Improved 18F labeling of peptides with a fluoride-aluminum-chelate complex

Abstract

We reported previously the feasibility to radiolabel peptides with fluorine-18 ((18)F) using a rapid one-pot method that first mixes (18)F(-) with Al(3+) and then binds the (Al(18)F)(2+) complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al(18)F NOTA complexes were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100 degrees C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide could be labeled up to 115 GBq/micromol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.

Figure 1

RP-HPLC analysis of IMP467, ¹⁸F-IMP467, and ¹⁹F-IMP467. The unlabeled peptide elutes as a single peak (A), but when radiolabeled with Al¹⁸F (¹⁸F-IMP467), two peaks are observed (B). ¹⁹F-IMP467 also showed 2 main peaks (C) [13.4 min peak is unlabeled peptide as seen in (A)]. The 14.9 min peak was isolated and evaluated over time (D-F), showing the equilibration of the isomeric counterpart (~13.0 min).

Figure 2

Serum stability of IMP467 as analyzed by RP-HPLC; (A) ¹⁸F-IMP467 in serum at time-zero (B) ¹⁸F-IMP467 in serum after 1 h, (C) ¹⁸F-IMP467 in serum after 4 h.

Figure 3

Binding of ¹⁸F-IMP467 to TF2 by SE-HPLC; (A) ¹⁸F-IMP467 after HLB purification, alone, or (B), mixed with TF2 anti-CEA x anti-HSG bsMAb.

Figure 4

In vivo stability of ¹⁸F-IMP467. SE- and RP-HPLC analysis of ¹⁸F-IMP467 before injection and in urine samples taken from mice 0.5 h and 1.5 h post injection. RP-HPLC shows the same elution profile in the original product and in the urine, while SE-HPLC was performed to show ¹⁸F-IMP467 eliminated in urine continued to retain binding to the TF2 anti-CEACAM5 x anti-HSG bsMAb.

Scheme 1

Synthesis of bis-t-butyl NOTA

Scheme 2

Synthesis of succinyl C-NETA ligand (4).