptor binding potential.
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Fluoretos/metabolismo , Radioisótopos de Flúor/metabolismo , Neoplasias/diagnóstico por imagem , Peptídeos Cíclicos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Receptores de Somatostatina/metabolismo , Silício/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Fluoretos/química , Fluoretos/farmacocinética , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacocinética , Humanos , Camundongos Nus , Modelos Moleculares , Neoplasias/diagnóstico , Neoplasias/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Silício/química , Silício/farmacocinéticaAssuntos
Neoplasias Hepáticas/diagnóstico por imagem , Tumores Neuroendócrinos/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Receptores de Peptídeos/química , Somatostatina/química , Nanomedicina Teranóstica/métodos , Idoso , Braquiterapia , Proliferação de Células , Radioisótopos de Flúor , Humanos , Radioisótopos de Índio , Neoplasias Hepáticas/secundário , Masculino , Metástase Neoplásica , Peptídeos/químicaRESUMO
Secondary lymphedema (SL)is a frequent and devastating complication of modern oncological therapy and filarial infections. A lack of a reliable preclinical model to investigate the underlying mechanism of clinical stage progression has limited the development of new therapeutic strategies. Current first line treatment has shown to be merely symptomatic and relies on lifetime use of compression garments and decongestive physiotherapy. In this study, we present the development of a secondary lymphedema model in 35 rats using pre- and intraoperative fluorescence-guided mapping of the lymphatics and microsurgical induction. In contrast to the few models reported so far, we decided to avoid the use of radiation for lymphedema induction. It turned out, that the model is nearly free of complications and capable of generating a statistically significant limb volume increase by water displacement measurements, sustained for at least 48 days. A translational, accurate lymphatic dysfunction was visualized by a novel VIS-NIR X-ray ICG-Clearance-Capacity imaging technology. For the first-time SL stage progression was validated by characteristic histological alterations, such as subdermal mast cell infiltration, adipose tissue deposition, and fibrosis by increased skin collagen content. Immunofluorescence confocal microscopy analysis suggested that stage progression is related to the presence of a characteristic α SMA+/HSP-47+/vimentin+ fibroblast subpopulation phenotype. These findings demonstrate that the in-vivo model is a reliable and clinically relevant SL model for the development of further secondary lymphedema therapeutic strategies and the analysis of the veiled molecular mechanisms of lymphatic dysfunction.
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Corantes Fluorescentes/química , Linfedema/patologia , Microcirurgia/efeitos adversos , Actinas/metabolismo , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Verde de Indocianina/química , Linfedema/etiologia , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos Lew , Pele/patologia , Vimentina/metabolismoRESUMO
DOTA (1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid), which forms extremely stable complexes with a large number of metal ions, is one of the most important and most commonly used chelators for in vivo applications such as cancer diagnosis and therapy. However, many of the published synthesis protocols for DOTA derivatives are complicated and give the products in low yields. Here we report improved synthesis routes for tris-tBu-DOTA, tris-benzyl-DOTA, and thiol-DOTA, and also describe the synthesis of the novel compound tris-4-nitro-benzyl-DOTA. In addition, we determined the applicability of the DOTA derivatives tris-tBu-DOTA, thiol-DOTA, tris-benzyl-DOTA, tris-4-nitrobenzyl-DOTA, tris-allyl-DOTA, DOTA-PFP-ester, and DOTA-PNP-ester for multimerization reactions using amino functionalized PAMAM dendrimers of different sizes. Thiol-DOTA was found to be the best compound for efficient reactions with dendritic scaffolds generating highly homogeneous DOTA-multimers. This DOTA derivative could be quantitatively conjugated to a 128-mer dendrimer.
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Compostos Heterocíclicos com 1 Anel/síntese química , Butanos/química , Cromatografia Líquida de Alta Pressão , Compostos Heterocíclicos com 1 Anel/química , Estrutura Molecular , Nitrobenzenos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Compostos de Sulfidrila/químicaRESUMO
18F-labeled non-sulfonylurea hypoglycemic agent (S)-2-(2-[(18)F]fluoroethoxy)-4-((3-methyl-1-(2-piperidin-1-yl-phenyl)-butylcarbamoyl)-methyl)-benzoic acid ([(18)F]repaglinide), a derivative of the sulfonylurea-receptor (SUR) ligand repaglinide, was synthesized as a potential tracer for the non-invasive investigation of the sulfonylurea 1 receptor status of pancreatic beta-cells by positron emission tomography (PET) in the context of type 1 and type 2 diabetes. [(18)F]Repaglinide could be obtained in an overall radiochemical yield (RCY) of 20% after 135 min with a radiochemical purity higher than 98% applying the secondary labeling precursor 2-[(18)F]fluoroethyltosylate. Specific activity was in the range of 50-60 GBq/micromol. Labeling was conducted by exchanging the ethoxy-moiety into a 2-[(18)F]fluoroethoxy group. To characterize the properties of fluorinated repaglinide, the affinity of the analogous non-radioactive (19)F-compound for binding to the human SUR1 isoform was assessed. [(19)F]Repaglinide induced a complete monophasic inhibition curve with a Hill coefficient close to 1 (1.03) yielding a dissociation constant (K(D)) of 134 nM. Biological activity was proven via insulin secretion experiments on isolated rat islets and was comparable to that of repaglinide. Finally, biodistribution of [(18)F]repaglinide was investigated in rats by measuring the concentration of the compound in different organs after i.v. injection. Pancreatic tissue displayed a stable accumulation of approximately 0.12% of the injected dose from 10 min to 30 min p.i. 50% of the radioactive tracer could be displaced by additional injection of unlabeled repaglinide, indicating that [(18)F]repaglinide might be suitable for in vivo investigation with PET.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carbamatos/farmacocinética , Ilhotas Pancreáticas/diagnóstico por imagem , Ilhotas Pancreáticas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Piperidinas/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Animais , Carbamatos/síntese química , Estudos de Viabilidade , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacocinética , Ilhotas Pancreáticas/patologia , Marcação por Isótopo/métodos , Taxa de Depuração Metabólica , Especificidade de Órgãos , Piperidinas/síntese química , Canais de Potássio Corretores do Fluxo de Internalização , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores de Droga , Receptores de Sulfonilureias , Distribuição TecidualRESUMO
For many years, the main application of [(18)F]F-DOPA has been the PET imaging of neuropsychiatric diseases, movement disorders, and brain malignancies. Recent findings however point to very favorable results of this tracer for the imaging of other malignant diseases such as neuroendocrine tumors, pheochromocytoma, and pancreatic adenocarcinoma expanding its application spectrum. With the application of this tracer in neuroendocrine tumor imaging, improved radiosyntheses have been developed. Among these, the no-carrier-added nucleophilic introduction of fluorine-18, especially, has gained increasing attention as it gives [(18)F]F-DOPA in higher specific activities and shorter reaction times by less intricate synthesis protocols. The nucleophilic syntheses which were developed recently are able to provide [(18)F]F-DOPA by automated syntheses in very high specific activities, radiochemical yields, and enantiomeric purities. This review summarizes the developments in the field of [(18)F]F-DOPA syntheses using electrophilic synthesis pathways as well as recent developments of nucleophilic syntheses of [(18)F]F-DOPA and compares the different synthesis strategies regarding the accessibility and applicability of the products for human in vivo PET tumor imaging.
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Di-Hidroxifenilalanina/análogos & derivados , Neoplasias Encefálicas/diagnóstico por imagem , Di-Hidroxifenilalanina/síntese química , Di-Hidroxifenilalanina/química , Di-Hidroxifenilalanina/uso terapêutico , Humanos , Transtornos Mentais/diagnóstico por imagem , Transtornos dos Movimentos/diagnóstico por imagem , Traçadores Radioativos , CintilografiaRESUMO
BACKGROUND: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the digestive tract. The GIST differ substantially from gastrointestinal carcinomas regarding tumor biology, treatment strategies and indications for surgery. Every surgeon involved in the treatment of GIST should be acquainted with these aspects. OBJECTIVES: The aims of this article are to discuss the value of positron emission tomography (PET) in the surgical treatment of patients with GIST and to provide an outlook on the development of molecular tracers specifically tailored for GIST. RESULTS: PET is an invaluable decision aid in the multimodal therapy of GIST and particularly for deciding on surgical indications. Specific scenarios in which PET is used are primary staging monitoring during neoadjuvant therapy and staging and response assessment in the metastatic setting. The routinely used tracer is 18F-fluorodeoxyglucose (18F-FDG) and uptake reliably correlates with the metabolism of GIST lesions. Compared to computed tomography and magnetic resonance imaging (CT/MRI), 18F-FDG-PET often allows a more timely and accurate response assessment. GIST-specific molecular tracers, which could provide a direct prognosis regarding response and development of resistance to treatment, are currently in preclinical development. However, pharmacokinetic and immunological issues still need to be resolved. A distant aim is the development of "theranostics", i.e. substances which serve both diagnostic and therapeutic purposes. DISCUSSION: PET has an established value in the multimodal treatment of GIST and is particularly useful for deciding on surgical indications.
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Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/cirurgia , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/cirurgia , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Terapia Combinada , Metabolismo Energético/fisiologia , Fluordesoxiglucose F18 , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/patologia , Humanos , Terapia Neoadjuvante , Prognóstico , Sensibilidade e Especificidade , Carga TumoralRESUMO
Recently, silicon fluoride building blocks (SiFA) have emerged as valuable and promising tools to overcome challenges in the labeling of peptides and proteins for positron emission tomography (PET). Herein, we report a fully automated synthesis of N-succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) by a commercially available Scintomics Hot Box 3 synthesis module, to be used as a prosthetic group for peptide and protein labeling. The drying of K2.2.2./K (18)F complex was performed according to the Munich method modified by our group (avoiding azeotropic drying) using oxalic acid to neutralize the base from the (18)F(-) containing QMA eluent. This K2.2.2./K (18)F complex was then used for SiFA (18)F-(19)F isotopic exchange followed by a fast purification by a solid-phase-extraction (SPE) to afford [(18)F]SiFB with an average preparative radiochemical yield (RCY) of 24±1% (non-decay corrected (NDC)) within a synthesis time of 30 min. The [(18)F]SiFB produced by automated synthesis was then used for the (18)F-labeling of rat serum albumin (RSA) as a proof of applicability.
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Radioisótopos de Flúor/química , Compostos de Organossilício/síntese química , Compostos Radiofarmacêuticos/síntese química , Succinimidas/síntese química , Animais , Fluoretos/química , Marcação por Isótopo/instrumentação , Marcação por Isótopo/métodos , Radioquímica/instrumentação , Radioquímica/métodos , Ratos , Albumina Sérica/química , Compostos de Silício/químicaRESUMO
Radiometal nuclides can serve as diagnostic markers in molecular imaging or can be used in therapeutic settings for a rising number of human afflictions. For the targeted delivery of these medically interesting ions, appropriate chelating agents forming stable complexes are of fundamental importance. For different metal ions exhibiting different physical and chemical properties, resulting in different coordination chemistries and therefore differing requirements on the chelator used, a broad variety of chelating agents has been developed over the years. Not only the chemical properties of the metal ion determine the choice of the chelator, but also the desired in vivo behavior of the resulting molecular imaging or therapeutic compound influences the choice of the complexation agent. Furthermore, the conjugation chemistry for the introduction of the chelator into the biologically active compound and the complexation reaction of the metal ion can affect the choice of the appropriate chelator. This review outlines chelating agents used in medicinal chemistry, their radiometal complexation behavior and their potential influence on the properties of the resulting drugs.
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Quelantes , Compostos Radiofarmacêuticos , Quelantes/química , Quelantes/uso terapêutico , Humanos , Imagem Molecular , Estrutura Molecular , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/uso terapêuticoRESUMO
Today the term "click chemistry" is often used equivalent with the copper-catalyzed 1,3-dipolar Huisgen cycloaddition. Originally, the concept was introduced in 2001 to describe reactions fulfilling a set of criteria that are most useful for chemical syntheses in drug research. In radiopharmaceutical chemistry where short lived radioisotopes are introduced into various different substance classes for in vivo imaging of biochemical processes, the expanding field of radioactive bioconjugation has become predominant. Labeled biomolecules such as peptides, proteins and oligonucleotides generated via bioconjugation of chelators for radiometal introduction as well as novel valuable secondary precursors for (18)F labeling have enriched the growing field of molecular imaging substantially. When introducing radioactive nuclides with a very short half-life into biomolecules, some of the typical criteria defined by click-chemistry are more crucial than others. Time is always the most important issue, whereas avoiding the formation of by-products that have to be removed without chromatography is of minor importance. The short-lived radionuclide (11)C for example has a physical half-life of only 20 min so that the labeling procedure cannot exceed 40-60 minutes (2-3 half-lifes). In this contribution, we outline reactions and molecules which meet the requirements of click chemistry reactions and are suitable for radiosyntheses of short lived SPECT ((99m)Tc: t(1/2) = 6 h, (111)In: t(1/2) = 2.81 d) and PET ((11)C: t(1/2) = 20.3 min to (64)Cu: t(1/2) = 12.7 h) radiotracers for in vivo imaging of biological processes and review the contributions in the field of radiochemical "click-reactions" - 1,3-dipolar Huisgen cycloadditions and beyond.
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Compostos Radiofarmacêuticos/química , Aminas/síntese química , Aminas/química , Radioisótopos de Carbono , Radioisótopos de Flúor , Humanos , Isocianatos/síntese química , Isocianatos/química , Marcação por Isótopo , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/química , Tomografia Computadorizada de EmissãoRESUMO
Fluorescent probes are of increasing interest in medicinal and biological applications for the elucidation of the structures and functions of healthy as well as tumour cells. The quality of these investigations is determined by the intensity of the fluorescence signal. High dye/carrier ratios give strong signals. However, these are achieved by the occupation of a high number of derivatisation sites and therefore are accompanied by strong structural alterations of the carrier. Hence, polyvalent substances containing a high number of fluorescent dyes would be favourable because they would allow the introduction of many dyes at one position of the compound to be labelled.A large number of different dyes have been investigated to determine the efficiency of coupling to a dendrimer scaffold and the fluorescence properties of the oligomeric dyes, but compounds that fulfil the requirements of both strong fluorescence signals and reactivities are rare. Herein we describe the synthesis and characterisation of dye oligomers containing dansyl-, 7-nitro-2,1,3-benzoxadiazol-4-yl- (NBD), coumarin-343, 5(6)-carboxyfluorescein and sulforhodamine B2 moieties based on polyamidoamine (PAMAM) dendrimers. The PAMAM dendrimers were synthesised by an improved protocol that yielded highly homogeneous scaffolds with up to 128 conjugation sites. When comparing the fluorescent properties of the dye oligomers it was found that only the dansylated dendrimers met the requirements of enhanced fluorescence signals. The dendrimer containing 16 fluorescent dyes was conjugated to the anti-epidermal-growth-factor receptor (EGFR) antibody hMAb425 as a model compound to show the applicability of the dye multimer compounds. This conjugate revealed a preserved immunoreactivity of 54%.We demonstrate the applicability of the dye oligomers to the efficient and applicable labelling of proteins and other large molecules that enables high dye concentrations and therefore high contrasts in fluorescence applications.
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Marcadores de Afinidade/química , Anticorpos Monoclonais/química , Corantes Fluorescentes/química , Poliaminas/química , Marcadores de Afinidade/síntese química , Sítios de Ligação , Linhagem Celular Tumoral , Dendrímeros , Receptores ErbB/imunologia , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Estrutura Molecular , Poliaminas/síntese química , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Estereoisomerismo , Fatores de TempoRESUMO
AIM: TGF-beta1 is an important local and systemic regulatory molecule during fracture healing. Various authors have shown differences in the systemic levels of TGF-beta1 over the time taken for bone healing in distraction osteogenesis and osteotomies. Previous studies have shown characteristic differences in the physiological levels of growth factors between normal fracture healing and delayed fracture union. The aim of the present study was to evaluate possible differences in sera levels of patients with normal and delayed union fracture healing. METHODS: Patients with long bone shaft fractures were recruited prospectively. Peripheral blood samples were collected over a period of 1 year using a standardized time schedule. At the end of the individual's investigation period, TGF-beta1 levels were determined. To achieve a homogeneous collective of patients, only those with a maximum of two fractures were included in the study. After matching for four criteria, we compared patients with normal fracture healing to patients with delayed unions. The fact of delayed union was accepted in case of failed consolidation 4 months after trauma. RESULTS: During a prospective study period of 1 year, 15 patients with normal fracture healing could be compared to 15 patients suffering from delayed union. By determining the absolute sera levels we found a typical increase of TGF-beta1 up to 2 weeks after fracture in both groups, with a subsequent decrease up to the sixth week after fracture. However, a decline in serum concentration occurred earlier in patients with delayed union, causing significantly lower TGF-beta1 levels in the non-union group 4 weeks after trauma (P=0.00006). CONCLUSION: Even with a relatively small number of patients, we could show a significant difference in serum concentrations of TGF-beta1 between the investigated groups. If these results can be verified within a larger collective, TGF-beta1 could be used as a predictive cytokine for delayed fracture healing.