Two-step methodology for high-yield routine radiohalogenation of peptides: ¹⁸F-labeled RGD and octreotide analogs

Routine application of ¹⁸F-labeled peptides for quantitative in vivo receptor imaging of receptor-expressing tissues and quantification of receptor status using PET is limited by the lack of appropriate radiofluorination methods for routine large-scale synthesis of ¹⁸F-labeled peptides. To satisfy this demand, a new ¹⁸F-labeling methodology based on the chemoselective oxime formation between an unprotected aminooxy-functionalized peptide and an ¹⁸F-labeled aldehyde or ketone was investigated and optimized with respect to peptide conjugation. Methods: 4-[¹⁸F]Fluorobenzaldehyde ([¹⁸F]FB-CHO) was prepared from the 4-formyl-N,N,N-trimethylanilinium precursor via direct no-carrier-added ¹⁸F-fluorination (dimethyl sulfoxide, 60°C, 15 min) and purified using a cation-exchange/reversed-phase cartridge system. Radiochemical yields (RCYs) of N-(4-[¹⁸F] fluorobenzylidene) oxime ([¹⁸F]FBOA) formation with various aminooxy-modified peptides such as minigastrin, RGD, and octreotate analogs were investigated as a function of reaction time and temperature, peptide concentration, and pH. Biodistribution studies were performed with an [ ¹⁸F]FBOA-RGD dimer ((c(RGDfE)-HEG)₂-K-Dpr-[ ¹⁸F]FBOA, 60 and 120 min after injection) and a gylcosylated [ ¹⁸F]FB-Tyr³-octreotate (Gluc-S-Dpr([¹⁸F]FBOA)- TOCA), 10 and 60 min after injection) using M21 and M21L human melanoma and AR42J rat pancreatic tumor-bearing nude mice, respectively. Results: [ ¹⁸F]FB-CHO was obtained in a nonoptimized RCY of 50% within 30 min. At low peptide concentrations (0.5 mmol/L), optimal [¹⁸F]FBOA- labeling efficiencies (60%-80%) were obtained within 15 min at 60°C and pH 2-3, independently of the peptide used, affording the [¹⁸F]FBOA- peptides in overall RCYs of up to 40% (from end of bombardment) after purification. Both (c(RGDfE)HEG)₂-K-Dpr-[¹⁸F]FBOA and Gluc-S-Dpr([¹⁸F]FBOA)TOCA showed pharmacokinetics suitable for early (≤60 min) high-contrast PET imaging, high tumor uptake (2.48 ± 0.15 %ID/g [RGD] and 21.8 ± 1.4 %ID/g [TOCA] at 60 min after injection, where %ID/g = percentage injected dose per gram), and tumor-to-organ ratios that compared well with the corresponding [¹⁸F]fluoropropionyl analogs [¹⁸F] Galacto-RGD and Gluc-Lys([¹⁸F]FP)TOCA, which are prepared via multistep procedures. Conclusion: Oxime formation between aminooxy-functionalized peptides and an ¹⁸F-labeled aldehyde or ketone - in this case, [¹⁸F]FB-CHO - combines fast 1-step, high-yield synthesis of an ¹⁸F-labeled prosthetic group stable against in vivo defluorination with rapid, 1-step chemoselective conjugation to unprotected peptides under mild conditions. Thus, it allows fast and straightforward large-scale production of ¹⁸F-labeled peptides for clinical routine PET application. Furthermore, it opens new perspectives to peptide radiohalogenation in general, permitting labeling of the same precursor both with diagnostic (¹⁸F, ¹²⁴I, ^120gI, ¹²³I) and therapeutic (²¹¹At, ¹³¹I) radiohalogens.