Evaluation of Organo [18F]Fluorosilicon Tetrazine as a Prosthetic Group for the Synthesis of PET Radiotracers

Abstract

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]6), providing [18F]fluoroalbumin ([18F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/µmol, confirmed the applicability of [18F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [18F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [18F]6 was used for the radiolabeling of albumin-TCO ([18F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging.

Metabolism of a Bioorthogonal PET Tracer Candidate [19F/18F]SiFA-Tetrazine in Mouse Liver Microsomes: Biotransformation Pathways and Defluorination Investigated by UHPLC-HRMS

“…We have previously reported the biological evaluation of [ 18 F]SiFA–Tz in healthy CD-1 mice revealing defluorination of the tracer candidate in vivo . 22 The fluorine-18 radiolabel detached from [ 18 F]SiFA–Tz accumulated significantly in the bone within 1 h after administration ( Figure S1 ). Therefore, in this study we decided to investigate this unexpected finding to further elucidate the biotransformation pathways of this proposed PET tracer to guide structural optimization.…”

“…Acetonitrile (ACN) and formic acid (FA) (both LC-MS grade) were purchased from Merck (LiChropur, Merck KGaA, Darmstadt, Germany). SiFA–Tz was synthesized in-house (purity >97%) 22 and dissolved in ACN (DNA synthesis quality) for HRMS studies (= analytical standard).…”

“…Livers of CD-1 mice (female, weight 25–33 g, 11 to 12 weeks, Charles River, n = 2) from a previously reported study were harvested after intravenous administration of [ 18 F]SiFA–Tz into the lateral tail vein. 22 The harvested organs were subjected to gamma counting ( t = 1 min per organ), followed promptly by storage in freezer (−20 °C) until further processing. For the metabolite analysis, the harvested organs were taken to ambient temperature from a freezer (−20 °C), homogenized with a hand-held VDI 12 homogenizer (VWR, West Chester, PA, USA) in cold ACN–water solution (80:20), and centrifuged (10 000 g , 10 min), and the supernatants were filtered with 0.22 μm sterile syringe filter (Millex-GV, Merck Millipore Ltd., Tullagreen Carrigtwohill, Co. Cork Ireland).…”

“…The animal experiment referenced in this study was conducted under a project license approved by the National Board of Animal Experimentation in Finland (license number ESAVI/12132/04.10.07/2017). 22 …”

“… 20 We recently reported biological evaluation of an oxime-linked SiFA-tetrazine (SiFA–Tz), [ 18 F]-( E / Z )-SiFA–Tz ( Figure 1 ) in healthy mice, which revealed rapid accumulation of radioactivity in both occipital bone and in tibia. 22 …”