Novel Radiolabeled Bisphosphonates for PET Diagnosis and Endoradiotherapy of Bone Metastases

Bone metastases, often a consequence of breast, prostate, and lung carcinomas, are characterized by an increased bone turnover, which can be visualized by positron emission tomography (PET), as well as single-photon emission computed tomography (SPECT). Bisphosphonate complexes of 99mTc are predominantly used as SPECT tracers. In contrast to SPECT, PET offers a higher spatial resolution and, owing to the 68Ge/68Ga generator, an analog to the established 99mTc generator exists. Complexation of Ga(III) requires the use of chelators. Therefore, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclododecane-1,4,7-triacetic acid), and their derivatives, are often used. The combination of these macrocyclic chelators and bisphosphonates is currently studied worldwide. The use of DOTA offers the possibility of a therapeutic application by complexing the β-emitter 177Lu. This overview describes the possibility of diagnosing bone metastases using [68Ga]Ga-BPAMD (68Ga-labeled (4-{[bis-(phosphonomethyl))carbamoyl]methyl}-7,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid) as well as the successful application of [177Lu]Lu-BPAMD for therapy and the development of new diagnostic and therapeutic tools based on this structure. Improvements concerning both the chelator and the bisphosphonate structure are illustrated providing new 68Ga- and 177Lu-labeled bisphosphonates offering improved pharmacological properties.

The development of radiometal-labeled bisphosphonate-based tracers requires the use of chelators for complexation of trivalent metals. Many research groups across the world are currently undertaking research into complexing bisphosphonate compounds to radionuclides using macrocyclic chelators and aim at identifying a labeled product that has high affinity for bone and offers a high thermodynamic and kinetic stability. For the complexation of Ga(III), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclododecane-1,4,7triacetic acid), as well as their derivatives, are commonly used.
For the treatment of disseminated bone metastases, there are two classes of therapeutic bone-seeking radiopharmaceuticals: calcimimetic-and phosphonate-based radiopharmaceuticals. The simplest bone binding radiopharmaceuticals for palliative endoradiotherapy, belonging to the class of calcium mimetics, for example, 89 Sr, 32 P, and 223 Ra. Their localization underlies the same mechanisms as calcium and, therefore, may be unpredictable [9].
Due to the short range of the α-rays emitted by 223 Ra, an impairment of the red bone marrow can be avoided, while allowing deposition of high-energy doses into the target tissue. The first successful clinical phase III studies showed a low haemotoxicity and prolonged survival in metastatic prostate cancer [10]. However, the consequences of the 223 Ra decay chain for the body, as well as the influence of the α-rays on the sensitive gastrointestinal tract, remain uncertain [9]. The longer half-lives of nuclides such as 89 Sr and 32 P have discouraged their use and have favored nuclides such as 153 Sm and 177 Lu with shorter half-lives and lower bone marrow toxicity. The use of 177 Lu is particularly promising due to its suitable decay properties (t 1/2 = 6.71 d, β − = 89%, E βmax = 0.5 MeV) and its carrier-free production route [11]. These trivalent nuclides reach regions of increased bone turnover in the form of complexes with phosphonate-containing chelators, like EDTMP (ethylenediamine tetra(methylene phosphonic acid)) ( Figure 1). These phosphonate-containing chelators exhibit high thermodynamic stabilities with trivalent nuclides, the acyclic ligands, however, possess lower kinetic stabilities [12]. Nevertheless, radiopharmaceuticals based on phosphonates like EDTMP and HEDP (1,1-hydroxyethylidene diphosphonate) (Figure ) show good results in palliative therapy of painful bone metastases in combination with 153 Sm, 177 Lu, 186 Re, and 188 Re [13]. 188 Re has also been shown to have good properties as a therapeutic nuclide due to its appropriate decay characteristics (t 1/2 = 0.7 d, E βmax = 2.12 MeV) and its generator-based production [14].
However, EDTMP complexes have shown low in vivo stability and an excess of the ligand is routinely applied in order to avoid decomplexation in vivo (>1.5 mg/kg body weight of EDTMP vs. approximately 0.05-0.250 mg BPAMD (4-{[bis-(phosphonomethyl))carbamoyl]methyl}-7, 10-bis(car-boxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid)) [12,15]. This excess may lead to a blocking of the biological target which could reduce the radiotracer uptake. Furthermore, high amounts of 152 Sm due to the production route of 153 Sm can cause a reduction of the dose rate deposited Pharmaceuticals 2017, 10, 45 3 of 12 on osseous metastases and, therefore, a lower therapeutic efficiency [16,17]. Using 177 Lu instead of 153 Sm increases the specific activity due to the carrier-free production [17], but the low kinetic stability of EDTMP complexes remains problematic. This review describes the concept of macrocyclic chelate-conjugated bisphosphonates, which are able to circumvent the disadvantages of open-chain chelators, and possible improvements concerning the chosen chelator, as well as the bisphosphonate structure, based on the DOTA bisphosphonate BPAMD.

Status Quo
During the last 10 years, the clinical application of bisphosphonates, especially for the treatment of patients with osseous metastases, distinctly increased. Bisphosphonates are analogs of naturally-occurring pyrophosphate. In contrast to pyrophosphate, they are resistant to chemical, as well as enzymatic, hydrolysis due to the substitution of the central oxygen atom by a carbon atom. Their effect is based on two characteristics: they show high affinity for bone material and inhibitory effects on osteoclasts [18]. Binding of bisphosphonates to bone material probably relies on bidentate or tridentate complexation of calcium atoms in the hydroxyapatite depending on the bisphosphonate structure [19].
The two side chains on the carbon atom are replaceable and responsible for the activity of the particular bisphosphonate ( Figure 2). Substitution of R1 by a hydroxyl or amino group enhances the affinity to hydroxyapatite. Varying the R2 side chain influences the antiresorptive potency [18]. Nitrogen atoms, especially aromatic nitrogen atoms, considerably raise the antiresorptive potency. This is linked to another hydrogen bond between the amine and the hydroxyapatite and the ability to act on biochemical activities, for example, the inhibition of farnesyl pyrophosphate synthase (FPPS) [18]. In SPECT tracers like [ 99m Tc]Tc-MDP ( 99m Tc-labeled methylene diphosphonate) the phosphonates are responsible for complexation of the radionuclide, as well as binding to the target tissue, which may lead to a decreased uptake in bone metastases [20]. This drawback can be circumvented by complete separation of the chelating unit and the targeting vector, using a macrocyclic chelator for complexation of the radiometal and a coupled bisphosphonate as targeting vector. Figure 3 shows the concept of the combination of a macrocyclic chelator with a bisphosphonate. Depending on the chelator various radionuclides can be complexed, also allowing the combination of diagnosis and therapy in one and the same compound. This theranostic concept already showed excellent results concerning diagnosis and treatment of neuroendocrine tumors using DOTA-TOC (1,4,7,10-tetraazacyclododecan-4,7,10-tricarboxy-methyl-1-yl-acetyl-D-Phe 1 -Tyr 3octreotide) radiolabeled with 68 Ga and 177 Lu [21]. Structures of MDP (methylene diphosphonate), HEDP (1,1-hydroxyethylidene diphosphonate), and EDTMP (ethylenediamine tetra(methylene phosphonic acid)).
This review describes the concept of macrocyclic chelate-conjugated bisphosphonates, which are able to circumvent the disadvantages of open-chain chelators, and possible improvements concerning the chosen chelator, as well as the bisphosphonate structure, based on the DOTA bisphosphonate BPAMD.

Status Quo
During the last 10 years, the clinical application of bisphosphonates, especially for the treatment of patients with osseous metastases, distinctly increased. Bisphosphonates are analogs of naturally-occurring pyrophosphate. In contrast to pyrophosphate, they are resistant to chemical, as well as enzymatic, hydrolysis due to the substitution of the central oxygen atom by a carbon atom. Their effect is based on two characteristics: they show high affinity for bone material and inhibitory effects on osteoclasts [18]. Binding of bisphosphonates to bone material probably relies on bidentate or tridentate complexation of calcium atoms in the hydroxyapatite depending on the bisphosphonate structure [19].
The two side chains on the carbon atom are replaceable and responsible for the activity of the particular bisphosphonate ( Figure 2). Substitution of R1 by a hydroxyl or amino group enhances the affinity to hydroxyapatite. Varying the R2 side chain influences the antiresorptive potency [18]. Nitrogen atoms, especially aromatic nitrogen atoms, considerably raise the antiresorptive potency. This is linked to another hydrogen bond between the amine and the hydroxyapatite and the ability to act on biochemical activities, for example, the inhibition of farnesyl pyrophosphate synthase (FPPS) [18]. This review describes the concept of macrocyclic chelate-conjugated bisphosphonates, which are able to circumvent the disadvantages of open-chain chelators, and possible improvements concerning the chosen chelator, as well as the bisphosphonate structure, based on the DOTA bisphosphonate BPAMD.

Status Quo
During the last 10 years, the clinical application of bisphosphonates, especially for the treatment of patients with osseous metastases, distinctly increased. Bisphosphonates are analogs of naturally-occurring pyrophosphate. In contrast to pyrophosphate, they are resistant to chemical, as well as enzymatic, hydrolysis due to the substitution of the central oxygen atom by a carbon atom. Their effect is based on two characteristics: they show high affinity for bone material and inhibitory effects on osteoclasts [18]. Binding of bisphosphonates to bone material probably relies on bidentate or tridentate complexation of calcium atoms in the hydroxyapatite depending on the bisphosphonate structure [19].
The two side chains on the carbon atom are replaceable and responsible for the activity of the particular bisphosphonate ( Figure 2). Substitution of R1 by a hydroxyl or amino group enhances the affinity to hydroxyapatite. Varying the R2 side chain influences the antiresorptive potency [18]. Nitrogen atoms, especially aromatic nitrogen atoms, considerably raise the antiresorptive potency. This is linked to another hydrogen bond between the amine and the hydroxyapatite and the ability to act on biochemical activities, for example, the inhibition of farnesyl pyrophosphate synthase (FPPS) [18]. In SPECT tracers like [ 99m Tc]Tc-MDP ( 99m Tc-labeled methylene diphosphonate) the phosphonates are responsible for complexation of the radionuclide, as well as binding to the target tissue, which may lead to a decreased uptake in bone metastases [20]. This drawback can be circumvented by complete separation of the chelating unit and the targeting vector, using a macrocyclic chelator for complexation of the radiometal and a coupled bisphosphonate as targeting vector. Figure 3 shows the concept of the combination of a macrocyclic chelator with a bisphosphonate. Depending on the chelator various radionuclides can be complexed, also allowing the combination of diagnosis and therapy in one and the same compound. This theranostic concept already showed excellent results concerning diagnosis and treatment of neuroendocrine tumors using DOTA-TOC (1,4,7,10-tetraazacyclododecan-4,7,10-tricarboxy-methyl-1-yl-acetyl-D-Phe 1 -Tyr 3octreotide) radiolabeled with 68 Ga and 177 Lu [21]. In SPECT tracers like [ 99m Tc]Tc-MDP ( 99m Tc-labeled methylene diphosphonate) the phosphonates are responsible for complexation of the radionuclide, as well as binding to the target tissue, which may lead to a decreased uptake in bone metastases [20]. This drawback can be circumvented by complete separation of the chelating unit and the targeting vector, using a macrocyclic chelator for complexation of the radiometal and a coupled bisphosphonate as targeting vector. Figure 3 shows the concept of the combination of a macrocyclic chelator with a bisphosphonate. Depending on the chelator various radionuclides can be complexed, also allowing the combination of diagnosis and therapy in one and the same compound. This theranostic concept already showed excellent results concerning diagnosis and treatment of neuroendocrine tumors using DOTA-TOC (1,4,7,10-tetraazacyclododecan-4,7,10-tricarboxy-methyl-1-yl-acetyl-D-Phe 1 -Tyr 3 -octreotide) radiolabeled with 68 Ga and 177 Lu [21]. One of these so-called macrocyclic bisphosphonates is BPAMD (Figure 4), which was initially able to show its high potential in terms of high bone accumulation in 68 Ga small animal PET experiments [22]. Later, it also showed good results in the first human applications [23] ( Figure 5). The bisphosphonate revealed very high target-to-soft tissue ratios combined with a fast renal clearance. SUVs (standardized uptake values) were comparable with those of the [ 18 F]NaF scan, and some metastases even showed higher accumulation of the bisphosphonate. These promising diagnostic examinations finally led to the first therapeutic applications using the β-emitter 177 Lu instead of 68 Ga.
[ 177 Lu]Lu-BPAMD was successfully applied in several patients ( Figure 6). It showed a comparable biodistribution as [ 68 Ga]Ga-BPAMD, including a good target-to-background ratio and a fast renal clearance. The radiopharmaceutical's long half-life in the metastases provided high tumor doses which led to a significant reduction in osteoblastic activity of the bone metastases. Furthermore, the therapy did not cause any significant adverse effects [21,24]. One of these so-called macrocyclic bisphosphonates is BPAMD (Figure 4), which was initially able to show its high potential in terms of high bone accumulation in 68 Ga small animal PET experiments [22]. One of these so-called macrocyclic bisphosphonates is BPAMD (Figure 4), which was initially able to show its high potential in terms of high bone accumulation in 68 Ga small animal PET experiments [22]. Later, it also showed good results in the first human applications [23] ( Figure 5). The bisphosphonate revealed very high target-to-soft tissue ratios combined with a fast renal clearance. SUVs (standardized uptake values) were comparable with those of the [ 18 F]NaF scan, and some metastases even showed higher accumulation of the bisphosphonate. These promising diagnostic examinations finally led to the first therapeutic applications using the β-emitter 177 Lu instead of 68 Ga.
Later, it also showed good results in the first human applications [23] ( Figure 5). The bisphosphonate revealed very high target-to-soft tissue ratios combined with a fast renal clearance. SUVs (standardized uptake values) were comparable with those of the [ 18 F]NaF scan, and some metastases even showed higher accumulation of the bisphosphonate. These promising diagnostic examinations finally led to the first therapeutic applications using the β-emitter 177 Lu instead of 68 Ga. [ 177 Lu]Lu-BPAMD was successfully applied in several patients ( Figure 6). It showed a comparable biodistribution as [ 68 Ga]Ga-BPAMD, including a good target-to-background ratio and a fast renal clearance. The radiopharmaceutical's long half-life in the metastases provided high tumor doses which led to a significant reduction in osteoblastic activity of the bone metastases. Furthermore, the therapy did not cause any significant adverse effects [21,24].  A comparative biodistribution study between [ 177 Lu]Lu-BPAMD and [ 177 Lu]Lu-EDTMP indicated higher bone uptake for [ 177 Lu]Lu-BPAMD, as well as a higher target-to-background ratio [25]. This may be attributed to the already-mentioned much higher amount of ligand used for the preparation of [ 177 Lu]Lu-EDTMP and the conceivable target blocking.
Despite those promising clinical results, there is still much potential for improvements with regard to radiosynthesis, raising the accumulation in bone metastases and reducing the uptake in healthy tissue. According to Figure 3, both the bisphosphonate and the chelator should be optimized.

Chelator
Chelators based on a polyamino polycarboxylic structure belong to the most efficient ligands and are widely used for the complexation of metal ions. They can be divided into two categories, open chain ligands, such as EDTA (ethylenediaminetetraacetic acid), and DTPA (diethylenetriaminepentaacetic acid) and macrocyclic chelates, such as DOTA or NOTA [26].  A comparative biodistribution study between [ 177 Lu]Lu-BPAMD and [ 177 Lu]Lu-EDTMP indicated higher bone uptake for [ 177 Lu]Lu-BPAMD, as well as a higher target-to-background ratio [25]. This may be attributed to the already-mentioned much higher amount of ligand used for the preparation of [ 177 Lu]Lu-EDTMP and the conceivable target blocking.
Despite those promising clinical results, there is still much potential for improvements with regard to radiosynthesis, raising the accumulation in bone metastases and reducing the uptake in healthy tissue. According to Figure 3, both the bisphosphonate and the chelator should be optimized.

Chelator
Chelators based on a polyamino polycarboxylic structure belong to the most efficient ligands and are widely used for the complexation of metal ions. They can be divided into two categories, open chain ligands, such as EDTA (ethylenediaminetetraacetic acid), and DTPA (diethylenetriaminepentaacetic acid) and macrocyclic chelates, such as DOTA or NOTA [26]. A comparative biodistribution study between [ 177 Lu]Lu-BPAMD and [ 177 Lu]Lu-EDTMP indicated higher bone uptake for [ 177 Lu]Lu-BPAMD, as well as a higher target-to-background ratio [25]. This may be attributed to the already-mentioned much higher amount of ligand used for the preparation of [ 177 Lu]Lu-EDTMP and the conceivable target blocking.
Despite those promising clinical results, there is still much potential for improvements with regard to radiosynthesis, raising the accumulation in bone metastases and reducing the uptake in healthy tissue. According to Figure 3, both the bisphosphonate and the chelator should be optimized.

Chelator
Chelators based on a polyamino polycarboxylic structure belong to the most efficient ligands and are widely used for the complexation of metal ions. They can be divided into two categories, open chain ligands, such as EDTA (ethylenediaminetetraacetic acid), and DTPA (diethylenetriaminepentaacetic acid) and macrocyclic chelates, such as DOTA or NOTA [26].
DOTA is the most commonly used macrocyclic chelator for PET applications. It is able to complex a variety of isotopes, e.g., 44/47 Sc, 111 In, 177 Lu, 86/90 Y, and 225 Ac. It is also used broadly with 67/68 Ga, which offers the possibility of a theranostic application, as already mentioned using the example of 68 Ga-and 177 Lu-labeled DOTA-TOC [27,28]. Nevertheless, DOTA has a comparatively low stability constant for gallium (log K = 21.3) resulting in temperatures of about 95 • C needed for radiolabeling. NOTA, by contrast, exhibits a smaller ring structure and a higher stability constant (log K = 31.0) due to the smaller gallium fitting cavity [28]. Figure 7 shows the fast and quantitative 68 Ga-labeling of the NOTA bisphosphonate NO2AP BP (Figure 4). Comparison with the DOTA-based BPAMD shows the expected faster labeling of NOTA derivatives.  [27,28]. Nevertheless, DOTA has a comparatively low stability constant for gallium (log K = 21.3) resulting in temperatures of about 95 °C needed for radiolabeling. NOTA, by contrast, exhibits a smaller ring structure and a higher stability constant (log K = 31.0) due to the smaller gallium fitting cavity [28]. Figure 7 shows the fast and quantitative 68 Ga-labeling of the NOTA bisphosphonate NO2AP BP (Figure 4). Comparison with the DOTA-based BPAMD shows the expected faster labeling of NOTA derivatives. Interestingly, using this NOTA derivative not only provides an improved radiosynthesis, it also exhibits a significantly higher femur accumulation in rats compared with the DOTA derivative BPAMD (Figure 8). This may be explained by differences in charge and physical properties of both complexes. It is a recurrent phenomenon that different chelators provide different in vivo properties with the same target vector [27].  [29]. Within this study, the NOTA-based bisphosphonate was able to underline its high diagnostic efficiency. Error! Reference source not found. 9 shows the PET and SPECT scans of a breast cancer patient. Generally, the PET tracers are able to detect more, as well as smaller, metastases. [ 68 Ga]Ga-NO2AP BP revealed a similar detection capability as the gold standard [ 18 F]NaF. In selected metastases bisphosphonate uptake was even higher. Interestingly, using this NOTA derivative not only provides an improved radiosynthesis, it also exhibits a significantly higher femur accumulation in rats compared with the DOTA derivative BPAMD (Figure 8). This may be explained by differences in charge and physical properties of both complexes. It is a recurrent phenomenon that different chelators provide different in vivo properties with the same target vector [27].  Interestingly, using this NOTA derivative not only provides an improved radiosynthesis, it also exhibits a significantly higher femur accumulation in rats compared with the DOTA derivative BPAMD (Figure 8). This may be explained by differences in charge and physical properties of both complexes. It is a recurrent phenomenon that different chelators provide different in vivo properties with the same target vector [27].  [29]. Within this study, the NOTA-based bisphosphonate was able to underline its high diagnostic efficiency. Error! Reference source not found. 9 shows the PET and SPECT scans of a breast cancer patient. Generally, the PET tracers are able to detect more, as well as smaller, metastases. [ 68 Ga]Ga-NO2AP BP revealed a similar detection capability as the gold standard [ 18 F]NaF. These positive results were also confirmed in a prospective patient study by Passah [29]. Within this study, the NOTA-based bisphosphonate was able to underline its high diagnostic efficiency. Figure 9 shows the PET and SPECT scans of a breast cancer patient. Generally, the PET tracers are able to detect more, as well as smaller, metastases.
[ 68 Ga]Ga-NO2AP BP revealed a similar detection capability as the gold standard [ 18 F]NaF. In selected metastases bisphosphonate uptake was even higher.  In addition to the well-established NOTA derivatives, there is another class of bifunctional chelators appropriate for labeling with 68 Ga. These so-called DATA chelators are based on 6-amino-1,4-diazepine-triacetic acid and enable more rapid quantitative radiolabeling under milder conditions [30]. The combination of this chelator with next-generation bisphosphonates is also conceivable and might provide a compound of high diagnostic efficiency, as well [31].

Pharmacophoric Group
As mentioned above, the side chains on the central carbon atom are responsible for the bisphosphonate's activity, i.e., in terms of affinity to hydroxyapatite. Using a hydroxybisphosphonate, bearing a hydroxyl group as R1, could lead to higher bone accumulation due to increased affinity for bone material. Furthermore, an aromatic nitrogen atom in the R2 side chain could cause building of another hydrogen bond and thereby also raise bone accumulation ( Figure 10) [18]. In addition to the well-established NOTA derivatives, there is another class of bifunctional chelators appropriate for labeling with 68 Ga. These so-called DATA chelators are based on 6-amino-1,4-diazepine-triacetic acid and enable more rapid quantitative radiolabeling under milder conditions [30]. The combination of this chelator with next-generation bisphosphonates is also conceivable and might provide a compound of high diagnostic efficiency, as well [31].

Pharmacophoric Group
As mentioned above, the side chains on the central carbon atom are responsible for the bisphosphonate's activity, i.e., in terms of affinity to hydroxyapatite. Using a hydroxybisphosphonate, bearing a hydroxyl group as R1, could lead to higher bone accumulation due to increased affinity for bone material. Furthermore, an aromatic nitrogen atom in the R2 side chain could cause building of another hydrogen bond and thereby also raise bone accumulation ( Figure 10) [18].
Such a bisphosphonate, like risedronate or zoledronate, would also influence biochemical processes. They possess an inhibiting effect on the FPPS and inhibition of this enzyme causes an increased apoptosis rate [18]. A DOTA-conjugated zoledronate (DOTA ZOL , Figure 4 ex vivo biodistribution studies in healthy Wistar rats ( Figure 13) [32]. Figure 14 shows its high bone accumulation, especially in the high metabolic epiphyseal plates and other joint regions.
conditions [30]. The combination of this chelator with next-generation bisphosphonates is also conceivable and might provide a compound of high diagnostic efficiency, as well [31].

Pharmacophoric Group
As mentioned above, the side chains on the central carbon atom are responsible for the bisphosphonate's activity, i.e., in terms of affinity to hydroxyapatite. Using a hydroxybisphosphonate, bearing a hydroxyl group as R1, could lead to higher bone accumulation due to increased affinity for bone material. Furthermore, an aromatic nitrogen atom in the R2 side chain could cause building of another hydrogen bond and thereby also raise bone accumulation ( Figure 10) [18]. Figure 10. Classification of bisphosphonates according to their adsorption affinity to hydroxyapatite (HAP). Figure 10.
Classification of bisphosphonates according to their adsorption affinity to hydroxyapatite (HAP). Such a bisphosphonate, like risedronate or zoledronate, would also influence biochemical processes. They possess an inhibiting effect on the FPPS and inhibition of this enzyme causes an increased apoptosis rate [18].  Figure  14 shows its high bone accumulation, especially in the high metabolic epiphyseal plates and other joint regions.   Such a bisphosphonate, like risedronate or zoledronate, would also influence biochemical processes. They possess an inhibiting effect on the FPPS and inhibition of this enzyme causes an increased apoptosis rate [18].  Figure  14 shows its high bone accumulation, especially in the high metabolic epiphyseal plates and other joint regions.  Such a bisphosphonate, like risedronate or zoledronate, would also influence biochemical processes. They possess an inhibiting effect on the FPPS and inhibition of this enzyme causes an increased apoptosis rate [18].  Figure 13) [32]. Figure  14 shows its high bone accumulation, especially in the high metabolic epiphyseal plates and other joint regions.  Such a bisphosphonate, like risedronate or zoledronate, would also influence biochemical processes. They possess an inhibiting effect on the FPPS and inhibition of this enzyme causes an increased apoptosis rate [18].  Figure 13) [32]. Figure  14 shows its high bone accumulation, especially in the high metabolic epiphyseal plates and other joint regions.   Considering the good results of both the 68 Ga-and the 177 Lu-labeled derivatives in small animal studies, 68 Ga-and 177 Lu-labeled DOTA ZOL seem to offer high potential for theranostic applications. This potential now needs to be proven in clinical studies. Figure 15 shows a comparison of [ 68 Ga]Ga-PSMA-11 and [ 68 Ga]Ga-DOTA ZOL in one and the same prostate cancer patient. Both tracers detected multiple skeletal lesions in thoracic and lumbar vertebrae, as well as in the pelvis. Comparison of SUVs revealed an approximately three-fold higher uptake of the bisphosphonate in bone metastases and an approximately three-fold lower uptake in normal tissue organs, exemplifying the bisphosphonate's better target-to-background ratio.  tetraazacyclododec-1-yl)-acetic acid) (Figures 11 and 12) [32]. [ 68 Ga]Ga-DOTA ZOL showed the highest bone accumulation and very low uptake in soft tissue. [ 177 Lu]Lu-DOTA ZOL revealed a comparable femur accumulation in ex vivo biodistribution studies in healthy Wistar rats ( Figure 13) [32]. Figure  14 shows its high bone accumulation, especially in the high metabolic epiphyseal plates and other joint regions.  Considering the good results of both the 68 Ga-and the 177 Lu-labeled derivatives in small animal studies, 68 Ga-and 177 Lu-labeled DOTA ZOL seem to offer high potential for theranostic applications. This potential now needs to be proven in clinical studies. Figure 15 shows a comparison of [ 68 Ga]Ga-PSMA-11 and [ 68 Ga]Ga-DOTA ZOL in one and the same prostate cancer patient. Both tracers detected multiple skeletal lesions in thoracic and lumbar vertebrae, as well as in the pelvis. Comparison of SUVs revealed an approximately three-fold higher uptake of the bisphosphonate in bone metastases and an approximately three-fold lower uptake in normal tissue organs, exemplifying the bisphosphonate's better target-to-background ratio.  Considering the good results of both the 68 Ga-and the 177 Lu-labeled derivatives in small animal studies, 68 Ga-and 177 Lu-labeled DOTA ZOL seem to offer high potential for theranostic applications. This potential now needs to be proven in clinical studies. Figure 15 shows a comparison of [ 68 Ga]Ga-PSMA-11 and [ 68 Ga]Ga-DOTA ZOL in one and the same prostate cancer patient. Both tracers detected multiple skeletal lesions in thoracic and lumbar vertebrae, as well as in the pelvis. Comparison of SUVs revealed an approximately three-fold higher uptake of the bisphosphonate in bone metastases and an approximately three-fold lower uptake in normal tissue organs, exemplifying the bisphosphonate's better target-to-background ratio. Considering the good results of both the 68 Ga-and the 177 Lu-labeled derivatives in small animal studies, 68 Ga-and 177 Lu-labeled DOTA ZOL seem to offer high potential for theranostic applications. This potential now needs to be proven in clinical studies. Figure 15 shows a comparison of [ 68 Ga]Ga-PSMA-11 and [ 68 Ga]Ga-DOTA ZOL in one and the same prostate cancer patient. Both tracers detected multiple skeletal lesions in thoracic and lumbar vertebrae, as well as in the pelvis. Comparison of SUVs revealed an approximately three-fold higher uptake of the bisphosphonate in bone metastases and an approximately three-fold lower uptake in normal tissue organs, exemplifying the bisphosphonate's better target-to-background ratio.  [ 177 Lu]Lu-DOTA ZOL has been used in ten patients with bone metastases for dosimetry studies (data to be published). Whole body scintigraphic images were acquired at different time points after injection. Within this study it showed a fast renal clearance and a high target-to-background ratio, and was able to confirm the results of the ex vivo biodistribution studies ( Figure 16). [ 177 Lu]Lu-DOTA ZOL has been used in ten patients with bone metastases for dosimetry studies (data to be published). Whole body scintigraphic images were acquired at different time points after injection. Within this study it showed a fast renal clearance and a high target-to-background ratio, and was able to confirm the results of the ex vivo biodistribution studies ( Figure 16).

Conclusions
The combination of novel bisphosphonates with macrocyclic chelators provides promising tracers for diagnosis, therapy, and also theranostics of bone metastases.
Currently, the most potent 68 Ga-bisphosphonate is [ 68 Ga]Ga-NO2AP BP , which enables quantitative radiolabeling and exhibits very high accumulation in bone metastases 30-60 min after injection, as well as a fast blood clearance and very low uptake in soft tissue. It is superior to [ 99m Tc]Tc-MDP and comparable to [ 18 F]NaF.
DOTA bisphosphonates are eminently suitable for labeling with 177 Lu. [ 177 Lu]Lu-BPAMD has proved valuable in clinical application. The low-energy β − emission hardly reaches the bone marrow and only a low or no haematotoxicity was observed. The good target-to-background ratio, that all examined bisphosphonates have in common, is also advantageous for therapeutic applications due to reduced radiation dose for non-target tissue.
Due to further developments regarding the chemical structure of these macrocyclic bisphosphonates, new 68 Ga-and 177 Lu-labeled bisphosphonates possessing improved pharmacological properties are expected. Zoledronate based bisphosphonates appear to be the most potent radiotracers with regard to bone lesions. Thus, DOTA ZOL for example may be a potent conjugate for theranostics of bone metastases. The target-to-background ratio is progressively better in later images and best at 24 h p.i.

Conclusions
The combination of novel bisphosphonates with macrocyclic chelators provides promising tracers for diagnosis, therapy, and also theranostics of bone metastases.
Currently, the most potent 68 Ga-bisphosphonate is [ 68 Ga]Ga-NO2AP BP , which enables quantitative radiolabeling and exhibits very high accumulation in bone metastases 30-60 min after injection, as well as a fast blood clearance and very low uptake in soft tissue. It is superior to [ 99m Tc]Tc-MDP and comparable to [ 18 F]NaF.
DOTA bisphosphonates are eminently suitable for labeling with 177 Lu. [ 177 Lu]Lu-BPAMD has proved valuable in clinical application. The low-energy β − emission hardly reaches the bone marrow and only a low or no haematotoxicity was observed. The good target-to-background ratio, that all examined bisphosphonates have in common, is also advantageous for therapeutic applications due to reduced radiation dose for non-target tissue.
Due to further developments regarding the chemical structure of these macrocyclic bisphosphonates, new 68 Ga-and 177 Lu-labeled bisphosphonates possessing improved pharmacological properties are expected. Zoledronate based bisphosphonates appear to be the most potent radiotracers with regard to bone lesions. Thus, DOTA ZOL for example may be a potent conjugate for theranostics of bone metastases.