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Introduction The gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood–brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [ 18 F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these 18 F-labeled compounds in brain of living rats. Methods We synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for 18 F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo . Results A palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The 18 F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5–15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the 18 F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo . Conclusions The novel small molecule 18 F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro , and may prove useful for quantitative autoradiographic studies in vitro . The compounds were permeable to the blood–brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain. [ABSTRACT FROM AUTHOR]

Introduction: Incorporation of fluorine-18 (18F) into radiotracers by capturing ionic [18F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [18F]fluoride (Al[18F]2+) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[18F]2+-radiolabeling of an αvβ6 integrin-targeted peptide (NOTA-PEG28-A20FMDV2) and its in vitro and in vivo evaluation. Methods: Aluminum [18F]fluoride was prepared at r.t. from [18F]fluoride (40 MBq–11 GBq) and introduced into NOTA-PEG28-A20FMDV2 (1) in sodium acetate (pH 4.1; 100°C, 15 min). The radiotracer Al[18F] NOTA-PEG28-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in αvβ6(+) and αvβ6(−) cells) and in vivo (paired αvβ6(+) and αvβ6(−) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites). Results: The radiotracer 2 was prepared in 90±6 min (incl. formulation; n= 3) in 19.3±5.4% decay corrected radiochemical yield (radiochemical purity: >99%; specific activity: 158±36 GBq/μmol) and was stable in PBS and serum (2 h). During in vitro cell binding studies, 2 showed high, αvβ6-targeted binding (αvβ6(+): 42.4±1.2% of total radioactivity, ratio (+)/(−) = 8.4/1) and internalization (αvβ6(+): 28.3±0.5% of total radioactivity, (+)/(−) = 11.7/1). In vivo, 2 maintained αvβ6-targeted binding (biodistribution; 1 h: αvβ6(+): 1.74±0.38% ID/g, (+)/(−) = 2.72/1; 4 h: αvβ6(+): 1.21±0.56% ID/g, (+)/(−) = 4.0/1; 11% intact 2 in tumor at 1 h), with highest uptake around the tumor edge (autoradiography). Most of the radioactivity cleared rapidly in the urine within one hour, but a significant fraction remained trapped in the kidneys (4 h: 229±44% ID/g). Conclusion: The Al[18F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good αvβ6-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [18F]-prosthetic groups (e.g., [18F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Click chemistry, particularly the Huisgen 1,3-dipolar cycloaddition of an alkyne with an azide, has quickly become popular for site-specific radiolabeling. Recently, strain-promoted click chemistries have been developed, eliminating the need for potentially toxic copper catalysts. This study presents radiolabeling of an αvβ6 integrin targeting peptide (A20FMDV2) via strain-promoted click using a fluorine-18 prosthetic group, and in vitro and in vivo evaluation. Methods: The radiotracer [18F]FBA-C6-ADIBON3-PEG7-A20FMDV2 (1) was prepared from [18F]FBA-C6-ADIBO (2) and N3-PEG7-A20FMDV2 (ethanol; 10min; 35–45°C). HPLC-purified and formulated radiotracer 1 was evaluated in vitro by cell binding (DX3puroβ6, αvβ6-positive; and DX3puro, αvβ6-negative control) and serum stability, and in vivo using PET/CT imaging and biodistribution studies in mice. Results: The radiotracer 1 was readily prepared and purified (from 2 : 40±4min including HPLC, 11.9±3.2% decay corrected isolated radiochemical yield, >99% radiochemical purity, n =4) and displayed good stability (1h: >99%, saline; 94.6%, serum). Strong αvβ6-targeted binding was observed in vitro (DX3puroβ6 cells, 15min: 43.2% binding, >6:1 for DX3puroβ6:DX3puro). In the mouse model DX3puroβ6-tumor binding was low (1h: 0.47±0.28% ID/g, 4h: 0.14±0.09% ID/g) and clearing from the bloodstream was via the renal and hepatobiliary routes (urine: 167±84% ID/g at 1h, 10.3±4.8% ID/g at 4h; gall bladder: 95±33% ID/g at 1h, 63±11% ID/g at 4h). Conclusion: Copper-free, strain-promoted click chemistry is an attractive, straightforward approach to radiolabeling. Although the [18F]FBA-C6-ADBIO-based prosthetic group did not interfere with αvβ6-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature. [Copyright &y& Elsevier]