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

Abstract

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.

Introduction

α_vβ₃-Integrin participates in angiogenesis and developmental neovascularization [1], [2], [3], [4]. Moreover, monoclonal antibodies or small peptides containing the Arg–Gly–Asp (RGD) amino acid sequence [5] direct α_vβ₃-integrin to block angiogenesis in human melanoma and breast tumor xenografts [6], [7], [8], [9]. Several reports have been issued on the development of radiolabeled RGD peptides for α_vβ₃-integrin receptor targeting [10], [11], [12], [13], [14], [15], [16], [17], [18]. In particular, ¹⁸F-labeled RGD peptides are ideal for clinical and research uses due to their ideal nuclear characteristics with regard to positron emission tomography that has high resolution and sensitivity [19], [20].

The labeling of peptides with ¹⁸F requires specially designed synthons because peptides cannot be directly labeled by nucleophilic fluorination. Several such synthons have been developed and successfully employed for the labeling of peptides and proteins [21], [22], [23], [24], [25], [26]. Among these synthons, N-succinimidyl 4-[¹⁸F]fluorobenzoic acid ([¹⁸F]SFB) is the most widely used for the labeling of many bioactive molecules because of their high in vivo stabilities and labeling yields. However, [¹⁸F]SFB synthesis requires a time-consuming three-step method [19].

Recently, we reported a straightforward and efficient method for preparing ¹⁸F-labeled human serum albumin (HSA) via hydrazone formation between 6-hydrazinonicotinamide-HSA (HYNIC-HSA) and 4-[¹⁸F]-fluorobenzaldehyde ([¹⁸F]4) [4-fluorobenzaldehyde (4)] [27].

In the present study, we evaluated the usefulness of this method for the ¹⁸F labeling of RGD peptide. The in vitro affinity of 4′-[¹⁸F]-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) ([¹⁸F]5) [4′-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) (5)] for α_vβ₃-integrin was investigated using competitive binding assays, and the in vivo behavior of [¹⁸F]5 was investigated in a murine ischemic hindlimb model, which has previously been described as an excellent angiogenic model [28], [29].