Synthesis and in Vitro Evaluation of¹⁸F- and¹⁹F-Labeled Insulin:  A New Radiotracer for PET-based Molecular Imaging Studies

Abstract

A new and regioselective strategy was developed for the preparation of fluorine-18-labeled insulin as a novel positron emission tomography (PET) tracer. [18F]-4-Fluorobenzoic acid (4-18FBA), which was produced in 83 +/- 8% yield (n = 10), through the use of succinimidyl [18F]-4-fluorobenzoate (4-(18)FSB), was conjugated through a short spacer (6-aminohexanoic acid, AHx) to the PheB1 residue of a protected form of insulin. 18FB-AHx-insulin (8b) was repeatedly prepared in practical quantities (10-20 mCi, 370-740 MBq) in good radiochemical yield (9 +/- 5%, n = 9) and in a specific activity of 7.8 mCi/micromol. The final product was characterized by comparing the radioHPLC and radioTLC of 8b with that of the 19F-analogue (19FB-AHx-insulin, 8a) and by analyzing a carrier-added synthesis by mass spectrometry. Dithiothreitol and endoproteinase Glu-C digestion experiments on 8a confirmed that the prosthetic group was in fact conjugated to the PheB1 residue. An insulin receptor (IR) phosphorylation assay using CHO-hIR cells overexpressing recombinant human insulin receptors indicated no statistical difference in the extent of autophosphorylation stimulated by 8a as compared to that for human insulin (EC50 values of 0.82 nM and 1.0 nM, respectively). The stimulation of 2-deoxyglucose uptake in 3T3-L1 mouse adipocytes utilizing 8a versus unmodified human insulin gave similar EC50 values of 0.68 nM and 0.41 nM, respectively. The IC50 values for 8a versus native insulin for the displacement of 125I-insulin from HEK-293 cells were also the same within experimental error (2.6 nM for 8a versus 2.4 nM for unmodified human insulin). These results support the use of the 18F-insulin analogue as a PET tracer for imaging the distribution of insulin in vivo.

Facile synthesis of N‐succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) for protein labeling

“…The substitution reaction is followed by hydrolysis of the ester protecting group using aqueous NaOH, 10,11 aqueous HCl, 4 or trifluoroacetic acid. 3,12,13 The final synthetic step employs activation by N,N,N 0 ,N 0 -tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU). When ethyl and tert-butyl esters were used as precursors, two purification steps were necessary: purification of the intermediate […”

“…18 F]fluorination reaction had been carried out in MeCN in earlier studies using the tert-butyl benzoate precursor 4 and the pentamethylbenzyl benzoate precursor 12 HSTU can also be used in place of TSTU to achieve similar radiochemical yields. In our hands, our one-pot procedure produced the same yield as those reported earlier 1 but in a shorter time.…”

Batch‐mode microfluidic radiosynthesis of N‐succinimidyl‐4‐[18F]fluorobenzoate for protein labelling

“…This is particularly relevant for radiolabelled molecular probes derived from short-lived isotopes (for example, 11 C or 18 F). 18 F is the most frequently used positron emitter, due to its availability via routine production on low-energy cyclotron and its intermediate half-life (109.7 min) providing flexibility in regard to chemistry. 3 The most common method of labelling proceeds via nucleophilic substitution involving [ 18 F]fluoride ion.…”

Two‐step radiosynthesis of [18F]N‐succinimidyl‐4‐fluorobenzoate ([18F]SFB)

“…Nevertheless, the process has been established on automated platforms. [14][15][16] The reagent 18 F-SFB has been used to radiolabel numerous peptides such as human C-peptide, 17 neurotensin(8-13), 18 annexin-V, 19 insulin, 20 bombesin, 21 vasoactive intestinal peptide, 22 and RGD peptides, [23][24] as well as oligonucleotides. 25 So far, one-step approaches using N-succinimidyl benzoate precursors have been attempted but had no success.…”