An osteoclast-targeting agent for imaging and therapy of bone metastasis

Abstract

A hybrid compound (DO3A-BP) featuring a radiometal bifunctional chelator (1,4,7,10-tetraazacyclotetradecane-N,N’,N”,N’”-tetraacetic acid, DOTA) and an osteoclast-targeting moiety (bisphosphonate) was designed and synthesized. The ¹¹¹In-labeled complex of DO3A-BP showed significantly elevated uptake in osteoclasts compared to the undifferentiated adherent bone marrow derived cells. Biodistribution studies revealed a favorable tissue distribution profile in normal mice with high bone uptake and long retention, and low or negligible accumulation in non-target organs.

A variety of cancers preferentially metastasize to the skeleton at their advanced stages. Whole-body scan using ^99mTc-MDP (MDP: methylene diphosphonate) is currently the standard clinic practice for the detection of bone metastases.^{1, 2} However, due to its low specificity a final diagnosis is often aided by other imaging modalities, such as X-ray radiography, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography, and/or bone marrow biopsy.^{3, 4} Of the clinical methods to treat bone metastases, therapies utilizing bisphosphonates (BPs) and radiopharmaceuticals play critical roles. To date, several BPs have been approved by the FDA⁵ and they are commonly used to treat skeletal complications caused by metastases and other bone diseases, including tumor-associated osteolysis,^{6, 7} hypercalcemia,⁸ Paget’s disease,^{9, 10} and osteoporosis.¹¹ As shown in Figure 1, bisphosphonates are a group of compounds with a chemical structure similar to that of the natural inorganic pyrophosphate (PPi), an endogenous regulator of bone mineralization, but differing in the central atom where BPs have a methylene carbon rather than an oxygen atom in PPi. This structural feature renders BPs resistant to hydrolysis under acidic conditions or by pyrophosphatases. Varieties of BPs could be obtained by tuning the R₂ side chain while leaving the R₁ group intact as either –OH or –H.¹² It has recently become clear that BPs first bind avidly to the bone mineral surface and are subsequently internalized selectively by osteoclasts, where they inhibit the osteoclastic activity and induce apoptosis. In addition, BPs have been found to inhibit tumor cell adhesion to mineralized bone as well as tumor cell invasion and proliferation.^{13, 14}

Figure 1.

Pyrophosphate and bisphophonate structures.

The binding of the hydroxyl groups of BPs to Ca²⁺ in hydroxyapitate of bone is responsible for the accumulation of BPs in bone. However, it reduces the coordination sites of ^99mTc-MDP in vivo and subsequently ^99mTc-MDP decomposes into ^99mTcO4 and BP components. Therefore the bone uptake of ^99mTc-MDP is mainly dependent on the osteoblastic activity and the purely osteolytic lesion is poorly detectable.¹⁵ DOTA (1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid) is a commonly used bifunctional chelator for radioimmunodiagnosis and radioimmuo-therapy because it is able to form thermodynamically stable and kinetically inert complexes with many divalent or trivalent metal ions.^16–18 To date, few conjugates of DOTA and BPs have been reported but recent research has focused on the improvement of the binding affinity of BP to bone for the applications as delivery vehicles of MRI contrast agents and palliation agents for certain bone diseases.^{19, 20} Given the high expression of osteoclasts in both osteoblastic and osteolytic lesions, here we report the design and synthesis of an osteoclast-targeting compound by conjugating DOTA to an osteoclast-targeting BP moiety through an ethylenediamine linker, and its in vitro and in vivo evaluation.

A convergent approach was chosen to synthesize the hybrid ligand (DO3A-BP), in which the derivatives of a bisphosphonate (3) and DOTA metal chelator (7) were synthesized separately as shown in Scheme 1 and Scheme 2.

Scheme 1.

Synthesis of derivatives of (a) a bisphosphonate and (b) DOTA.

Scheme 2.

Synthesis of DO3A-BP (9)

The carboxylate acid substituted bisphosphonate, 3-bis-(dibenzyloxyphosphoryl)propanoic acid (3), was synthesized by a literature procedure with an overall yield of 27% (Scheme 1a).²¹ The synthesis of 7 was previously reported.²² However, an alternative route was used in this work (Scheme 2), in which a commercially available intermediate, 5 (1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl)acetate: DO3A-tBu-ester) was used as starting material instead of cyclen and a higher overall yield (77%) was achieved. Alkylation of 5 with methyl chloroacetate afforded 6, and then 7 was obtained by reacting 6 with excess ethylene diamine. Subsequent conjugation of 3 with 7 via the standard DCC/HOBt procedure (DCC: dicyclo-hexylcarbodiimide; HOBt: 1-hydroxybenzotriazol) (Scheme 2) afforded an orthogonally protected DO3A-BP (8) in a yield of 50%. Compound 8 was obtained with high chemical purity via the column chromatography eluted with CHCl₃/MeOH (10:1). The ¹H NMR spectrum of 8 showed two well-separated peaks between 8.6 – 9.0 ppm, which can be ascribed to the amide protons on the linkage between the DOTA and BP moieties. A broad peak between 1.8 – 3.5 ppm was observed for the ethylene protons of the macrocycle and it overlapped with the methylene proton signals on the pendent arms. The product, 9 (DO3A-BP), was obtained in nearly quantitative yield after a two-step deprotection, where the t-butyl and benzyl protecting groups were removed by trifluoroacetic acid and Pd/C-catalyzed hydrogenation, respectively. Bisphosphonates are typically protected in the form of tetraalkyl bisphosphonate esters and deprotection was conducted by either acid hydrolysis or silylation-dealkylation, however low yields were commonly seen due to the decomposition under the acidic condition of the hydrolysis.^{19, 23} Our choice of using benzyl rather than alkyl as the BP protecting group enabled us to selectively deprotect 8 in quantitative yield by two separate procedures, which is advantageous to the formation of macrocyclic metal complexes; and provide a UV chromophore for the monitoring of the synthetic procedures. ²⁴

The radiolabeling of DO3A-BP with ¹¹¹In was carried out in 0.4 M NH4OAc buffer, pH 7.5, and monitored by radio-TLC every 4 h via reversed-phase C18 TLC eluted with 10% NH₄OAc/MeOH (v/v: 3:1). Under this TLC condition, free ¹¹¹In stays at the origin, while the labeled complex migrates with the mobile phase to a certain distance. Although it is well documented that DOTA and its derivatives can be labeled in nearly quantitative radiochemical yields within 2 h under mild conditions,^{25, 26} such a yield (> 95%) could only be obtained after 44 h under similar conditions in the formation of ¹¹¹In-DO3A-BP.²⁷ The slow kinetics of DO3A-BP is likely due to the ¹¹¹In binding competition between the bisphosphonate and the DOTA moieties.²⁰ An efficient radiolabeling method should be developed for the further application of this agent.

We found that the ability of bisphosphonate to chelate metal ions was dramatically reduced at low pH values due to the protonation of the phosphonate groups, which is consistent with the literature reports.^{28, 29} Based on this pH-dependent binding manner of BP to metal ions, the ¹¹¹In-labeled complex was challenged by reducing the pH from 7.5 to 4.0. The low pH was maintained for 3 days, no free ¹¹¹In was observed. This clearly indicates that the radiometal ion, ¹¹¹In(III), was bound to the DOTA moiety.

The octanol-water partition coefficient (log P) of ¹¹¹In-DO3A-BP was determined to be −4.05 ± 0.44 (n = 10), indicative of the highly hydrophilic nature of the compound. The in vitro serum stability of ¹¹¹In-DO3A-BP was evaluated out to 48 h by radio-TLC. The concentration of ¹¹¹In-DO3A-BP was 0.05 mM, while the protein concentration in rat serum was in large excess. The ¹¹¹In-DO3A-BP complex remained nearly 100% intact within two days of incubation with rat serum at 37°C (n = 4). The high in vitro stability of ¹¹¹In-labeled complex warrants further in vitro and in vivo evaluation. ²⁷

The osteoclast-targeting property of ¹¹¹In-DO3A-BP was evaluated on mouse bone marrow derived cells grown for 7 days in the presence or absence of receptor activator for nuclear factor κ B (RANK) and macrophage colony-stimulating factor (M-CSF) ligands according to an established method.^{30, 31} Osteoclasts are large multinucleate cells formed by the fusion of cells of the monocyte-macrophage lineage in the presence of RANK ligand and M-CSF, which are characterized by high expression of tartrate resistant acid phosphatase (TRAP) and cathepsin K. After incubation of 5 – 8 days, osteoclasts were formed as seen by cell morphology microscope and TRAP staining (Figure 2. Right). In contrast, bone marrow adherent cells showed negative response to TRAP staining (Figure 2. Left).

Figure 2.

TRAP staining of bone marrow adherent cells (left) and osteoclasts (right).

For comparison, the uptake in original bone marrow adherent cells (BMCs) and bone marrow macrophages (BMMs) were used as controls. Bone marrow macrophages are considered as the intermediates between BMC and OCs and are formed by treatment with M-CSF only. After a one-hour incubation of ¹¹¹In-DO3A-BP with the cells, the cells were lysed and the lysates were counted by a γ-counter. The cell uptake of ¹¹¹In-DO3A-BP was normalized to protein content in each well, which was determined by a commercially available kit. Shown in Figure 3, the osteoclast uptake of ¹¹¹In-DO3A-BP was about two fold higher than either BMC or BMM, demonstrating its preferential uptake by osteoclasts.³²

Figure 3.

Comparative uptake of ¹¹¹In-DO3A-BP by osteoclasts (OC), bone marrow adherent cell (BMC) and bone marrow macrophages (BMM) at 1 h incubation. Data were obtained from five independent experiments (n > 6) and are presented as uptake ratios versus BMC. Gamma counts of the cell lysates were normalized to the protein content of each well for the uptake ratio calculation.

The biodistribution of ¹¹¹In-DO3A-BP in normal Balb/c mice is presented in Table 1.³³ As anticipated, the ¹¹¹In-DO3A-BP complex showed high accumulation within 1 h post-injection (p.i.) in the bone and long residence, which is reflected by only a 20% decrease over 48 h.

Table 1.

Biodistribution of ¹¹¹In-labeled DO3A-BP in normal Balb/c mice (n = 4). Data are presented as %ID/g ± standard deviation. B/B: Bone/Blood; B/M: Bone/Muscle.

	1 h p.i.	4 h p.i.	24 h p.i.	48 h p.i.
Blood	0.34 ± 0.08	0.01 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Lung	0.52 ± 0.06	0.16 ± 0.04	0.08 ± 0.02	0.08 ± 0.02
Liver	0.41 ± 0.04	0.33 ± 0.05	0.16 ± 0.01	0.14 ± 0.01
Spleen	0.23 ± 0.02	0.15 ± 0.04	0.08 ± 0.01	0.09 ± 0.01
Kidney	4.61 ± 0.42	3.93 ± 1.01	1.16 ± 0.16	0.70 ± 0.09
Muscle	0.49 ± 0.05	0.04 ± 0.01	0.03 ± 0.01	0.05 ± 0.01
Bone	3.39 ± 0.49	2.66 ± 0.81	2.02 ± 0.59	2.60 ± 0.56
B/B	10.12	393.12	644.90	816.49
B/M	6.94	72.91	69.90	49.06

In agreement with the reported BP-derivatized compounds,³⁴ the radiolabeled DO3A-BP showed a relatively high renal uptake, which, however, was rather efficiently cleared. The rapid blood clearance was a result of the high affinity of BPs for bone mineral in vivo.^{35, 36} The low liver uptake and efficient clearance reflects the high in vivo stability of ¹¹¹In-DO3A-BP. Impressively, this compound showed no significant uptake in other non-target organs (e.g. lungs, spleen, heart, or brain) and remained intact in the excreted urine within 48 h p.i. as determined by radio-TLC. The high bone/blood and bone/muscle contrasts further demonstrate the potential of this hybrid compound as a bone-seeking agent.

Taken together, we have synthesized an osteoclast-targeting compound by conjugating DOTA and a BP moiety via an ethylenediamine linker in high overall yields. Our preliminary in vitro and in vivo evaluation results indicate that this compound may find applications in osteoclast-targeted radiotherapy and nuclear imaging of bone diseases, which over-expresses osteoclasts relative to osteoblasts or osteoblastic activity, such as multiple myeloma.