Validation of the standardization framework SSTR-RADS 1.0 for neuroendocrine tumors using the novel SSTR­targeting peptide [18F]SiTATE.

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.

Biodistribution and first clinical results of 18F-SiFAlin-TATE PET: a novel 18F-labeled somatostatin analog for imaging of neuroendocrine tumors.

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.

Next Generation of SiFAlin-Based TATE Derivatives for PET Imaging of SSTR-Positive Tumors: Influence of Molecular Design on In Vitro SSTR Binding and In Vivo Pharmacokinetics.

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.

Next Step toward Optimization of GRP Receptor Avidities: Determination of the Minimal Distance between BBN(7-14) Units in Peptide Homodimers.

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.

6-[18F]fluoro-L-DOPA: a well-established neurotracer with expanding application spectrum and strongly improved radiosyntheses.

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.

Automated radiosynthesis of N-succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) for peptides and proteins radiolabeling for positron emission tomography.

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.

Chelating agents and their use in radiopharmaceutical sciences.

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.

Click-chemistry reactions in radiopharmaceutical chemistry: fast & easy introduction of radiolabels into biomolecules for in vivo imaging.

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.

PAMAM structure-based multifunctional fluorescent conjugates for improved fluorescent labelling of biomacromolecules.

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.

Antibody-dendrimer conjugates: the number, not the size of the dendrimers, determines the immunoreactivity.

Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect.

Improved syntheses and applicability of different DOTA building blocks for multiply derivatized scaffolds.

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.