Noninvasive imaging of osteoclasts in parathyroid hormone-induced osteolysis using a ⁶⁴Cu-labeled RGD peptide

Bone diseases are often a result of increased numbers of osteoclasts, or bone-resorbing cells. Bone metastases are a significant cause of morbidity in many types of cancer. An imaging agent targeting osteoclasts, which are upregulated in osteolytic lesions, may facilitate earlier follow-up in patients with osteolytic or mixed bone metastases. Osteoclasts express high levels of α_vβ₃ integrin, to which peptides containing the Arg-Gly-Asp (RGD) sequence are known to bind. We proposed that radiolabeled RGD peptides could be used to detect osteoclasts in lytic bone lesions. Methods: The cross-bridged macrocyclic chelator 4,11-bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) was conjugated to c(RGDyK) for radiolabeling with ⁶⁴Cu (t_1/2, 12.7 h; β⁺, 17.4%; E_β+max, 656 keV; β^-, 39%; E _β2max, 573 keV). The in vitro affinity of Cu(II)-CB-TE2A- c(RGDyK) for α_vβ₃ and α_vβ ₅ was evaluated in a heterologous competitive binding assay. Ex vivo uptake was examined in osteoclasts prepared from bone marrow macrophages. As a proof of principle, biodistribution and imaging studies were performed on parathyroid hormone (PTH)-induced osteolysis in the calvarium. Results: Cu-CB-TE2A-c(RGDyK) was shown to have a 30-fold higher affinity for α_vβ₃ than for α_vβ ₅. Osteoclasts were shown to specifically take up ⁶⁴Cu-CB-TE2A-c(RGDyK). However, bone marrow macrophages showed only nonspecific uptake. PTH treatment increased calvarial uptake of ⁶⁴Cu-CB-TE2A-c(RGDyK), compared with uptake in mice receiving a sham treatment. In addition, calvarial uptake correlated linearly with the number of osteoclasts on the bone surface. Conclusion: These results suggest that ⁶⁴Cu-CB-TE2A-c(RGDyK) selectively binds α_vβ ₃ on osteoclasts and may potentially be used to identify increased numbers of osteoclasts in osteolytic bone diseases such as osteolytic bone metastasis and inflammatory osteolysis.