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

Osteoclasts are multinucleated cells formed by the fusion of mononuclear progenitors of the monocyte/macrophage family. They are the principal cells involved in bone resorption. Bone diseases, both nonneoplastic and neoplastic, often involve increased numbers of osteoclasts. For example, the periarticular osteolysis that complicates rheumatoid arthritis is due to aggressive osteoclast recruitment. The skeleton is one of the most common sites of cancer metastasis (1). It has been reported that in 75% of patients with metastatic breast cancer, metastatic bone lesions—which can be osteolytic, osteoblastic, or mixed—will develop (2). Breast cancer cells in bone stimulate recruitment of osteoclasts, resulting in osteolysis (3). Bone metastases cause significant morbidity, including pain, pathologic fracture, spinal cord compression, and hypercalcemia (1,2). In addition, microscopic bone tumor metastases appear to be an independent predictor of poor outcome in breast cancer even among lymph node–negative patients with primary tumors smaller than 2 cm (4). The most common method for detecting bone metastases is γ-camera imaging using 99mTc-methylene diphosphonate (MDP), commonly referred to as a bone scan (2). Bone scans rely on osteoblast activity for incorporation of the 99mTc-MDP radiopharmaceutical into bone (5). Therefore, purely osteolytic lesions are poorly detectable (6), and bone tumors responding to anticancer therapy, with resultant bone healing involving osteoblast-mediated bone deposition, can exhibit a false-positive flare response for several months after treatment (2,7,8). PET with 18F-FDG has been used for bone metastasis detection (5), but cells involved in inflammation and bone healing at metastatic foci may also have a high glucose uptake (9,10). Anatomic imaging methodologies such as radiography, MRI, and CT have also been used to detect or monitor bone metastases. However, metastases to the bone become apparent on radiographs only after the loss of more than 50% of the bone mineral content at the site of disease (11). The loss of bone density must be significant for CT to be able to detect osteolytic bone lesions (11), and MRI detects tumor in the bone marrow but does not readily image bone degradation (12). Dual-energy x-ray absorptiometry scans are frequently used in osteoporosis to monitor changes in bone density and the response to antiresorptive therapy. These can be difficult to interpret in the case of compression fractures and osteoarthritis, which can occur in osteopenic populations. Therefore, imaging agents that identify osteoclasts in vivo are urgently needed to quantify the burden of osteolytic bone metastases, evaluate the response of osteolytic bone metastases to therapy, and identify which patients will respond to antiresorptive agents. Cyclic pentapeptides containing an Arg-Gly-Asp (RGD) consensus sequence have been investigated as high-affinity αvβ3 ligands with good specificity (13). The integrin αvβ3 has increasingly become a target for tumor and angiogenesis imaging using radiolabeled RGD peptides (14,15). Osteoclasts express the highest levels of αvβ3 of any cell in the body (16), and αvβ3 ligands have been shown to inhibit osteoclast attachment to and absorption of bone (17,18). Therefore, αvβ3 integrin was selected as the target protein for osteoclast imaging in osteolytic bone disease. Many tumor cells also express αvβ3 (19). For clinical imaging, detection of both tumor cells and associated osteoclasts would improve the sensitivity of an osteoclast-imaging agent targeting αvβ3. However, for validating an αvβ3 imaging agent for direct detection of osteoclasts, a model of pharmacologically induced osteolysis is appropriate to differentiate tumor cell–mediated and osteoclast-mediated uptake in bone lesions. Parathyroid hormone (PTH) induces osteoclast-mediated osteolysis when serially injected subcutaneously at the calvarium (20). This pharmacologic model of PTH-induced osteolysis will allow investigation of osteoclast-mediated bone uptake independent of tumor cells. 64Cu (t1/2, 12.7 h) is suitable for both PET and targeted radiotherapy because of its decay characteristics (β+, 17.4%; Eβ+max, 656 keV; β−, 39%; Eβ−max, 573 keV) (21,22) and because it can be produced at a high specific activity on a small biomedical cyclotron (23). Peptides and proteins are radiolabeled with 64Cu using a covalently linked bifunctional chelator. Recent studies have shown that commonly used bifunctional chelators such as 1,4,7,10-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) are unstable in vivo relative to the cross-bridged chelator 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) (24). Therefore, CB-TE2A is a better candidate for copper-based PET, resulting in lower liver and blood uptake and higher target-to-background signals than can be obtained with conventional bifunctional chelators (25). Here, we have conjugated c(RGDyK) (26) to CB-TE2A for radiolabeling with 64Cu (Fig. 1). Osteoclasts were shown to selectively take up 64Cu-CB-TE2A-c(RGDyK) in vitro. In a proof-of-principle study, a mouse model of PTH-induced osteolysis was used to validate 64Cu-CB-TE2A-c(RGDyK) for osteoclast imaging in vivo. FIGURE 1 Structure of CB-TE2A-c(RGDyK).