Synthesis and characterization of ¹⁸F‐labeled active site inhibited factor VII (ASIS)

Abstract

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress.

Quantitative PET Imaging of Tissue Factor Expression Using 18F-Labeled Active Site–Inhibited Factor VII

“…At 1 h, the images show high retention of 18 F-FVIIai in the vasculature and highly perfused organs such as liver and kidneys, with comparatively low retention in the tumor regions. 18 F-FVIIai is cleared predominantly through the kidneys, and an increased tumor-to-tissue contrast was evident at 2 h. At 4 h most of the tracer was cleared whereas the uptake in the tumors remained high, giving rise to a high tumor-to-tissue contrast. In addition, 18 F-FVIIai accumulated in joint regions throughout the mice.…”

“…Dried 18 F-SFB was produced using a 3-step 1-pot procedure as previously described (17,18). FVIIai (1.0 mg in 400 mL of N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) buffer, pH 7.5) was added to the dried 18 F-SFB and stirred for 1 h at room temperature to yield 18 F-FVIIai.…”

“…Static images were acquired 1, 2, and 4 h (acquisition time, 480, 600 and 1,200 s, respectively) after intravenous injection of 18 F-FVIIai (7.3-9.3 MBq) in 160 mL. Animals received about 37 mg of 18 F-FVIIai and were anesthetized by sevoflurane during injections and imaging sessions. Data were acquired in list-mode, and the images were reconstructed using the 3-dimensional maximum a posteriori algorithm with a voxel size of 0.3 · 0.3 · 0.8 mm and a spatial resolution at the center of the field of view of 1.2 mm.…”

“…Here we report the development and in vivo testing of a new PET tracer for specific imaging of TF using FVIIai labeled with 18 F. FVIIai (molecular weight, 50 kDa), which binds to TF with an affinity approximately 5-fold higher than FVIIa (16), was radiolabeled with 18 F by N-succinimidyl 4-18 F-fluorobenzoate ( 18 F-SFB; molecular weight, 237 Da). The in vivo properties of 18 F-FVIIai for PET imaging were evaluated in a mouse model of human pancreatic cancer using small-animal PET/CT.…”

“…The in vivo properties of 18 F-FVIIai for PET imaging were evaluated in a mouse model of human pancreatic cancer using small-animal PET/CT. The uptake of 18 F-FVIIai measured by PET was correlated with TF expression measured ex vivo to confirm specific imaging of tumor TF expression.…”