Well-Designed Bone-Seeking Radiolabeled Compounds for Diagnosis and Therapy of Bone Metastases

Bone-seeking radiopharmaceuticals are frequently used as diagnostic agents in nuclear medicine, because they can detect bone disorders before anatomical changes occur. Furthermore, their effectiveness in the palliation of metastatic bone cancer pain has been demonstrated in the clinical setting. With the aim of developing superior bone-seeking radiopharmaceuticals, many compounds have been designed, prepared, and evaluated. Here, several well-designed bone-seeking compounds used for diagnostic and therapeutic use, having the concept of radiometal complexes conjugated to carrier molecules to bone, are reviewed.

It has been known that strontium (Sr) acts as calcium mimic and accumulates in high osteoblastic activity lesions since strontium is one of the alkaline earth metals [5]. 89 Sr has a physical half-life of 50.5 days and emits beta particles with a maximum energy of 1.46 MeV (Table 1). Strontium-89 chloride ( 89 SrCl 2 , Metastron) was the first radiopharmaceutical approved for the palliation of metastatic bone pain by the US Food and Drug Administration (FDA). 89 SrCl 2 for the palliation of metastatic bone pain for breast cancer patients and prostate cancer patients showed a pain relief rate of 57-92%. These studies are summarized in reviews [6][7][8][9].
Samarium-153 ( 153 Sm) has a physical half-life of 46.3 hours and emits beta particles with a maximum energy of 0.81 MeV (20%), 0.71 MeV (49%), and 0.64 MeV (30%) and a 28% abundance of gamma rays with energy of 103 keV (Table 1). 153 Sm-ethylenediaminetetramethylene phosphonic acid (EDTMP, Quadramet) is a complex of 153 Sm and EDTMP (Figure 1(c)), which has high affinity for bone mineral. 153 Sm-EDTMP was approved and has been widely used in the United States for palliation of metastatic bone pain. The biodistribution of 153 Sm-EDTMP is similar to that of bone scintigraphic agents such as 99m Tc-MDP (methylene diphosphonate) [10]. Accordingly, it was reported that the dosimetry of 153 Sm-EDTMP could be predicted using 99m Tc-MDP bone scintigraphy [11]. 153 Sm-EDTMP showed a pain relief rate in 62-84% of patients with metastatic bone pain. These studies are also summarized in reviews [6][7][8]. Meanwhile, in a study of comparison between the effects of the 89 SrCl 2 and 153 Sm-EDTMP to patients with bone metastases, there was no statistical difference in response rates [12].
Zoledronic acid (Zometa), which is a bisphosphonate compound, has been widely used for the prevention of skeletal complications. Lam et al. combined zoledronic acid and 153 Sm-EDTMP to treat hormone-refractory prostate cancer patients [13]. It was concluded that zoledronic acid  treatment does not influence 153 Sm-EDTMP skeletal uptake and combined treatment is feasible and safe. The therapeutic bone-seeking radiopharmaceutical radium-223 chloride ( 223 RaCl 2 ) was approved by FDA and European Medicines Agency (EMA) in 2013 based on data from a phase III randomized trial (the Alpharadin in Symptomatic Prostate Cancer Patients: ALSYMPCA). Surprisingly, 223 RaCl 2 significantly improved overall survival in patients with castration-resistant prostate cancer with bone metastases in the ALSYMPCA study [14,15]. In addition, because it is the first radiopharmaceutical emitting alpha particles approved for clinical use, 223 RaCl 2 is currently attracting much attention.
99m Tc-MDP, 99m Tc-HMDP, 89 SrCl 2 , 153 Sm-EDTMP, and 223 RaCl 2 are milestones in the development of bone-seeking radiopharmaceuticals for clinical use (Table 2). Although developing superior bone-seeking compounds is difficult, we reviewed the promising well-designed bone-seeking compounds for diagnosis and therapy of bone metastases in basic research.  yet been optimized from a chemical and pharmaceutical perspective, because these complexes are not well-defined singlechemical species but are mixtures of short-chain and longchain oligomers [16]. Moreover, the phosphonate groups in 99m Tc-MDP and 99m Tc-HMDP are used both as ligands for coordination and as carriers to hydroxyapatite (HA) in bone [17], which may decrease the inherent affinity of MDP and HMDP for bone. To overcome these drawbacks, a more logical design strategy has been proposed on the basis of the conjugation of a stable radiometal complex to a carrier molecule to bone. This drug design allows the ligand and carrier function to work independently and effectively. In 2002, Verbeke et al. described 99m Tc-l,l-ethylenedicysteine (EC) complex, a renal tracer agent known to have rapid renal excretion, conjugated to bisphosphonate ( 99m Tc-EC-AMDP, Figure 2(a)) [18]. 99m Tc-EC-AMDP showed faster blood clearance and a higher bone/blood ratio compared with 99m Tc-MDP in animal experiments.
In 2006, we reported 99m Tc-mercaptoacetylglycylglycylglycine-(MAG3-) conjugated bisphosphonate ( 99m Tc-MAG3-HBP, Figure 2(b)) and 99m Tc-6-hydrazinonicotinic acid (HYNIC) with tricine and 3-acetylpyridine as coligands conjugated to bisphosphonate ( 99m Tc-HYNIC-HBP, Figure 2(c)) [19]. In in vitro HA binding experiments, the binding rates of 99m Tc-MAG3-HBP and 99m Tc-HYNIC-HBP to HA were higher than those of 99m Tc-HMDP. In a biodistribution study in rats, 99m Tc-MAG3-HBP and 99m Tc-HYNIC-HBP showed higher accumulation in bone compared with 99m Tc-HMDP reflecting the in vitro findings. The blood clearance of 99m Tc-MAG3-HBP was delayed because of the high rate of protein binding in blood and the bone/blood ratio of 99m Tc-MAG3-HBP was lower than that of 99m Tc-HMDP. In contrast, the blood clearance of 99m Tc-HYNIC-HBP was as rapid as that of 99m Tc-HMDP and its bone/blood ratio was higher.
Liu et al. reported findings on 99m Tc-HYNIC-conjugated bisphosphonate ( 99m Tc-HYNIC-AMDP, Figure 2(d)) in 2011 [20]. The authors found that 99m Tc-HYNIC-AMDP had a higher bone uptake and higher bone/blood and bone/muscle ratios at an early time point after injection as compared with 99m Tc-MDP. In that study, 99m Tc-HYNIC-AMDP showed favorable biodistribution as a bone-seeking agent, but the bone accumulation of 99m Tc-MDP, a bone scintigraphy agent as a control, appeared to be too low. Two tricine molecules are used as coligands in 99m Tc-HYNIC-AMDP. However, as it has been reported, the complex [ 99m Tc](HYNIC)(tricine) 2 is not stable and exists in multiple forms, and the pharmacokinetics could be affected by the exchange reaction between tricine and protein in the plasma and tissues [21][22][23]. The pharmacokinetics of 99m Tc-HYNIC-AMDP may be improved by exchanging one tricine molecule for another molecule, such as one of the pyridine derivatives. were higher than that of 99m Tc-MDP at 4 hours after injection because of their fast clearance. The difference in bone accumulation among 99m Tctricarbonyl complex-conjugated bisphosphonate compounds could be derived from the introduction of a hydroxyl group at the central carbon of the bisphosphonate because bisphosphonates containing the hydroxyl group have been reported to have higher affinity for bone minerals [26][27][28].
As mentioned above, certain 99m Tc-complex-conjugated bisphosphonate compounds have shown favorable biodistribution as bone imaging agents and higher bone/blood ratios compared with those of 99m Tc-MDP or 99m Tc-HMDP. Consequently, these results suggest that the strategy of developing stable 99m Tc-complex-conjugated bisphosphonates is promising.

Radiogallium-Complex-Conjugated
Bisphosphonate Compounds as Bone Imaging Agents for Positron Emission Tomography (PET) 68 Ga is a practical and interesting radionuclide for clinical PET because of its radiophysical properties, particularly as a 68 Ge/ 68 Ga generator-produced radionuclide has a half-life ( 1/2 ) of 68 minutes (Table 1) [30]. It does not require an on-site cyclotron and can be eluted on demand. Indeed, in principle, the long half-life of the parent nuclide 68 Ge ( 1/2 = 270.8 days) provides a generator with a long lifespan. The above-mentioned drug design concept, which is a stable complex-conjugated bisphosphonate, could also be applicable to gallium complexes. With the aim of developing a superior bone imaging PET tracer, some types of radiogallium complex-conjugated bisphosphonate compounds have been reported.
In 2011, we also reported 67 Ga-DOTA complexconjugated bisphosphonate ( 67 Ga-DOTA-Bn-SCN-HBP, Figure 4(d)) [34]. Although the aim was to develop a superior 68 Ga-labeled bone imaging agent for PET, in the initial basic studies 67 Ga was used because of its longer half-life. In biodistribution experiments in normal mice, 67 Ga-DOTA-Bn-SCN-HBP rapidly and highly accumulated in bone but was rarely observed in tissues other than bone. As a result, the bone/blood ratio of 67 Ga-DOTA-Bn-SCN-HBP was comparable to that of 99m Tc-HMDP, which is a gold standard for a bone scintigraphy agent.
These results suggest that the drug design concept of radio gallium complex-conjugated bisphosphonate could be useful for the development of 68 Ga PET imaging agents for bone disorders such as bone metastases.

Re-Complex-Conjugated Bisphosphonate for Palliation of Bone Metastases
Gamma ray emitter radionuclide and positron emitter radionuclide-labeled bone-seeking agents are used for the diagnosis of bone metastases. A prominent symptom caused by bone metastases is pain, which has a significant impact on the patients' quality of life. Bone-seeking agents labeled with high-energy beta particle emitter radionuclides and alpha particle emitter radionuclides are used for palliation of pain caused by bone metastases. Rhenium, which has similar chemical properties to technetium, because they are members of family VIIA of the periodic table, has two useful radionuclides, 186 Re and 188 Re, which are useful for radionuclide therapy [36]. Both rhenium radionuclides emit not only beta particles for therapy but also gamma rays, which are suitable for diagnoses: 186 Re ( 1/2 = 3.78 days, − max = 1.07 MeV, = 137 keV) and 188 Re ( 1/2 = 16.98 hours, − max = 2.12 MeV, = 155 keV) ( Table 1). In addition, 188 Re has a further advantage for clinical use because it is obtained from an in-house alumina-based 188 W/ 188 Re generator, similar to a 99 Mo/ 99m Tc generator [37].
When considering the use of rhenium in bone-seeking agents for palliation, similar to 99m Tc-MDP and 99m Tc-HMDP, it is known that rhenium coordinates with some bisphosphonate derivatives. 186/188 Re-1-hydroxyethylidene-1,1-diphosphonate ( 186/188 Re-HEDP, Figure 1(d)), which has high affinity for bone, has been prepared and used for clinical research [9,38,39]. Although the chemical properties of rhenium are similar to those of technetium as mentioned above, rhenium is more easily oxidized than technetium [40], and it has been reported that 186 Re-HEDP is not as stable as 99m Tc-bisphosphonate complexes [41]. Some studies reported that 186 Re-HEDP showed unexpected gastric uptake in patients with bone metastases [42,43]. This may be derived from the accumulation of free rhenium (perrhenate: ReO 4 − ) in the stomach due to the instability of 186 Re-HEDP [40,44]. Moreover, as with 99m Tc-MDP and 99m Tc-HMDP, the phosphonate groups in 186/188 Re-HEDP are used as both ligands for coordination and as carrier to HA in bone, which may decrease the inherent affinity of HEDP for bone.
To overcome these problems, designing a stable 186/188 Recomplex-conjugated bisphosphonate would be useful. Therefore, we studied 186 Re-monoaminemonoamidedithiol- Previous studies suggested that the hydroxyl group affects affinity for bone minerals [26,27]. We evaluated the therapeutic potential of 186 Re-MAG3-HBP for the palliation of metastatic bone pain using an animal model of bone metastasis [47]. The planar image of 186 Re-MAG3-HBP showed high accumulation of radioactivity in bone metastasis lesion. 186 Re-MAG3-HBP was more effective for palliation and was compared with 186 Re-HEDP using the hind paw withdrawal response to stimulation with von Frey filaments. Moreover, although 186 Re-HEDP did not affect tumor growth, 186 Re-MAG3-HBP significantly inhibited tumor growth. CpTR-Gly)-conjugated bisphosphonate [48]. [ 186 Re]CpTR-Gly-APD showed characteristics superior to those of 186 Re-HEDP, such as higher stability in plasma, a higher binding rate for HA, higher bone accumulation, and lower plasma protein binding. When [ 186 Re]CpTR-Gly-APD with HEDP (9.0 mg/kg) was administered to mice, the accumulation of radioactivity in bone significantly decreased and the blood clearance was delayed. Therefore, the authors concluded that the specific activity of 186 Re-labeled bisphosphonate compounds is very important to bone accumulation and blood clearance.
These results indicate that the concept of the stable 186 Recomplex-conjugated bisphosphonates could be more useful and that novel 186 Re-complex-conjugated bisphosphonate complexes could be attractive candidates as palliative agents in metastatic bone pain.

Aspartic Acid Peptides as Carriers of Radionuclides to Bone
Several major noncollagenous bone proteins, such as osteopontin and bone sialoprotein, have repeating sequences of acidic amino acids (Asp or Glu) in their structures, offering potential HA binding sites [50][51][52]. It has been reported that polyglutamic and polyaspartic acids have a high affinity for HA and could be used as carriers for drug delivery to bone [53][54][55].
In 2013, we reported 67 Ga-DOTA-conjugated l-Asp peptides ( 67 Ga-DOTA-(Asp) , Figure 7(c)), which had varying peptide lengths ( = 2, 5, 8, 11, or 14) [57]. Binding affinity to HA of 67 Ga-DOTA-(Asp) increased with an increase in These results indicate that not only bisphosphonate molecules but also acidic amino acid peptide sequences could be useful as carriers of radionuclides to bone metastases lesions. Moreover, radiometal complex-conjugated acidic amino acid peptides may provide slightly different information from radiometal complex-conjugated bisphosphonates.

Carbon-11 Labeled Cathepsin K Inhibitors
Cathepsin K is a member of the papain family of cysteine peptidases with a primary physiological function of cleavage of type I and type II collagen [58]. The enzyme is highly expressed in activated osteoclasts, and a change in the number of the osteoclast is related to bone diseases such as osteoporosis [59]. Therefore, it could be useful to determine the changes in osteoclast numbers in such disease states by imaging cathepsin K. Because many inhibitors of cathepsin K have been synthesized and evaluated both in vitro and in vivo, their derivatives may be candidates as mother compounds for cathepsin K imaging agents. The possibility of targeting cathepsin K in in vivo imaging, using a near-infrared reporter probe, was confirmed in a previous report [60].  Figure 8(b)) in 2014 [61]. Nonradioactive counterparts of [ 11 C]1 and [ 11 C]2 were reported in 2007 [62]. In that study, because the pyrimidine core structure docked into the cathepsin K active site, many types of derivatives based on a pyrimidine scaffold were synthesized and evaluated as cathepsin K inhibitors. Among them, the nonradioactive counterparts showed greater affinity and selectivity for cathepsin K. For inhibition of cathepsin K, cathepsin L, and cathepsin S, IC 50 values of compound 1 were 0.022, 0.17, and 0.7, and those of compound 2 were <0.003, 1.2, and 0.9, respectively. [ 11 C]1 and [ 11 C]2 were radiosynthesized by standard reaction conditions used for alkylation reactions with [ 11 C]methyl iodide. In vivo -PET imaging experiments showed that [ 11 C]1 and [ 11 C]2 inhibitors have a higher uptake in actively growing bone regions, such as distal ulnar, carpal, distal and proximal humeral, distal femur, and proximal tibia, than in nontarget regions such as muscle. The uptake in specific bone regions was based on specific binding to cathepsin K because the uptake was inhibited by pre-or coinjection of an excess amount of ligands. These results indicated that radiolabeled cathepsin K inhibitors could have potential as in vivo imaging agents to determine a change in the number of osteoclasts.

Dual-Modality Single Photon Emission Computed Tomography/Near-Infrared (SPECT/NIR) Fluorescent Probe
Recently, multimodality molecular imaging combining several imaging techniques has attracted much attention in basic scientific and clinical research. Nuclear medical imaging can detect deep tissues in the body with high sensitivity, but there are some problems such as relatively poor spatial resolution [63]. Optical imaging is a relatively new imaging modality that offers real-time and nonradioactive and high-resolution imaging of fluorophores in lesion sites, but it is difficult to detect a deep tissue using this technique [64]. Fluorescence imaging with near-infrared (NIR, 700-900 nm wavelength) light reveals relatively low tissue absorption. IRDye78 is a heptamethine indocyanine-type NIR fluorophore with peak absorption at 771 nm and peak excitation emission at 806 nm. Pam78 (Figure 9(a)), a IRDye78-conjugated pamidronate (one of the bisphosphonate derivatives), has been reported as a NIR fluorescence imaging probe targeted to HA [65]. HA is considered to be a good marker for some diseases because calcification (HA deposition) occurs during the processes of cancer and atherosclerosis.
Bhushan et al. reported the trifunctional diagnostic agent Pam-Tc/Re-800 (Figure 9(b)) in 2008 [66]. Pam-Tc/Re-800 possesses a radiometal complex as a nuclear imaging probe, a fluorescent site as a fluorescence imaging probe, and bisphosphonate having high affinity for HA as a carrier to bone in a molecule. In an in vitro experiment, Pam-Tc/Re-800 showed specific and selective binding to HA. In the fluorescence imaging of microcalcified breast cancer rat model, Pam-Re-800 detected breast cancer microcalcifications. In SPECT/CT imaging, Pam-Tc-800 showed not only accumulation in normal bone but also highly sensitive detection of breast cancer microcalcifications. In biodistribution experiments, the total body clearance of Pam-Tc-800 at 4 hours after injection was comparable to that of 99m Tc-MDP. Moreover, Pam-Tc-800 showed a higher uptake in bone and tumor than 99m Tc-MDP. These results indicated that the novel trifunctional agent could provide simultaneous imaging by SPECT and NIR fluorescence. Dual-modality imaging may compensate for the drawbacks of the other modalities.

Summary
In this paper, several well-designed bone-seeking compounds were reviewed. They are chemically well-characterized and different from 99m Tc-MDP and 99m Tc-HMDP. Some demonstrated superior biodistribution characteristics. The mechanism by which all the compounds (except the carbon-11 labeled cathepsin K inhibitors) accumulate in bone is derived due to a high affinity for HA. We estimate that 68 Ga-DOTA-conjugated bisphosphonate compounds, such as 68 Ga-BPAMD and 68 Ga-DOTA-Bn-SCN-HBP, are the most promising diagnostic agents for bone metastases because they show superior biodistribution characteristic and 68 Ga is a useful PET radionuclide in clinical. Moreover, as DOTA ligand could form a complex with not only 67/68 Ga but also 177 Lu and 90 Y, the palliation therapy is applicable using the same ligand. Namely, this system is "theranostics", which is a combination of diagnosis and therapy.
Thus, the information from imaging data and the type of bone metastasis susceptible to treatment should be similar to those for existing bone-seeking radiopharmaceuticals. We hope that novel bone-seeking compounds that possess a different accumulation mechanism will be developed in the near future.