Novel Radiolabeled Bisphosphonates for PET Diagnosis and Endoradiotherapy of Bone Metastases

Abstract

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.

1. Introduction

Malignant tumors, especially carcinomas of the breast, prostate, and lung, often lead to painful bone metastases. Since complications, like severe bone pain, pathological fractures, spinal cord compression, or hypercalcaemia, distinctly influence the quality of life and, therefore, result in a shorter survival, diagnosis of bone metastases at an early stage, as well as subsequent therapy is of great importance for patients [1,2]. Bone-seeking radiopharmaceuticals are currently used for diagnostic and therapeutic purposes [3].

Bone lesions are characterized by an increase in bone turnover. This increased metabolism of the bone material can be visualized via both single-photon emission computed tomography (SPECT) and positron emission tomography (PET). In contrast to SPECT, PET offers a higher spatial, as well as temporal, resolution [4]. As a SPECT nuclide, ^99mTc is predominantly used in the form of ^99mTc-labeled bisphosphonate complexes. For PET [¹⁸F]NaF is used as bone-seeking compound [5,6].

The ⁶⁸Ge/⁶⁸Ga generator system provides a distinguished PET analog of the established ^99mTc generator. The daughter nuclide ⁶⁸Ga offers appropriate decay properties (t_1/2 = 67.7 min, β⁺ = 89%, E_βmax = 1.9 MeV) and the generator ensures a long shelf-life with a continuous supply of ⁶⁸Ga [7]. In addition to [¹⁸F]NaF, ⁶⁸Ga-labeled PSMA radioligands have emerged recently and are currently used for diagnosis of bone metastases as a consequence of prostatic cancer [8]. However, further ⁶⁸Ga-based bone-seeking PET-radiopharmaceuticals have not been established clinically.

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-1,4,7-triacetic 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, ⁸⁹Sr, ³²P, and ²²³Ra. 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 ²²³Ra, 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 ²²³Ra 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 ⁸⁹Sr and ³²P have discouraged their use and have favored nuclides such as ¹⁵³Sm and ¹⁷⁷Lu with shorter half-lives and lower bone marrow toxicity. The use of ¹⁷⁷Lu 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 ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, and ¹⁸⁸Re [13]. ¹⁸⁸Re 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 ¹⁵²Sm due to the production route of ¹⁵³Sm can cause a reduction of the dose rate deposited on osseous metastases and, therefore, a lower therapeutic efficiency [16,17]. Using ¹⁷⁷Lu instead of ¹⁵³Sm 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.

2. Design and Development of Radiolabeled Bisphosphonates

2.1. 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 [^99mTc]Tc-MDP (^99mTc-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-Phe¹-Tyr³-octreotide) radiolabeled with ⁶⁸Ga and ¹⁷⁷Lu [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 ⁶⁸Ga 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 [¹⁸F]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 ¹⁷⁷Lu instead of ⁶⁸Ga. [¹⁷⁷Lu]Lu-BPAMD was successfully applied in several patients (Figure 6). It showed a comparable biodistribution as [⁶⁸Ga]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 [¹⁷⁷Lu]Lu-BPAMD and [¹⁷⁷Lu]Lu-EDTMP indicated higher bone uptake for [¹⁷⁷Lu]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 [¹⁷⁷Lu]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.

2.2. 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., ^44/47Sc, ¹¹¹In, ¹⁷⁷Lu, ^86/90Y, and ²²⁵Ac. It is also used broadly with ^67/68Ga, which offers the possibility of a theranostic application, as already mentioned using the example of ⁶⁸Ga- and ¹⁷⁷Lu-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 ⁶⁸Ga-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].

These positive results were also confirmed in a prospective patient study by Passah et al. comparing [⁶⁸Ga]Ga-NO2AP^BP, [¹⁸F]NaF, and [^99mTc]Tc-MDP in female breast cancer patients with osseous metastases [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. [⁶⁸Ga]Ga-NO2AP^BP revealed a similar detection capability as the gold standard [¹⁸F]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 ⁶⁸Ga. 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].

2.3. 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) was already labeled with ⁶⁸Ga and ¹⁷⁷Lu and examined in in vitro and ex vivo biodistribution studies, as well as small animal PET and SPECT studies. [⁶⁸Ga]Ga-DOTA^ZOL was compared to [¹⁸F]NaF and a known DOTA-α-H-bisphosphonate ([⁶⁸Ga]Ga-BPAPD (⁶⁸Ga-labeled (4-{[(bis–phosphonopropyl)carba-moyl]methyl}-7,10-bis-(carboxy-methyl)-1,4,7,10-tetraazacyclododec-1-yl)-acetic acid) (Figure 11 and Figure 12) [32]. [⁶⁸Ga]Ga-DOTA^ZOL showed the highest bone accumulation and very low uptake in soft tissue. [¹⁷⁷Lu]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 ⁶⁸Ga- and the ¹⁷⁷Lu-labeled derivatives in small animal studies, ⁶⁸Ga- and ¹⁷⁷Lu-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 [⁶⁸Ga]Ga-PSMA-11 and [⁶⁸Ga]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.

[¹⁷⁷Lu]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).

3. 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 ⁶⁸Ga-bisphosphonate is [⁶⁸Ga]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 [^99mTc]Tc-MDP and comparable to [¹⁸F]NaF.

DOTA bisphosphonates are eminently suitable for labeling with ¹⁷⁷Lu. [¹⁷⁷Lu]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 ⁶⁸Ga- and ¹⁷⁷Lu-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.