Introduction

Radionuclide bone imaging is the most common clinical investigation in nuclear medicine. At present the complexes of 99mTc–MDP [1] and 99mTc–HMDP [2] are the most widely used tracer agents for clinical radioisotopic bone scanning, whereas they have a number of suboptimal properties. A considerable time interval is required after injection of the radiopharmaceutical before bone scanning can be started. This necessary delay between injection and imaging is a serious inconvenience for the nuclear medicine personnel and for the patients, especially for children. To enable imaging at an earlier time after injection, a radiopharmaceutical with higher affinity for bone is required.

Bisphosphonate analogs accumulate in bone because their phosphonate groups bind to the Ca2+ of hydroxyapatite crystals [3]. So the diphosphonate group is necessary both for the complexation of 99mTc and the uptake of the complex in the skeleton [1]. In the case of 99mTc–MDP and 99mTc–HMDP, the phosphonate groups coordinate with technetium [4], which might reduce the ability of binding to the bone surface.

In order to solve this problem, many efforts have been done recently [5–10]. These studies mainly focus on the development of a “new generation” bone imaging agents based on the concept of bifunctional radiopharmaceuticals. The bisphosphonate part of these complexes can be freed and its affinity to the bone can been improved. In this study, 6-hydrazinopyridine-3-carboxylic acid (HYNIC) was chosen as chelating sites because they have been widely used for 99mTc labeling of proteins, peptides [11–15] and some small molecular compounds [16, 17] on the basis of different coligands and conjugated to aminomethylenediphosphonic acid (AMDP). 99mTc–HYNIC–AMDP was prepared by coordination with 99mTc, and their properties in vitro and in vivo were studied and evaluated as a potential bone imaging agent. In this complex, HYNIC (6-hydrazinopyridine-3-carboxylic acid) was using as a bifunctional chelating agent, so the bisphosphonate part of the complex can be freed and its affinity to the bone can be improved. To the best of our knowledge, this is the first report using the HYNIC–AMDP in the preparation of 99mTcO complex as bone imaging agent.

Experimental

Materials

1,3-Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) and trimethylsilyl bromide (TMSBr) were obtained from Aldrich Chemical Co. Other chemicals were purchased from Acros Chemical Co. 99mTc–MDP was prepared by reconstitution with a conventional MDP labeling kit (Beijing Shihong Pharmaceutical Center) with a 99mTcO4 − solution. 1H-NMR spectra were collected on a Bruker 500 MHz FT-NMR spectrometer. ESI–MS were performed on API-3000 LC/MS. Na99mTcO4 was obtained from a commercial 99Mo/99mTc generator, Beijing Atomic High-tech Co. ICR mice (weighing 20–25 g) was obtained from the Animal Center of Peking University.

Synthesis of HYNIC–AMDP

6-(Tert-butoxycarbonyl)-hydrazinopyridine-3-carboxylic acid (Boc-HYNIC) and tetraethyl ester of aminomethylenediphosphonic acid (AMDPE) were synthesized according to procedures described elsewhere [18, 19].

Tetraethyl ester of aminomethylenediphosphonic acid (AMDPE, 0.61 mg, 2.0 mmol) was dissolved in 10 mL dry DMF, then 6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carboxylic acid (Boc-HYNIC) (0.66 g, 2.6 mmol) and 1-hydroxybenzotriazole (HOBt) (0.35 g, 2.6 mmol) were added. The solution was cooled to 0 °C and 1,3-dicyclohexylcarbodiimide (DCC) (0.54 g, 2.6 mmol) was added. The reaction mixture was stirred for 30 min at 0 °C and then over night at room temperature. White precipitate was removed by filtration. Next, the solvent was evaporated under reduced pressure giving the crude product. After purification by column chromatography using a dichloromethane : methanol (30:1, v/v) solvent system, the solvent was removed under reduced pressure to provide pure 1-[6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carbonyl]amino-1,1-diphosphonate tetraethyl ester (Boc-HYNIC–AMDPE, 0.48 g, 45%). 1H-NMR (CDCl3): δ 8.64 (s, 1H), 8.06 (s, 1H), 7.89–7.71 (m, 1H), 7.46–7.37 (m, 1H), 6.79 (s, 1H), 5.35 (m, 1H), 4.24–4.20 (m, 8H), 1.47 (s, 9H), 1.38–1.29 (m, 12H); MS (ESI): [M + H]+ 539.4.

To a solution of 1-[6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carbonyl]amino-1,1-diphosphonate tetraethyl ester (Boc-HYNIC–AMDPE, 0.22 g, 0.4 mmol) in CH2Cl2 (10 mL) at 0 °C was added trimethylsilyl bromide (TMSBr, 1 mL, 7.5 mmol). After stirring at room temperature for 72 h, the reaction mixture was evaporated, and to the residue was added methanol (10 mL). After stirring during 1.5 h, the methanol was removed by evaporation, and the residue was dried in a vacuum over P2O5 to obtain crude 1-[6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carbonyl]amino-1,1- diphosphonic acid (Boc-HYNIC–AMDP). After addition of a mixture of methanol (5 mL) and 3 M HCl (1 mL), the mixture was stirred during 30 min. The solvent was evaporated, and the residual solid was stirred in methanol (10 mL). The white precipitate was filtered off, washed with methanol, and dried under a stream of nitrogen to yield 1-(6-hydrazinopyridine-3-carbonyl)amino-1,1-diphosphonic acid (HYNIC–AMDP, 0.095 g, 71%) as white crystals. 1H-NMR (D2O): δ 8.02(s,1H), 7.81 (d, 1H), 6.74 (d, 1H), 5.15 (m, 1H); MS (ESI): [M + H]+ 327.1.

Labeling of HYNIC–AMDP

The radiolabeling of HYNIC–AMDP with 99mTc using tricine as coligand was performed by adding 0.5 mL solution of tricine (100 mg/mL in 0.9% saline) and 0.5 mL of SnCl2 solution (0.5 mg/mL in 0.1 N HCl) to a clean 10 mL vial containing 100 μL of HYNIC–AMDP solution (10 mg/mL in 0.9% saline), and the pH value was adjusted to 8 with 0.1 N HCl and 0.1 N NaOH. Then 1 mL freshly eluted 99mTcO4 − (15 MBq) from a commercial generator was added to that vial and it was kept at 100 °C for 15 min.

Radiochemical purity analysis

The final complexes were analyzed by TLC and HPLC for the radiochemical purity (RCP). The TLC was performed on a polyamide strip and eluted with saline. The reaction mixture was spotted on the start line, then the strip was developed using saline as a developing system. The strips were removed, dried, cut to 1 cm segments, and assayed for the radioactivity using a well-type γ-scintillation counter.

HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 × 4.6 mm 2), Shimadzu SCL-10AVP series. The flow rate was 1 mL/min. The mobile phase was isocratic with 80% solvent A (0.2 M phosphate buffer, pH 6.0) and 20% solvent B (methanol) at 0–5 min, followed by a gradient mobile phase going from 20% solvent B at 5 min to 25% solvent B at 15 min and to 30% solvent B at 25 min.

Stability test

The stability of 99mTc–HYNIC–AMDP was studied by measuring the RCP of the final complexes by HPLC at different times after preparation.

Determination of the partition coefficient (log P) for the complex

The octanol/buffer partition coefficient was measured according to the procedure described by Mukherjee et al. [20–22]. The partition coefficient was determined by mixing the complex with an equal volume of 1-octanol and phosphate buffer (0.025 mol/L, pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5000 rpm for 5 min. The two layers were separated and from each phase 0.1 mL of the aliquot was removed and counted separately in a well γ-counter. Each measurement was repeated three times. Care was taken to avoid cross contamination between the phases. The partition coefficient P was calculated using the following equation:

$$ P = \left( {{\text{cpm in octanol}} - {\text{cpm in background}}} \right)/\left( {{\text{cpm in buffer}} - {\text{cpm in background}}} \right) $$

Usually the final partition coefficient value was expressed as log P.

Biodistribution studies in mice

Biodistribution studies were carried out in ICR mice weighing 20–25 g. The final 99mTc complex solution (99mTc–HYNIC–AMDP, 99mTc–AMDP or 99mTc–MDP) was diluted to a concentration of 7.4 MBq/mL with saline. Then 0.1 mL of the diluted tracer solution was injected via a tail vein of mice (four groups each of five mice). The mice were sacrificed at 0.5, 1, 2, 3 h post injection and the organs of interested were weighted and counted in a NaI well-type γ-counter. The results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g). Corrections were made for background radiation and physical decay during experiment. All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation.

Results and discussion

Synthesis of HYNIC–AMDP

HYNIC–AMDP, the precursor of 99mTc–HYNIC–AMDP, was synthesized through a multiple-step reaction. The Boc-HYNIC–AMDPE was prepared by the coupling of the carboxyl group of Boc-HYNIC with the amino group of AMDPE. Following that, phosphonate esters were removed by addition of trimethylsilyl bromide (TMSBr) for 72 h to give Boc-HYNIC–AMDP, and HYNIC–AMDP was obtained according to the subsequent deprotection of the Boc group by addition of 3 M HCl/CH3OH for 30 min.The reaction was schematically shown in Scheme  1. All the intermediates and product HYNIC–AMDP were characterized by 1HNMR and MS. AMDP was obtained according to acid hydrolysis of AMDPE. The AMDP was characterized by 1H NMR and MS. 1H NMR (D2O) δ:3.40 (t,1H, J = 17.9 Hz, P-CH-P). MS (ESI): [M]+ 227.6.

Scheme 1
scheme 1

Synthesis of HYNIC–AMDP and Preparation procedure of 99mTc–HYNIC–AMDP

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Labeling of HYNIC–AMDP

99mTc–HYNIC–AMDP was prepared by a one-pot reaction of HYNIC–AMDP with 99mTcO4− and tricine in the presence of SnCl2. The radiochemical purity of 99mTc–HYNIC–AMDP was more than 95%.

99mTc–MDP was prepared by reconstitution with a conventional MDP labeling kit (Beijing Shihong Pharmaceutical Center) with a 99mTcO4− solution. AMDP was directly radiolabeled with 99mTc using SnCl2 as reducing agent at room temperature.

Radiochemical purity analysis

The radiochemical purity of the 99mTc–HYNIC–AMDP was routinely checked by TLC and HPLC. By TLC, in saline, the free 99mTcO4 − and 99mTcO2·nH2O remained near the origin with R f = 0.0–0.1, while 99mTc–HYNIC–AMDP migrated with the solvent front with R f = 0.8–1.0. An HPLC radiochromatogram was presented in Fig. 1. It was observed that the retention time of 99mTcO4 − was 3.5 min, while that of 99mTc–HYNIC–AMDP was found to be 12.7 min. Single peak suggested only one product (99mTc–HYNIC–AMDP) was formed. The mean radiochemical purity of the product was over 95% immediately after the preparation.

Fig. 1
figure 1

HPLC radiochromatogram of 99mTc–HYNIC–AMDP

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The radiochemical purity of 99mTc–AMDP and 99mTc–MDP was determined by TLC on polyamide strip eluted with saline. 99mTcO2·nH2O and 99mTcO4 − remained at the origin while 99mTc–AMDP/99mTc–MDP migrated with the front. The radiochemical purity of 99mTc–AMDP and 99mTc–MDP were both over 95% after preparation.

Stability test

The stability of 99mTc–HYNIC–AMDP was studied by measuring the RCP at different times after preparation. Its radiochemistry purity was more than 90% after 6 h, which indicated the complex possesses a great stability in the reaction mixture at room temperature.

Determination of the partition coefficient (log P) for the complex

The partition coefficient (log P) values of 99mTc–HYNIC–AMDP and 99mTc–AMDP at pH 7.4 were −2.77 and −1.52 respectively, suggesting two complexes were highly hydrophilic and 99mTc–HYNIC–AMDP was more hydrophilic than 99mTc–AMDP.

Biodistribution studies in mice

The results of biodistribution of 99mTc–HYNIC–AMDP, 99mTc–AMDP and 99mTc–MDP were presented in Tables  1, 2 and 3 separately.

Table 1 Biodistribution of 99mTc–HYNIC–AMDP in ICR mice (%ID/g)
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Table 2 Biodistribution of 99mTc–AMDP in ICR mice (%ID/g)
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Table 3 Biodistribution of 99mTc–MDP in ICR mice (%ID/g)
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As described in Table 1, 99mTc–HYNIC–AMDP had a high bone uptake and good target/non-target ratios. The B/B and B/M ratios increased from 1–3 h post injection. The kidney uptake was much higher than the hepatic. Early renal activity reflects urinary elimination of this complex. The uptakes of the tracer in other organs (muscle, lung, heart, and spleen) were within the normal values. As compared with 99mTc-AMDP, significantly higher bone uptake, B/B and B/M ratios of 99mTc–HYNIC–AMDP were observed. That indicated that the phosphonate groups maybe do not coordinate with technetium when labeling of HYNIC–AMDP using tricine as coligand, which could be responsible for high bone uptake of 99mTc–HYNIC–AMDP. Of course, the structure of 99mTc–HYNIC–AMDP was required to further study. Also, other factors may be influence the bone uptake, so further study could be required to illustrate the mechanism for bone localization.

As compared with 99mTc–MDP, 99mTc–HYNIC–AMDP had much higher bone uptake and B/B and B/M ratios. The comparison of biodistribution of 99mTc–HYNIC–AMDP with 99mTc–MDP at 1 h post injection was shown in Fig. 2. As seen from Fig. 2, the bone uptake and the ratios of B/B and B/M of 99mTc–HYNIC–AMDP is obviously much higher than the results of 99mTc–MDP at 1 h post injection, and the uptakes of 99mTc–HYNIC–AMDP in other organs (muscle, lung, heart, and spleen) were similar to the 99mTc–MDP. The comparison of blood clearance of 99mTc-HYNIC-AMDP with 99mTc-MDP was presented in Fig. 3. As seen from Fig. 3, the blood clearance of 99mTc–HYNIC–AMDP (from 0.70 ± 0.01 at 0.5 h to 0.38 ± 0.02%ID/g at 1 h post injection) was slightly faster than that of 99mTc–MDP(from 0.47 ± 0.13 at 0.5 h to 0.28 ± 0.05%ID/g at 1 h post injection) at early time after injection. By comparison, 99mTc–HYNIC–AMDP is superior to 99mTc–MDP with regard to the bone uptake and the B/M and B/B ratios at early time after injection, which suggested that it could be potentially useful for bone imaging at an earlier time after injection according to further investigations of the biological behavior of this complex.

Fig. 2
figure 2

The comparison of biodistribution of 99mTc–HYNIC–AMDP and 99mTc–MDP at 1 h post injection

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Fig. 3
figure 3

Blood clearance curves of 99mTc–HYNIC–AMDP and 99mTc–MDP

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The comparison of biodistribution of 99mTc–HYNIC–AMDP with some reported 99mTc–chelate–conjugated bisphosphonate derivatives was shown in Table 4. Although the animal species used in biodistribution studies of the 99mTc-complexes listed in Table 4 were slightly different, the amount of bone uptake and the ratio of B/B can be seen roughly. As seen from Table 4, 99mTc–HYNIC–AMDP exhibited the highest bone uptake (35.08 ± 4.92%ID/g at 1 h p. i.) and B/B ratio (92.32 at 1 h p. i.) at early time after injection among the four complexes. A decrease of B/B ratio in the order was observed: 99mTc–HYNIC–AMDP > 99mTc–MAG3–HBP > 99mTc–EC–AMDP > 99mTc–Gem/BP. And the order of the bone uptake was 99mTc–HYNIC–AMDP > 99mTc–Gem/BP > 99mTc–EC–AMDP > 99m Tc–MAG3–HBP. Compared with 99mTc–EC–AMDP (using EC as bifunctional chelator), the bone uptake (35.08 ± 4.92%ID/g at 1 h p. i.) and B/B ratio (92.32 at 1 h p. i.) of 99mTc–HYNIC–AMDP (using HYNIC as bifunctional chelator) were obviously much higher. Also, the biodistribution of 99mTc–HYNIC–AMDP in normal mice pointed to the possibility of the use of this tracer as bone imaging agent. However, many biological studies are required to establish these findings as the examination of the tracer in the skeletal lesions and normal bone and the quantitative determination of the tissue uptake of this tracer.

Table 4 Comparison of biodistribution of 99mTc–HYNIC–AMDP with some reported 99mTc-chelate-conjugated bisphosphonate derivatives
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Conclusion

In summary, the novel HYNIC-conjugated aminomethylenediphosphonate ligand HYNIC–AMDP has been successfully synthesized and 99mTc–HYNIC–AMDP was prepared in high yields using tricine as coligand. The high bone uptake, good retention and high target to non-target activity ratios of the 99mTc–HYNIC–AMDP in normal mice exhibited favorable properties, especially 99mTc–HYNIC–AMDP had a higher bone uptake and bone/blood ratio at early time after injection than 99mTc–MDP, suggesting that it could be potentially useful for bone imaging at an earlier time after injection according to further investigations of the biological behavior of this complex.