An improved method of ¹⁸F peptide labeling: hydrazone formation with HYNIC-conjugated c(RGDyK)

Radiolabeled α_vβ₃-integrin antagonists are increasingly investigated as a means of imaging angiogenesis. Several methods of labeling α_vβ₃-integrin binding peptide with ¹⁸F have been reported recently. In the present study, we devised a straightforward means for labeling Arg-Gly-Asp (RGD) peptide with ¹⁸F via hydrazone formation between c(RGDyK)-hydrazinonicotinic acid (HYNIC) (3) and 4-[¹⁸F]-fluorobenzaldehyde ([¹⁸F]4). The resulting reaction mixture was purified by HPLC to give 4′-[¹⁸F]-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) ([¹⁸F]5). The conjugation efficiency of 3 and 4 to form [¹⁸F]5 was 95.2%, and the radiochemical purity of [¹⁸F]5 after purification was >99%. The specific activity of [¹⁸F]5 estimated by radio-HPLC was 20.5 GBq/μmol (end of synthesis). Competitive binding assay of c(RGDyK) (1) and 5 was performed using [¹²⁵I]iodo-c(RGDyK) as a radioligand, and K_i values were found to be 2.8 and 21.7 nM, respectively. For the biodistribution study, the angiogenic mouse model was established by inducing unilateral ischemia on the left hindlimbs of ICR mice after femoral artery ablation. Seven days after inducing ischemia, [¹⁸F]5 was administered to the mice through the tail vein. Ischemic muscle uptake of [¹⁸F]5 was significantly higher than that of normal muscle (P<.01). Specific uptake was confirmed by coinjection of 1 with [¹⁸F]5. Here, we successfully labeled RGD peptide with ¹⁸F via hydrazone formation between 3 and 4, resulting to [¹⁸F]5. [¹⁸F]5 was found to have high affinity for α_vβ₃-integrin and to accumulate specifically in ischemic hindlimb muscle of mice. We suggest that ¹⁸F labeling via formation of hydrazone between HYNIC peptide and [¹⁸F]4 is a useful method for labeling c(RGDyK), which can be applied for imaging angiogenesis.