Advances in Drug Design of Radiometal-Based Imaging Agents for Bone Disorders

Nuclear medicine bone imaging has been the optimum diagnosis for the detection of bone disorders because the lesion could be detectable before the appearance of symptomatic and radiographic changes. Over the past three decades, 99mTc-MDP and 99mTc-HMDP have been used as bone scintigraphic agents because of their superior biodistribution characteristics, although they are far from optimal from a chemical and pharmaceutical point of view. Recently, a more logical drug design has been proposed as a concept of bifunctional radiopharmaceuticals in which the carrier molecules (bisphosphonates) and radiometal chelating groups are separated within a molecule, specifically, 99mTc-mononuclear complex-conjugated bisphosphonate. Some of the 99mTc-mononuclear complex-conjugated bisphosphonate compounds showed superior biodistribution in preclinical studies. Moreover, the drug design concept could be applied to 68Ga PET bone imaging agents. These studies would provide useful information for the development of radiometal-based imaging and therapeutic agents for bone disorders such as bone metastases.


Background
The skeleton is one of the most common organs to be affected by metastatic cancer. Carcinomas of the breast, lung, prostate, kidney, and thyroid have a tendency to easily metastasize to bone [1]. Although there has been significant advancement in imaging technologies, such as CT and MR, nuclear medicine bone imaging has been the optimum diagnosis for the detection of bone disorders, such as bone metastases, because of its high sensitivity. Namely, bone-seeking radiopharmaceuticals usually localize in skeletal lesions before the appearance of symptomatic and radiographic changes and the resulting easy evaluation of the entire skeleton [2]. This paper reviews currently available 99m Tc radiopharmaceuticals for bone scintigraphy and advances in drug design of radiometal-based bone-targeted compounds. sequence instead of the P-O-P sequence of pyrophosphate. Although these two chemical structures are similar, the P-C-P bond angles are smaller (117 degrees) than the P-O-P bond angles (128.7 degrees) and the P-C interatomic distance (1.79Å) is longer than that of P-O (1.63Å) [9]. Bisphosphonate bonding is very stable chemically and affords greater resistance to in vivo phosphatase hydrolysis. As a result, 99m Tc-HEDP exhibited more rapid clearance from blood and higher uptake in bone. In clinical study, 99m Tc-HEDP showed significantly higher lesion-to-normal bone ratios when compared with either 99m Tc-pyrophosphate or 99m Tc-polyphosphate [10].
After introduction of 99m Tc-methylene diphosphonate (MDP, Figure 1(c)) by Subramanian et al. [11] in 1975 and 99m Tc-hydroxymethylene diphosphonate (HMDP, Figure 1(d)) by Bevan et al. [12] in 1980, 99m Tc-MDP, and 99m Tc-HMDP, which showed superior biodistribution compared to 99m Tc-HEDP, have been used as radiopharmaceuticals for bone scintigraphy for over thirty years [13][14][15]. 99m Tc-MDP is postulated to form a bidentate-bidentate bridge with hydroxyapatite while the presence of the hydroxyl group in 99m Tc-HMDP would convert the ligand to form a bidentate-tridentate bridge and is expected to enhance the hydroxyapatite affinity of the 99m Tc complex [12,16]. However, the lower bone accumulation of 99m Tc-HEDP is not well understood because 99m Tc-HEDP should also form bidentate-tridentate binding. Increasing steric hindrance associated with the methyl group at the central carbon atom of HEDP, the difference in solubility, and differences in molecular size as well as in the bisphosphonate polymeric complexes themselves have all been suggested [12,17].
The accumulation of 99m Tc-bisphoshonate complexes in bone must be derived from the coordination of bisphosphonate to calcium in the hydroxyapatite of bone, but the mechanism of high uptake to lesion sites in bone has not been completely elucidated. One factor should be the increased vascularity and regional distribution of blood flow that results from disease. However, it has been shown that regional bone blood flow alone does not account for the increased uptake of radiopharmaceuticals [18]. Other factors are involved in their binding and interaction with bone. It is generally assumed that 99m Tc-bisphoshonate complexes accumulate at sites of active bone metabolism, that is to say, at areas of new bone formation or calcification [19,20]. It has also been reported that the accumulation mechanisms might be both adsorption onto the surface of hydroxyapatite in bone and incorporation into the crystalline structure of hydroxyapatite [21]. Newly formed bone has a much larger surface area than does stable bone. That is, the crystalline structure of hydroxyapatite in newly formed bone is amorphous and has a greater surface area than that in normal bone [22]. An in vitro study demonstrated that bisphosphonate compounds have significantly higher adsorption on amorphous calcium phosphate than on crystalline calcium phosphate [17].
Bisphosphonate compounds form multiple complexes with reduced 99m Tc. By using high-performance liquid chromatography (HPLC), the relative composition of 99m Tc-bisphosphonate complexes in a reaction mixture has been found to vary with pH and with technetium, and with oxygen concentrations [23]. It has been postulated that 99m Tc-bisphosphonate complexes would be a mixture of monomers, oxo-bridged dimers, and oligomeric clusters with varying technetium-oxo core configurations, oxidation states, and ligand coordination numbers [24]. These radiolabeled species have different biodistribution properties. It was reported that the smallest, low-charged, mononuclear 99m Tc-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 [25]. Thus, the exact structures and mechanisms of the action of 99m Tc-labeled bisphosphonate remain uncertain.

New Drug Design Concept of 99m Tc-Labeled Bisphosphonate ( 99m Tc Complex-Conjugated Bisphosphonate Compounds)
As mentioned above, despite over three decades of clinical use of 99m Tc-bisphosphonate complexes, these radiopharmaceuticals are far from optimal from a chemical and pharmaceutical point of view. For example, their structures and compositions remain unknown because they cannot be obtained as a well-defined single-chemical species, but as mixtures of short-chain and long-chain oligomers. The biological behavior of this type of tracer is also affected by the different degrees of ionization and by variations in the relative amount of oligomers after preparation [23]. In addition, in clinical studies, an interval of 2 to 6 hours is required between an injection of 99m Tc-labeled bisphosphonates and obtaining bone images [15]. Shortening this interval would lessen the burden to patients in terms of total examination length and radiation dose absorbed. To enable imaging at an earlier time after injection, a radiopharmaceutical with higher affinity for bone might be advantageous. Although the accumulation of bisphosphonate compounds in bone is achieved by binding the phosphonate groups with the Ca 2+ of hydroxyapatite crystals [26], the phosphonate groups in 99m Tc-MDP and 99m Tc-HMDP serve as both coordinating ligands and Ca 2+ binding functional groups [27], which might decrease the inherent accumulation of MDP and HMDP in bone.
Recently, to improve the 99m Tc-labeled bisphosphonates currently used, a more logical drug design has been proposed based on the concept of bifunctional radiopharmaceuticals in which the carrier molecules (bisphosphonate) and radiometal chelating groups are separated within the molecule so that they can each function independently and effectively.
In particular, 99m Tc-mononuclear complex-conjugated bisphosphonate compounds have been reported [28][29][30][31]. It was hypothesized that the bone affinity of 99m Tc labeled bisphosphonate would be enhanced by conjugating a stable mononuclear 99m Tc chelating group with a bisphosphonate moiety so that the conjugation does not impair the inherent chemical and biological properties of the bisphosphonate compounds. 99m Tc-L,L-ethylene dicysteine (EC), 99m Tc-mercaptoacetylglycylglycylglycine (MAG3), 99m Tc-6-hydrazinonicotinic acid (HYNIC), 99m Tc-tricarbonyl anchored by pyrazolyl-(pz-) containing ligand, and 99m Tc-tricarbonyl dipicolylamine (DPA) were selected as 99m Tc chelating molecules, and were conjugated with bisphosphonate compounds, ( 99m Tc-ECAMDP, 99m Tc-MAG3-HBP, 99m Tc-HYNIC-HBP, [ 99m Tc(CO) 3 (κ 3 -pz-BPOH)] + , and 99m Tc(CO) 3 -DPA-alendronate, resp., Figure 2). In the drug design of the 99m Tc-mononuclear complexconjugated bisphosphonate compounds, since these ligands contain a bisphosphonate site, there is a possibility that 99m Tc coordinates not with the proposed metal coordination moiety, such as EC, MAG3, and HYNIC but with the bisphosphonate moiety. To ascertain whether 99m Tc is chelated with only the proposed metal coordination moiety, some experiments were performed. For example, in the case of 99m Tc-HYNIC-HBP, 99m Tc-HYNIC-HBP was also prepared by the coupling of 99m Tc-HYNIC previously complexed with the bisphosphonate site (prelabel method). RP-HPLC analysis revealed the 99m Tc-HYNIC-HBP by the prelabel method to be identical to that obtained from the labeling of HYNIC-HBP with 99m Tc. These findings exclude the possibility of complexation between technetium and the bisphosphonate structure, and indicate the chelation of 99m Tc with the HYNIC moiety in HYNIC-HBP.
In these new compounds, 99m Tc-MAG3-HBP, 99m Tc-HYNIC-HBP, and 99m Tc(CO) 3 -DPA-alendronate were investigated for in vitro hydroxyapatite binding as an index of bone affinity. 99m Tc-MAG3-HBP and 99m Tc-HYNIC-HBP showed a significantly higher rate of binding to hydroxyapatite than did 99m Tc-HMDP. 99m Tc(CO) 3 -DPA-alendronate showed a higher affinity to hydroxyapatite than did 99m Tc-MDP. At the same time, all new 99m Tc-mononuclear complex-conjugated bisphosphonate compounds exhibited high bone uptake in in vivo animal experiments. 99m Tc-EC-AMDP and 99m Tc-HYNIC-HBP showed especially superior results; 99m Tc-EC-AMDP and 99m Tc-HYNIC-HBP showed significantly higher bone-to-blood ratios of radioactivity than did 99m Tc-MDP and 99m Tc-HMDP.

Radiogallium-Labeled Compounds as
Bone Imaging Agents for PET 68 Ga is one of the greatest practical and interesting radionuclides for clinical positron emission tomography (PET) because of its radiophysical properties (T 1/2 = 68 min) [32]. 68 Ga is a generator-produced nuclide and can be eluted at any time on demand. Specifically, it does not require an on-site cyclotron. In principle, the long half-life of the parent nuclide 68 Ge (T 1/2 = 270.8 days) provides a long life-span generator.
Investigations of 68 Ga-labeled compounds for bone imaging were previously reported in the 1970s [33,34]. In these reports, gallium was labeled with tripolyphosphate or ethylenediamine tetramethylene phosphonate (EDTMP) or diethylenetriamine pentamethylene phosphonate (DTPMP). These complexes showed high uptakes in bone. However, since use of the PET camera generally did not spread in the 1970s and the quality of PET cameras was not high, the attention given to 68 Ga PET imaging agents was not so high.
For the last decade, 68 Ga as a nuclide has been considered a useful radionuclide for PET imaging. Thus, many 68 Galabeled compounds have been developed. Recently, 68 Ga-EDTMP was also reevaluated by Mitterhauser et al. [35]. However, they stated that the advantage of 68 Ga-EDTMP over [ 18 F]-fluoride was not apparent and that the future clinical prospect of 68 Ga-EDTMP remained speculative.
The above-mentioned drug concept of stable mononuclear complex-conjugated bisphosphonate could be applicable to not only technetium complex radiopharmaceuticals but also to gallium radiopharmaceuticals. To develop a new PET tracer with radiogallium for imaging bone disorders such as bone metastases, 1,4,7,10-tetraazacyclododecane-1,4, 7,10-tetraacetic acid (DOTA) was chosen as a chelating site because it has been well known that Ga forms a stable complex with DOTA. Therefore, Ga-DOTA-conjugated bisphosphonate compounds ( 67 Ga-DOTA-Bn-SCN-HBP and 68 Ga-BPAMD, Figure 3) have been developed [36,37]. Actually, in biodistribution experiments, 67 Ga-DOTA-Bn-SCN-HBP rapidly accumulated in bone but was rarely observed in tissues other than bone. In addition, PET/CT imaging of bone metastases with 68 Ga-BPAMD showed high uptake in osteoblastic metastases of human (Figure 4). The maximal standardized uptake was 77.1 and 62.1 in the 10th thoracic and L2 vertebra versus 39.1 and 39.2 for 18 F-fluoride PET, respectively. These results suggest that the drug design concept of radiogallium complex-conjugated bisphosphonate could be useful for the development of 68 Ga PET imaging agents for bone disorders such as bone metastases. 18 F-fluoride was initially reported by Blau et al. in 1962 [38]. After the development of 99m Tc-labeled bone scintigraphy agents, such as 99m Tc-MDP, 18 F-fluoride was replaced by them because the physical characteristics of 99m Tc were more convenient for imaging with conventional gamma cameras in those days. However, in the last decade, PET and PET/CT have evolved significantly and become widespread. The situation has similarly changed for 68 Ga-labeled compounds. The changes caused the reemergence of 18 F-fluoride bone imaging with PET because current PET cameras have higher spatial resolution and greater sensitivity than conventional gamma cameras.

18 F-Fluoride as Bone Imaging Agent for PET
Like 99m Tc-MDP as mentioned above, it is known that the distribution of 18 F-fluoride in bone also reflects both blood flow in bone and osteoblastic activity. Once 18 F-fluoride reaches the surface of the newly formed hydroxyapatite crystals, fluoride anions are isomorphously exchanged with the hydroxyl group in hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) and fluorapatite (Ca 10 (PO 4 ) 6 F 2 ) is formed [39]. A previous paper reported that electron probe X-ray fluorescence studies on the topographical distribution of fluoride at the microscopic level in the iliac bone of an osteoporotic patient being treated with fluoride [40]. The results indicate that the distribution of 18 F-fluoride in newly mineralized bone is similar to that of 99m Tc-MDP.
There is an important difference between 18 F-fluoride and 99m Tc-MDP in terms of their protein binding rates. 18 Ffluoride barely binds to serum protein [41] whereas 99m Tc-MDP shows significant protein bindings. The difference in protein binding causes a difference in blood clearance between 18 F-fluoride and 99m Tc-MDP. Hence, an interval of 2-3 hours is needed between an injection of 99m Tc-MDP and bone imaging. In contrast, bone imaging can be performed less than 1 hour after an injection of 18 F-fluoride. Another difference between 18 F-fluoride and 99m Tc-MDP is in terms of uptake to blood cells. 18 F-fluoride is taken up by red blood cells. The erythrocyte concentration of 18 F-fluoride is approximately 45-50% of the plasma concentration, namely, approximately 30% of total blood concentration. However, 18 F-fluoride is freely diffusible from red blood cells to the bone surface, so the uptake of 18 F-fluoride to red blood cells should not interfere with the accumulation of 18 F-fluoride in bone [41].
In clinical use in oncology, some papers reported on a comparison between 18 F-fluoride PET and planar 99m Tc-MDP scintigraphy in the detection of bone metastases [42,43]. 18 F-fluoride PET was more sensitive in detecting bone metastases than planar 99m Tc-MDP scintigraphy. However, the cause of increased sensitivity-whether it was derived from 18 F-fluoride itself as a tracer or because of the improved performance of the PET camera-could not be determined.
Specifically, 18 F-fluoride PET has two important advantages in diagnosis imaging over planar 99m Tc-MDP scintigraphy: superior sensitivity and a shorter interval between injection of a tracer and bone imaging. Thus, the use of 18 Ffluoride could become common in the future.

Conclusion
Over the past three decades, 99m Tc-MDP and 99m Tc-HMDP have been used for detecting bone metastases, although their mechanisms of accumulation remain uncertain. Recent efforts of chelate-conjugated bisphosphonates and their derivatives have provided chemically well-characterized new 99m Tc-labeled bone-seeking tracers. Furthermore, the drug design concept could be applied to 68 Ga PET bone imaging agents. These studies would provide useful information for the development of radiometal-based imaging and therapeutic agents for bone disorders such as bone metastases.