Biocomplexes in radiochemistry

2.1 Diagnostic bone-seeking radiopharmaceuticals

Bisphosphonates are synthetic pyrophosphate analogs that are stable in vivo because of their P-C-P central structure, rather than the P-O-P configuration of pyrophosphates, which affords greater resistance to phosphatase hydrolysis (Fig. 1a). Because bisphosphonates inhibit osteoclast-mediated bone resorption and bone turnover, they have been used in the treatment of skeletal disorders, such as osteoporosis, metastatic bone cancer, and Paget’s disease [1, 2]. Bisphosphonates have a high affinity to bones, especially to hydroxyapatite, which is a mineral present in bones. Bisphosphonates also have been used as carriers of radioisotopes to bones. For many years, two ^99mTc-bisphosphonate complexes, ^99mTc-methylenediphosphonate (^99mTc-MDP, Fig. 1b) and ^99mTc-hydroxymethylenediphosphonate (^99mTc-HMDP, Fig. 1c), have been clinically used in nuclear medicine for diagnosis of bone disorders, such as metastatic bone cancer [3–5], because their high sensitivity can detect bone disorders before the occurrence of anatomical changes. Bone metastases are classified as osteolytic, osteosclerotic, or mixed types that reflect osteolytic or osteosclerotic changes caused by the highly activated osteoclasts or osteoblasts that occur in bone metastases. In addition, technetium-99m (^99mTc) is one of the most important radionuclides in nuclear medicine. ^99mTc has frequently been clinically used because (1) it has adequate physical half-life (T_1/2 = 6.01 h) for clinical use, (2) the gamma ray energy it emits (141 keV) is appropriate for SPECT imaging, and (3) it can be produced from the radionuclide generator ⁹⁹Mo/^99mTc, which enables generation of ^99mTc on demand.

Fig. 1

Chemical structures of bisphosphonates analogs (a) pyrophosphate, (b) MDP, (c) HMDP, (d) EDTMP, and (e) HEDP.

The ^99mTc-bisphosphonate complex accumulates in bones because of its high affinity for hydroxyapatite in the bisphosphonate structure. It is assumed that ^99mTc-MDP forms a bidentate – bidentate bridge with hydroxyapatite, whereas ^99mTc-HMDP must form a bidentate – tridentate bridge because of the presence of an additional hydroxyl group on the central carbon of the C-P-C structure in HMDP and is expected to enhance the hydroxyapatite affinity of the ^99mTc complex [6, 7].

The uptake mechanisms of ^99mTc-bisphoshonate complexes in bone metastases have not been completely elucidated. One of the factors related to the higher tracer uptake at the metastatic sites is increased vascularity and regional distribution of blood flow associated with the disease. However, regional bone blood flow alone does not explain the increased uptake of ^99mTc-bisphoshonate [8]. Other factors are also related to the binding and interaction between the complexes and the bones. It is known that ^99mTc-bisphoshonate complexes accumulate at sites of new bone formation or calcification [9, 10]. Kanishi has reported that the accumulation mechanisms might involve both adsorption onto the surface of hydroxyapatite in the bone and incorporation into the crystalline structure of hydroxyapatite [11]. The crystalline structure of hydroxyapatite in newly formed bone is amorphous and has a greater surface area than that in normal bone [12]. Bisphosphonate compounds show significantly higher in vitro adsorption onto amorphous calcium phosphate than onto crystalline forms [8].

^99mTc is supplied from the ⁹⁹Mo/^99mTc generator as ^99mTcO₄^–. The oxidation state of ^99mTc in ^99mTcO₄^– is +7. Bisphosphonate compounds form multiple complexes with reduced ^99mTc. By using high-performance liquid chromatography (HPLC), the relative composition of ^99mTc-bisphosphonate complexes in a reaction mixture has been found to vary with pH, ⁹⁹Tc carrier, and oxygen concentrations [13]. Wilson et al. have assumed that ^99mTc-bisphosphonate complexes would be a mixture of monomers, oxobridged dimers, and oligomeric clusters with various technetium-oxo core configurations, oxidation states, and ligand coordination numbers [14]. These ^99mTc-bisphosphonate complex species have different biodistribution properties in rats. Pinkerton et al. has reported that the smallest, low charge, mononuclear ^99mTc-bisphosphonate complex has the greatest uptake in bone lesions and the highest lesion-to-muscle and lesion-to-normal bone ratios in experiments using each isolated complex by HPLC [15]. Although these studies were performed over a quarter century ago, the exact structures and mechanisms of action of ^99mTc-bisphosphonate complexes remain unclear.

2.2 Development of novel diagnostic bone-seeking technetium complexes

Although ^99mTc-MDP and ^99mTc-HMDP are most frequently used as bone scintigraphy agents, their chemical and pharmaceutical properties have not been optimised. As mentioned above, these complexes are not well-defined single-chemical species but rather mixtures of short-chain and long-chain oligomers [13]. In ^99mTc-bisphosphonate complexes, the phosphonate groups are used both as ligands for complexation and as carriers of the radionuclide to bone [16], which could decrease the inherent affinity of bisphosphonate for bone. To develop superior bone-seeking radiopharmaceuticals, a more logical design strategy based on conjugation of a stable ^99mTc complex with a carrier like bisphosphonate has been proposed by our research group and other groups. I will now discuss some studies that used this drug design, which allowed the ligand and carrier function to operate independently and effectively.

Verbeke et al. have designed and evaluated a ^99mTc-L,L-ethylenedicysteine (EC) complex, a renal tracer agent known to have rapid renal excretion, conjugated to bisphosphonate (^99mTc-EC-AMDP, Fig. 2a) [17]. ^99mTc-EC-AMDP showed a faster blood clearance and a higher bone/blood ratio, which is an index signal/noise (S/N) ratio, relative to those of ^99mTc-MDP in animal experiments.

Fig. 2

Chemical structures of ^99mTc-complex-conjugated bisphosphonate compounds (a) ^99mTc-EC-AMDP, (b) ^99mTc-MAG3-HBP, (c) ^99mTc-HYNIC-HBP, (d) [^99mTc(CO)₃(PzNN-BP)], (e) [^99mTc(CO)₃(PzNN-ALN)], (f) [^99mTc(CO)₃(PzNN-PAM)], and (g) ^99mTc(CO)₃-DPA-alendronate.

Our research group developed stable ^99mTc-complex-conjugated bisphosphonate compounds: ^99mTc-mercaptoacetylglycylglycylglycine (MAG3)-conjugated bisphosphonate (^99mTc-MAG3-HBP, Fig. 2b) and ^99mTc-6-hydrazinonicotinic acid (HYNIC), with tricine and 3-acetylpyridine as co-ligands conjugated to bisphosphonate (^99mTc-HYNIC-HBP, Fig. 2c) [18]. In hydroxyapatite-binding experiments in vitro, the binding affinities of ^99mTc-complex-conjugated bisphosphonate compounds to hydroxyapatite were higher than that of ^99mTc-HMDP. In animal experiments, ^99mTc-complex-conjugated bisphosphonate compounds showed higher accumulation in bone than did ^99mTc-HMDP, which reflected the findings of hydroxyapatite binding experiments in vitro. However, the blood clearance of ^99mTc-MAG3-HBP was delayed because its protein-binding rate in blood was high. Thus, the bone/blood ratio of ^99mTc-MAG3-HBP was lower than that of ^99mTc-HMDP. The blood clearance of ^99mTc-HYNIC-HBP was similar to that of ^99mTc-HMDP. The bone/blood ratio of ^99mTc-HYNIC-HBP was higher than that of ^99mTc-HMDP.

Palma et al. developed a ^99mTc-tricarbonyl complex, which is anchored by a pyrazolyl (Pz)-containing ligand, conjugated to the bisphosphonate compounds ([^99mTc(CO)₃(PzNN-BP)], [^99mTc(CO)₃(PzNN-ALN)], and [^99mTc(CO)₃(PzNN-PAM)], Figs. 2d–2f) [19, 20]. The ligands N₂S₂ and N₃S have classically been some of the most useful ligands for complexation of ^99mTc complexes. The ^99mTc complexes have a (TcO)³⁺ core with technetium in its +5 oxidation state. In contrast, the ^99mTc-tricarbonyl complex has a [Tc(CO)₃]⁺ core with technetium in its +1 oxidation state. ^99mTc-tricarbonyl complexes, which are compact and kinetically inert, could be formed from the [Tc(CO)₃]⁺ core with suitable ligands [21]. Palma et al. have confirmed the structures of ^99mTc-tricarbonyl-complex-conjugated bisphosphonate compounds by using reversed-phase HPLC analyses. The analyses showed that the compounds had retention times identical to those of the corresponding nonradioactive rhenium (Re) complexes, which revealed the structural analogies because nonradioactive technetium does not exist (⁹⁹Tc is also a radionuclide). In animal experiments [^99mTc(CO)₃(PzNN-BP)] showed moderate bone uptake, but the uptake was lower than that of ^99mTc-MDP. In contrast, the bone accumulations of [^99mTc(CO)₃(PzNN-ALN)] and [^99mTc(CO)₃(PzNN-PAM)] were high and comparable to that of ^99mTc-MDP. At 4 h after injection of tracers, [^99mTc(CO)₃(PzNN-ALN)] and [^99mTc(CO)₃(PzNN-PAM)] showed higher bone/blood and bone/muscle ratios because they had faster clearance than that of ^99mTc-MDP. The differences in bone accumulation among the ^99mTc-tricarbonyl complex-conjugated bisphosphonate compounds could be derived from the existence of a hydroxyl group at the central carbon of the P-C-P structure in bisphosphonate compounds because some authors have described that bisphosphonate compounds containing the hydroxyl group have higher affinity for bone [22–24].

A ^99mTc-tricarbonyl complex-conjugated bisphosphonate that has a structure similar to that of [^99mTc(CO)₃(PzNN-ALN)] but with dipicolylamine (DPA) was developed by de Rosales et al. and used as a ligand for complexation [^99mTc(CO)₃-DPA-alendronate, Fig. 2g] [25]. ^99mTc(CO)₃-DPA-alendronate showed higher affinity for hydroxyapatite than did ^99mTc-MDP in in vitro experiments. In animal experiments, the bone accumulation of ^99mTc(CO)₃-DPA-alendronate was as high as that of ^99mTc-MDP.

As mentioned above, some ^99mTc-complex-conjugated bisphosphonate compounds have shown superior biodistribution of radioactivity as bone imaging agents relative to that of the existing bone scintigraphic agents, ^99mTc-complex, ^99mTc-MDP, and ^99mTc-HMDP. Consequently, the strategy and concept of ^99mTc-complex-conjugated bisphosphonates could be promising for the diagnosis of bone disorders, such as bone metastases.

2.3 Development of novel diagnostic bone-seeking gallium complexes for PET

Presently, ⁶⁸Ga has attracted much attention as a radionuclide for PET because its radiophysical properties are useful clinically, particularly as a ⁶⁸Ge/⁶⁸Ga generator-produced radionuclide with a half-life (T_1/2 ) of 68 min [26]. Its production does not require an expensive cyclotron, and ⁶⁸Ga can be produced on demand in a hospital. Indeed, because the half-life of the parent nuclide ⁶⁸Ge is long (T_1/2 = 270.8 d), the lifespan of the ⁶⁸Ge/⁶⁸Ga generator also must be long.

The above-mentioned concept of a stable complex-conjugated bisphosphonate could also be applicable to ⁶⁸Ga complexes. To develop superior PET tracers for diagnosis of bone disorders, some kinds of radiogallium complex-conjugated carriers for delivery to bone have been reported.

We developed a ⁶⁷Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) complex-conjugated bisphosphonate (⁶⁷Ga-DOTA-Bn-SCN-HBP, Fig. 3a) because DOTA forms a stable complex with gallium [27]. Although the aim was to develop a superior bone-seeking ⁶⁸Ga-labeled agent for PET, ⁶⁷Ga was used in the initial basic study because of its longer half-life (T_1/2 = 78 h). In animal experiments, ⁶⁷Ga-DOTA-Bn-SCN-HBP accumulated rapidly and highly in bone but was rarely observed in tissues other than bone. Consequently, the bone/blood ratio of ⁶⁷Ga-DOTA-Bn-SCN-HBP was comparable to that of ^99mTc-HMDP.

Fig. 3

Chemical structures of ^67/68Ga complex-conjugated bisphosphonate compounds (a) ⁶⁷Ga-DOTA-Bn-SCN-HBP, (b) ⁶⁸Ga-BPAMD, (c) ⁶⁸Ga-NOTA-BP, and (d) ⁶⁷Ga-DOTA-(Asp)_n.

Fellner et al. have reported a human study of ⁶⁸Ga-DOTA-conjugated bisphosphonate (⁶⁸Ga-BPAMD, Fig. 3b) [28]. ⁶⁸Ga-BPAMD showed high uptake in osteoblastic metastatic lesions in a prostate cancer patient. The maximum standardised uptake values (SUV_max) were 77.1 and 62.1 in the 10^th thoracic and L2 vertebra, respectively, for ⁶⁸Ga-BPAMD; the respective values were 39.1 and 39.2 for ¹⁸F-fluoride, which is a typical bone imaging agent for PET. Basic experiments on ⁶⁸Ga-BPAMD using µ-PET and a bone metastasis rat model have been also reported [29]. ⁶⁸Ga-BPAMD showed higher accumulation in metastatic bone lesions than in healthy bone of the same animal (contrast factor = 3.97 ± 1.82).

Furthermore, Suzuki et al. have developed ⁶⁸Ga-1,4,7-triazacyclononane-1,4, 7-triacetic acid (NOTA)-conjugated bisphosphonate (⁶⁸Ga-NOTA-BP, Fig. 3c) [30]. Triazamacrocyclic ligands may be more suitable for gallium complexation because of their high conformational and size selectivity. Actually, it has been reported that NOTA forms highly stable chelates and allows faster incorporation of gallium at lower temperatures than DOTA [31]. In animal experiments using Wistar rats, ⁶⁸Ga-NOTA-BP showed faster clearance and a higher bone/blood ratio than did ^99mTc-MDP and ¹⁸F-fluoride. Moreover, in a PET study using a mouse model of bone metastasis, ⁶⁸Ga-NOTA-BP highly accumulated in osteolytic lesions in the tibia.

Next, we investigated acidic amino acids as carriers of radionuclides to bone. Several major noncollagenous bone proteins, such as osteopontin and bone sialoprotein, contain repeating sequences of acidic amino acids, such as Asp or Glu, in their structures [32–34]. It has been reported that polyglutamic and polyaspartic acids have high affinities for hydroxyapatite and could be used as carriers for drug delivery to bones [35–37]. Recently, we reported ⁶⁷Ga-DOTA-conjugated L-Asp peptides [⁶⁷Ga-DOTA-(Asp)_n, Fig. 3d] with varying peptide lengths (n = 2, 5, 8, 11, or 14) [38]. The binding affinities for hydroxyapatite of ⁶⁷Ga-DOTA-(Asp)_n depended on their peptide lengths; longer peptides had higher affinities for hydroxyapatite. In biodistribution experiments in normal mice, ⁶⁷Ga-DOTA-(Asp)₈, ⁶⁷Ga-DOTA-(Asp)₁₁, and ⁶⁷Ga-DOTA-(Asp)₁₄ showed selectively high accumulation in bones (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively). Although the bone accumulation of ⁶⁷Ga-DOTA-(Asp)_n was lower than that of ⁶⁷Ga-DOTA-Bn-SCN-HBP, which suggested that bisphosphonates have higher affinities for bone than do polyaspartic acids, ⁶⁷Ga-DOTA-(Asp)_n showed faster blood clearance than did ⁶⁷Ga-DOTA-Bn-SCN-HBP. Accordingly, the bone/blood ratios of ⁶⁷Ga-DOTA-(Asp)₁₁ and ⁶⁷Ga-DOTA-(Asp)₁₄ were comparable to that of ⁶⁷Ga-DOTA-Bn-SCN-HBP.

These results suggest that not only bisphosphonate molecules but also acidic amino acid peptide sequences could be useful as carriers of radionuclides to bones. Moreover, the use of radiometal complex-conjugated carrier molecules for delivery to bones could be a useful approach for the development of ⁶⁸Ga PET tracers for bone disorders such as bone metastases.

2.4 Bone-seeking radiopharmaceuticals for palliative therapy of bone metastases

Gamma ray emitting radionuclide-and positron emitting radionuclide-labeled bone-seeking agents are used for the diagnosis of bone disorders such as bone metastases. Bones are one of the most common organs affected by metastatic cancer because of the presence of numerous growth factors in them [39, 40]. Especially, certain cancers, such as breast cancer, lung cancer, and prostate cancer, have a tendency to metastaize to bones. Most cancer patients in late phases of bone metastases suffer severe pain, but the pathophysiology is not well understood, and multiple mechanisms have been postulated. Control of metastatic bone pain is very important for improving patients’ quality of life [41–43]. As palliative treatments, nonsteroidal anti-inflammatory drugs (NSAIDs) are the first option for most patients, followed by progression to opioids as the intensity of pain rises by using a three-step “ladder” proposed by the WHO for cancer pain relief. However, it is difficult to control pain in patients with bone metastases. After the initial standard palliative treatment, about half of these patients continue to suffer from substantial bone pain [44]. Moreover, although efforts are made to decrease the side effects of drugs used for pain palliation, these drugs can produce unwanted side effects, such as gastrointestinal ulceration, neutropenia, enhanced bleeding, and disruptions in renal function in the case of NSAIDs, and nausea, sedation, and constipation in the case of opioids. Localised radiation therapy from an external source is an effective method for palliation of metastatic bone pain [45]. Localised radiation therapy decreases metastatic bone pain in many patients. However, because most patients with bone metastases have multiple metastatic lesion sites, treating patients, bone metastases with multiple metastatic lesions with localised radiation therapy is not easy. In that case, internal radiotherapy using bone-seeking compounds labeled with high-energy beta or alpha particle-emitter radionuclides has been shown to be an effective alternative that has fewer side effects than those associated with other treatments [46]. Pain palliation is achieved from beta or alpha particles emitted from radionuclides, but the mechanism of this pain palliation has not been elucidated. Because beta or alpha particles cause damage to tumour cells, palliation effects might occur because of a reduction of mechanical pressure. However, the palliative effects are usually observed before the tumour mass is reduced. Because it is known that lymphocytes, which are radiation-sensitive cells, secrete many kinds of cytokines related to pain, palliation effects could be derived from not only reduction of tumour cells but also damage to lymphocytes in the tumour tissue.

I will next discuss the approved radiopharmaceuticals and recent research related to radiolabeled compounds for palliative therapy. ⁸⁹SrCl₂ (Metastron®) was the first radiopharmaceutical approved by the FDA for the palliation of metastatic bone pain. Strontium (Sr) and calcium (Ca) are alkaline earth metals that are members of family IIA on the periodic table. It is known that the characteristics of Sr and Ca are similar and that Sr accumulates at sites of high osteoblastic activity through incorporation into mineralizing collagen during new bone formation [47]. ⁸⁹Sr has a long physical half-life of 50.5 d and emits high-energy beta particles, with a maximum energy of 1.46 MeV, and 0.01% gamma rays, with an energy of 910 keV. The usual dose of ⁸⁹SrCl₂ is 148 MBq (4 mCi) or 1.5–2.2 MBq/kg body weight. It has been reported that there is no dose-dependence for pain relief [48]. There is a lot of data on the palliative effects of ⁸⁹SrCl₂ for breast and prostate cancer patients with metastatic bone pain. Review articles have reported pain relief rates ranging from 57% to 92% [49–51]. Palliation effects are usually observed within 6 weeks after intravenous injection of ⁸⁹SrCl₂, and the mean duration of pain relief is approximately 6 months [52].

Samarium-153 (¹⁵³Sm) emits beta particles with maximum energies of 0.81 MeV (20%), 0.71 MeV (49%), and 0.64 MeV (30%) and a 28% abundance of gamma rays with an energy of 103 keV and can be used for imaging, unlike ⁸⁹Sr. ¹⁵³Sm has a physical half-life of 46.3 h. ¹⁵³Sm-EDTMP (Quadramet®), which has also been approved by the FDA, is a complex of ¹⁵³Sm with ethylenediaminetetramethylene phosphonic acid (EDTMP: lexidronam, Fig. 1d), which is a tetraphosphonate chelator with high affinity for bone. Because the biodistribution of ¹⁵³Sm-EDTMP is similar to that of ^99mTc-MDP, the dosimetry of ¹⁵³Sm-EDTMP could be predicted using ^99mTc-MDP bone scintigraphy [53]. The blood clearance of ¹⁵³Sm-EDTMP is very rapid, and the compound is excreted via the kidney into the urine [54]. The blood clearance is applicable to a biexponential model with an estimated half-life of 5.5 ± 1.1 and 65.4 ± 9.6 min [55]. About 50% of the injected dose is excreted into the urine at 6–7 h after injection. Most of the remaining dose is accumulated in bones. There is little accumulation in soft tissue, such as the liver, and less than 1% of the injected dose remains in the blood. The usual dose of ¹⁵³Sm-EDTMP is 37 MBq/kg (1 mCi/kg). Review articles have reported pain relief rates ranging from 62% to 84% [49–51]. When ⁸⁹SrCl₂– and ¹⁵³Sm-EDTMP-treated groups of prostate cancer patients with bone metastases were compared, no statistical differences were observed in response rates and side effects [56].

Radium-223 chloride (²²³RaCl₂), in which ²²³Ra is the alpha particle-emitting radionuclide, was approved by the FDA and European Medicines Agency (EMA) in 2013. Radium (Ra) also is an alkaline earth metal and a member of family IIA in the periodic table, as are Ca and Sr. Among Ra isotopes, ²²³Ra has a suitable half-life of 11.4 d for therapy and decays through a chain of daughter nuclides with an emitted total energy of approximately 28 MeV, with most of the energy released as alpha particles. In a phase III randomised trial (Alpharadin in Symptomatic Prostate Cancer Patients: ALSYMPCA), surprisingly, ²²³RaCl₂ significantly improved overall survival in castration-resistant prostate cancer patients with bone metastases [57, 58]. Moreover, ²²³RaCl₂ is associated with a low incidence of myelosuppression, which is assumed to be the major side effect and could be the dose-limiting factor. ²²³RaCl₂ is the first alpha particle-emitting radiopharmaceutical approved for clinical use and has been demonstrated to be an effective therapy for bone metastases; therefore, it is currently attracting much attention.

2.5 Development of novel bone-seeking complexes for palliation therapy of bone metastases

Rhenium has chemical properties similar to those of technetium because both elements are members of family VIIA in the periodic table. Of the rhenium isotopes, there are two radionuclides, ¹⁸⁶Re and ¹⁸⁸Re, that are useful for radionuclide therapy [59]. Both rhenium radionuclides emit not only high-energy beta particles for therapy but also gamma rays for imaging: ¹⁸⁶Re (T_1/2 = 3.7 d, β^–_max = 1.07 MeV, γ = 137 keV) and ¹⁸⁸Re (T_1/2 = 17.0 h, β^–_max = 2.12 MeV, γ = 155 keV). Furthermore, because ¹⁸⁸Re is a daughter nuclide of ¹⁸⁸W (T_1/2 = 60 d), ¹⁸⁸Re is obtained from an in-house alumina-based ¹⁸⁸W/¹⁸⁸Re generator, similar to a ⁹⁹Mo/^99mTc generator [60].

In the case of the use of rhenium radionuclides for bone-seeking radiophar-maceuticals in a manner similar to that of ^99mTc-MDP and ^99mTc-HMDP, it has been reported that rhenium forms complexes with some bisphosphonate derivatives as ligands. ^186/188Re-1-hydroxyethylidene-1, 1-diphosphonate (^186/188Re-HEDP), which is a ^186/188Re-complex with HEDP (Fig. 1e), has been evaluated in clinical research [61–63]. Although the chemical characteristics of rhenium and technetium are similar, rhenium is more easily oxidised than is technetium [64], and the stability of ¹⁸⁶Re-HEDP is lower than that of ^99mTc-bisphosphonate complexes [65]. Some studies have shown that gastric accumulation of radioactivity was observed after injection of ¹⁸⁶Re-HEDP in patients with bone metastases [66, 67]. It is known that ^186/188ReO₄^–accumulates in the stomach, as does ^99mTcO₄^–, and it is thought that this occurs because of the in vivo instability of ¹⁸⁶Re-HEDP [68]. Moreover, similar to the phosphonate groups in ^99mTc-MDP and ^99mTc-HMDP, those in ^186/188Re-HEDP are used as both a ligand for complexation and a carrier to bone, which may reduce the inherent affinity of HEDP to bones.

To develop superior ^186/188Re-labeled bone-seeking radiopharmaceuticals, I assumed that the above-mentioned concept of a stable complex-conjugated bisphosphonate would be more useful. Therefore, our research group designed and evaluated ¹⁸⁶Re-monoaminemonoamidedithiol (MAMA)- and ¹⁸⁶Re-mercaptoacetylglycylglycylglycine (MAG3)-conjugated bisphosphonate compounds (¹⁸⁶Re-MAMA-BP, ¹⁸⁶Re-MAMA-HBP, ¹⁸⁶Re-MAG3-HBP; Figs. 4a–c) and reported our findings [24, 69–72]. When ¹⁸⁶Re-complexes were incubated in buffered solution at 37°C, the Re-complex-conjugated bisphosphonate compounds, ¹⁸⁶Re-MAMA-HBP, ¹⁸⁶Re-MAMA-BP, and ¹⁸⁶Re-MAG3-HBP, were more stable than ¹⁸⁶Re-HEDP, as expected. In biodistribution experiments in mice, little gastric accumulation of radioactivity was observed after injection of ¹⁸⁶Re-MAMA-HBP, ¹⁸⁶Re-MAMA-BP, and ¹⁸⁶Re-MAG3-HBP. The drug design of Re-complex-conjugated bisphosphonates led to better stability in vitro and in vivo. Among these ¹⁸⁶Re-complex-conjugated bisphosphonate compounds, ¹⁸⁶Re-MAG3-HBP showed the most favourable biodistribution characteristics as a bone-seeking agent, such as high and selective bone accumulation. These favourable biodistribution characteristics could be related to high hydrophilicity (log P value:, –2.68 ± 0.01) and the introduction of a hydroxyl group to the central carbon of the bisphosphonate P-C-P structure. The therapeutic potential of ¹⁸⁶Re-MAG3-HBP for palliation of metastatic bone pain in a rat model of bone metastasis was evaluated. Compared with untreated control group rats, ¹⁸⁶Re-MAG3-HBP-treated rats showed significant palliation effects, as assessed by the hind paw withdrawal response to stimulation with von Frey filaments [73], and the palliation effects of ¹⁸⁶Re-MAG3-HBP tended to be higher than those of ¹⁸⁶Re-HEDP. Although ¹⁸⁶Re-HEDP did not inhibit tumour growth, ¹⁸⁶Re-MAG3-HBP significantly inhibited tumour growth.

Because one of the ^99mTc-complex-conjugated bisphosphonate compounds, ^99mTc(CO)₃-DPA-alendronate, has been introduced above, the same ligand was used to design and evaluate ¹⁸⁸Re(CO)₃-DPA-alendronate by the same group (Fig. 4d) [74]. ¹⁸⁸Re(CO)₃-DPA-alendronate also showed higher in vitro stability than did ¹⁸⁸Re-HEDP, which oxidised to ¹⁸⁸ReO₄– (up to 75%) after incubation in PBS for 48 h at 37°C. In imaging experiments, ¹⁸⁸Re(CO)₃-DPA-alendronate showed superior biodistribution of radioactivity than did ¹⁸⁸Re-HEDP, i.e. ¹⁸⁸Re(CO)₃-DPA-alendronate highly accumulated in metabolically active bone, such as joints with low soft-tissue uptake.

Fig. 4

Chemical structures of ^186/188Re complex-conjugated bisphosphonate compounds (a) ¹⁸⁶Re-MAMA-BP, (b) ¹⁸⁶Re-MAMA-HBP, (c) ¹⁸⁶Re-MAG3-HBP, and (d) ¹⁸⁸Re(CO)₃-DPA-alendronate.

These results indicate that the concept of stable ¹⁸⁶Re-complex-conjugated bisphosphonates could be very useful and that novel ¹⁸⁶Re-complex-conjugated bisphosphonate complexes could be attractive candidates as palliative agents in metastatic bone pain.

3.1 Technetium-labeled annexin A5

Phosphatidylserine (PS) exists on the intracellular face of the cell membranes of normal cells. When apoptosis occurs, the lipid distribution of the plasma membrane changes so that PS is exposed to the outside of the cell membrane. Therefore, PS could be a target for imaging of apoptosis, and the typical compound of PS-targeted carriers is annexin A5, which is a 36-kDa human protein with a nanomolar affinity for membrane-bound PS [75–77]. For imaging of apoptosis, many researchers have developed and evaluated radiolabeled annexin A5 compounds.

3.2 ^99mTc-4, 5-bis(thioacetamido)pentanoyl-annexin A5 (^099mTc-BTAP-annexin A5)

As mentioned above, ^99mTc is an ideal radionuclide for clinical use because of its physical properties. However, because most proteins and polypeptides do not possess chelation sites for formation of technetium complexes, a ligand for complexation with technetium should be introduced into proteins and polypeptides. Tetradentate chelators, such as N₃S and N₂S₂ coordination molecules, could form stable square pyramidal ^99mTc complexes with a [Tc=O]³⁺ core in which technetium is in the oxidation state +V. Among the ^99mTc-labeled annexin A5 compounds for imaging of apoptosis, ^99mTc-4, 5-bis(thioacetamido)pentanoyl (BTAP)-annexin A5 (Fig. 5a), which has a N₂S₂ ligand for complexation with ^99mTc, was reported to be the first ^99mTc-labeled annexin A5 compound. ^99mTc-BTAP-annexin A5 was used as an imaging agent for transplant rejection of cardiac transplantation by Narula et al. When ^99mTc-BTAP-annexin A5 was injected into cardiac allograft recipients, ^99mTc-BTAP-annexin A5 accumulated in the hearts of some recipients who showed at least moderate transplant rejection. These results suggested that ^99mTc-labeled annexin A5 was useful for noninvasive imaging of apoptosis [78]. However, N₂S₂ ligands, such as BTAP, should require harsh conditions for preparation of ^99mTc complexes because the protecting groups for thiol must be deprotected just before radiolabeling. Thus, ^99mTc-BTAP-annexin A5 was prepared by a preformed-chelate approach. The preformed-chelate approach means that the conjugation between the ^99mTc-BTAP complex and annexin A5 is performed after the complexation of ^99mTc with the BTAP ligand. The preformed-chelate approach is complicated and has low radiochemical yields because multiple steps and purification are needed. Easy ^99mTc labeling of annexin A5 is required for routine clinical use.

Fig. 5

Structures of (a) ^99mTc-BTAP-annexin A5, (b) ^99mTc-HYNIC-annexin A5, and (c) ^99mTc-C₃(BHam)₂-annexin A5.

3.3 ^99mTc-HYNIC-annexin A5

In 1998, Blankenberg et al. reported ^99mTc-HYNIC-annexin A5 (Fig. 5b), for imaging of apoptosis [79]. HYNIC is one of the most familiar ligands for labeling of peptides and proteins with ^99mTc. HYNIC forms a mixed ligand complex with ^99mTc and the proper coligands. In the complex, it has been reported that HYNIC works as a monodentate or bidentate ligand [80]. Several coligands, such as glucoheptonate, tricine, ethylene diamine diacetic acid (EDDA), and ternary ligand systems containing tricine and water-soluble phosphines or tricine and imine-N-containing heterocycles have been reported. Among these coligands, (tricine)₂ has been frequently used for ^99mTc labeling of proteins because the ^99mTc-(HYNIC)(tricine)₂ complex can be prepared under mild conditions with high radiochemical yields in a short reaction time; specifically, ^99mTc-(HYNIC) (tricine)₂ has been obtained without any purification after a one-step reaction [81, 82]. Presently, ^99mTc-HYNIC-annexin A5 with (tricine)₂ as coligands is the gold standard among agents used for imaging of apoptosis in nuclear medicine. Because easy labeling has helped researchers with imaging of apoptosis in studies using ^99mTc-HYNIC-annexin A5, many of these studies have been conducted in the clinic and as basic research [83]. However, ^99mTc-HYNIC-annexin A5 has some disadvantages because of the instability of the ^99mTc-(HYNIC)(tricine)₂ complex; in particular, ^99mTc-HYNIC-annexin A5 shows high uptake and long retention in nontarget tissues, such as the kidney and liver [84].

In basic research, accumulation of ^99mTc-HYNIC-annexin A5 in apoptosis-induced Jrukat T-cell lymphoblasts has shown a correlation with the percentage of FITC-labeled annexin A5 labeled cells (r² = 0.922) [85]. Moreover, many researchers have demonstrated that ^99mTc-HYNIC-annexin A5 has high affinity for apoptotic cells and can enable visualisation of apoptosis in various animal models [84, 86–88].

In a clinical study in 2007, ^99mTc-HYNIC-annexin A5 imaging before and after initiation of platinum anticancer agent-based chemotherapy in non-small-cell lung cancer patients was reported. All patients who showed increased uptake of ^99mTc-HYNIC-annexin A5 in tumours achieved complete or partial responses. The uptake of ^99mTc-HYNIC-annexin A5 by tumours correlated with treatment outcome (r² = 0.86; P = 0.0001) [89].

3.4 ^99mTc-labeled annexin A5 constructed with histidine residues

In 2002, preparation of mutant annexin A5 with N-terminal extensions constructed with three or six histidine residues for 99mTc labeling was reported [90]. A mutant annexin A5 with six histidine residues in the N-terminal showed higher radiochemical yield, radiochemical purity, and bioactivity than did the mutant annexin A5 with three histidine residues in the N-terminal. This study demonstrated that it was possible to successfully construct annexin A5 with histidine residues in the N-terminal to form specific chelation sites for the 99mTc-carbonyl complex without altering its high affinity.

3.5 ^99mTc-C₃(BHam)₂-annexin A5

A novel ^99mTc-labeled annexin A5, ^99mTc-C₃(BHam)₂-annexin A5, which has a bis(hydroxamamide) derivative as a bifunctional chelating agent to achieve low uptake and retention in nontarget tissues, was recently reported by my research group (Fig. 5c) [91]. It has been reported that a bis(hydroxamamide) derivative, N, N’-trimethylenedibenzohydroxamide ligand [C₃(BHam)₂] forms a stable ^99mTc complex over a wide pH range under mild reaction conditions within short reaction times [92]. The radiochemical yield is very high even at ligand concentrations as low as 2.5 × 10 ⁶ M. In mice, the radioactivity in liver was not residual after intravenous injection of ^99mTc-C₃(BHam)₂-IgG [93], which indicated that the radiometabolite of ^99mTc-C₃(BHam)₂-IgG could be not residual in metabolic organs. In the case of annexin A5, I assumed that there would be less residual radioactivity in metabolic organs after injection of ^99mTc-C₃(BHam)₂-annexin A5 and therefore, designed ^99mTc-C₃(BHam)₂-annexin A5. The bioactivity of ^99mTc-C₃(BHam)₂-annexin A5 was comparable to that of ^99mTc-HYNIC-annexin A5. In mouse biodistribution, the uptake by the kidney was lower for ^99mTc-C₃(BHam)₂-annexin A5 than for ^99mTc-HYNIC-annexin A5. Moreover, the radioactivity of metabolic organs, such as the liver and kidney, after injection of ^99mTc-C₃(BHam)₂-annexin A5 gradually decreased, whereas there was residual radioactivity in metabolic organs after the injection of ^99mTc-HYNIC-annexin A5. In therapeutic experiments, tumour growth in mice treated with 5-fluorouracil (5-FU) was significantly inhibited. Accumulation of ^99mTc-C₃(BHam)₂-annexin A5 in tumours significantly increased at 24 h after 5-FU treatment. The accumulation of ^99mTc-C₃(BHam)₂-annexin A5 correlated positively with the counts of terminal dUTP nick-end labeling (TUNEL)-positive cells. Moreover, the intratu-moral accumulations of ^99mTc-C₃(BHam)₂-annexin A5 by autoradiography significantly correlated with TUNEL-staining positive cells in corresponding sections (r = 0.716, P < 0.001, Fig. 6).

Fig. 6

Representative (a) autoradiographic images and (b, c) TUNEL-staining images for adjacent tumour sections from mice treated with 5-FU. (d) Correlation between the number of TUNEL-positive cells in each grid (0.45 mm × 0.55 mm) of a tumoural section and ^99mTc-C₃(BHam)₂-annexin A5 accumulation (%dose) determined by autoradiography in each corresponding grid of an adjacent section from mice treated with 5-FU.

3.6 ⁶⁸Ga-labeled annexin A5

A protein labeling system using ⁶⁸Ga and a sulfhydryl-derivatised chelator, 2, 2′-(7-(1-carboxy-4-(2-mercaptoethylamino)-4-oxobutyl)-1, 4,7-triazonane-1, 4-diyl)diacetic acid (NODA-GA-T), was reported in 2011 [94]. In that study, ⁶⁸Ga-labeled annexin A5 was prepared within only 15 min and accumulated in the apoptotic area of myocardial infarctions in a PET study using an animal model. In the same year, site-specific ⁶⁸Ga-labeled annexin A5 compounds, which are variants of annexin A5 containing a single cysteine residue at a position of 2 or 165 (Cys2-annexin A5 and Cys165-annexin A5, respectively), have been reported [95]. ⁶⁸Ga-Cys2-annexin A5 and ⁶⁸Ga-Cys165-annexin A5 were prepared within approximately 55 min with a 25% radiochemical yield (43% if corrected for decay). Both ⁶⁸Ga-labeled annexin A5 compounds preserved bioactivity and showed high in vitro stability. The uptakes of ⁶⁸Ga-Cys2-annexin A5 and ⁶⁸Ga-Cys165-annexin A5 by tumours were not high but were significantly increased by the treatment of cyclophosphamide and radiation therapy in a tumour model.

Radiolabeled annexin A5 seems to require some time before imaging of apoptosis after injection of radiotracers can be performed. Accordingly, although ⁶⁸Ga is a promising radionuclide for PET imaging, the half-life of ⁶⁸Ga may be too short if annexin A5 is used as a carrier to apoptotic cells.

3.7 Summary of radiolabeled annexin A5 for imaging of apoptosis

Quantitative imaging agents for apoptosis are useful tools because imaging enables early determination of therapeutic effects on diseases, even before anatomical changes occur at the lesion site. Selection of an appropriate therapy by using imaging data from an individual patient may be possible. Although some radiolabeled-annexin A5 compounds are in use, their biodistributions are not necessarily ideal. I hope that novel apoptosis imaging agents using other carriers to apoptotic cells that enable superior imaging to that currently available will be developed in future.