A novel facile method of labeling octreotide with (18)F-fluorine

Abstract

Several methods have been developed to label peptides with (18)F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of (18)F-aluminum fluoride (Al(18)F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Cys]-Throl (MH(+) 1305) [IMP466]) with (18)F.

Methods: Octreotide was conjugated with the NOTA chelate and labeled with (18)F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice and compared with that of (68)Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired.

Results: IMP466 was labeled with Al(18)F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al(18)F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37 degrees C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the (19)F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of (18)F-IMP466 (28.3 +/- 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 +/- 90), which could be blocked by an excess of unlabeled peptide (8.6 +/- 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of (68)Ga-IMP466 was similar to that of (18)F-IMP466. (18)F-IMP466 was stable in vivo, because bone uptake was only 0.4 +/- 0.2 %ID/g, whereas free Al(18)F accumulated rapidly in the bone (36.9 +/- 5.0 %ID/g at 2 h after injection). Small-animal PET/CT scans showed excellent tumor delineation and high preferential accumulation in the tumor.

Conclusion: NOTA-octreotide could be labeled rapidly and efficiently with (18)F using a 2-step, 1-pot method. The compound was stable in vivo and showed rapid accretion in somatostatin receptor subtype 2-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds with (18)F.

Conflict of interest statement

Disclosure: WJM and DMG are employed or have financial interest in Immunomedics, Inc. PL, RMS, WJGO and OCB declare no conflicts.

Figures

Figure 1

Preparation of “Al18F” and chelation with NOTA-octreotide.

Figure 2

RP-HPLC chromatograms of the IMP466 18F-labeling mix (panel A) and the purified 18F-IMP466 (panel B). Red traces represents radioactivity(left y-axis) and blue traces represents UV signal (right y-axis). In the HPLC chromatogram of the crude mixture, unbound “Al18F” eluted with the void volume (Rt = 0.8 min). Two radioactive peaks correspond to the stereoisomers of radiolabeled peptide times (Rt = 17.4 and Rt = 19.8 min). Finally, the unlabeled IMP466 was present in the UV channel (Rt = 21.4 min). After purification, only two radioactive peptide peaks are observed, indicating the formation of two stereoisomers.

Figure 3

Competitive binding assay (apparent IC50) of 19F-IMP466, 69Ga-IMP466 and 115In-DTPA-octreotide determined on AR42J tumor cells. Values on the y-axis represent binding expressed as a precentage of the binding without competitor.

Figure 4

HPLC chromatograms of purified 18F-IMP466 before injection (panel A) and a urine sample 30 min p.i. (panel B). Red traces represents radioactivity(left y-axis) and blue traces represents UV signal (right y-axis). The HPLC traces of the two samples are very similar, indicating that the excreted product is intact 18F-IMP466.

Figure 5

Biodistribution of 18F-IMP466 and 68Ga-IMP466 at 2 h p.i. in AR42J tumor-bearing mice (n=5/group). As a control, mice in separate groups (n=3/group) received an excess of unlabeled octreotide to demonstrate receptor specificity. Tumors weighed 0.04 – 0.33 g.

Figure 6

Biodistribution of 18F-IMP466 and unbound “Al18F” at 2 h p.i. in AR42J tumor-bearing mice (n=5/group). Tumors weighed 0.07 – 0.36 g.

Figure 7

Anterior 3D volume-rendering projections of fused PET and CT scans of mice with a s.c. AR42J tumor on the right flank injected with 18F-IMP466 (A), 18F-IMP466 in the presence of an excess of unlabeled IMP466 (B), and 68Ga-IMP466 (C). Arrows indicate tumors. Scans were recorded at 2 h p.i.