Introduction

Diphosphonates (DPs) are one of the important class of clinical drugs for bone disease (Vasireddy et al., 2003) and have been used in the therapy of early cancer treatment to prevent adverse effects of therapies on the bone health (Robert et al., 2011). They have experienced three generations. ZL is one typical third-generation DP and the most potent among the tested diphosphonates. For example, in preclinical models of bone resorption, ZL is at least 100 times more potent than either clodronate or pamidronate and it is at least 1,000 times more potent than etidronate (Smith, 2008).

As well known, DPs are good targeting ligands to serve as bone imaging agents (Jurisson et al., 1999). Up to date, a plenty of 99mTc-labeled DPs, such as 99mTc-MDP (Subramanian et al., 1975), 99mTc-HMDP (Bevan et al., 1980), and 99mTc-EHDP (Subramanian et al., 1972), have been widely used for many years in the clinical bone scanning. However, 99mTc-labeled DPs also present a set of clinical and chemical limitations (Vasireddy et al., 2003), such as the low specificity and relatively slow clearance from the blood and soft tissues, which usually leads to false negatives and delaying interval (2–6 h) between injecting and performing bone scanning (Love et al., 2003). To address these problems, a radiopharmaceutical with higher affinity for bone, lower tissue uptake, and more rapid clearance from the blood is required accordingly. Considering that the nature of ligand, DPs is a key factor in determining the performance of the bone imaging agent (Ogawa et al., 2006), so a novel diphosphonic acid was designed and synthesized to enable imaging at an earlier time.

To date, a number of 99mTc-labeled MDP analogs have been prepared and evaluated in animals (Pauwels et al., 2001; Love et al., 2003). We have also been involved for several years in the synthesis and biological evaluation of novel 99mTc-diphosphonate complexes for developing novel bone imaging agents (Luo et al., 2005; Niu et al., 2008; Chen et al., 2009; Lin et al., 2011). Previous studies have shown that optimization of the alkyl substituent in the imidazole ring of ZL or the carbon chain between the imidazolyl and geminal diphosphonate groups can bring significant influence on the biological properties of 99mTc-labeled complexes, including the bone uptake, blood, and soft tissues clearance. In a continuing effort to look for better bone imaging agent, therefore, a novel ZL derivative was designed and synthesized, 1-hydroxy-2-(2-butyl-1H-imidazole-1-yl) ethylidene-1,1-diphosphonic acid (BIDP, see Scheme 1). It was further labeled by the radionuclide technetium-99m. The in vivo biological performances of 99mTc-BIDP, such as the biodistribution, pharmacokinetics, and bone imaging, were investigated systematically.

Scheme 1
scheme 1

Chemical structures of the DP ligands

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Materials and methods

Reagents, instruments, and animals

All analytical chemical reagents were purchased from commercial sources and used without further purification. Mouse serum (MS) (through the centrifugal homemade) and human serum (HS) (supplied by Jiangyuan Hospital of Jiangsu Institute of Nuclear Medicine) were used for in vitro stability study. Technetium-99m was eluted as Na99mTcO4 from 99Mo/99mTc generator (radiochemical purity: 99.99 %; Jiangyuan Hospital of Jiangsu Institute of Nuclear Medicine).

Melting points were measured on Yanaco MP-500 melting point apparatus (Shimadzu, Japan). Electron spray ion mass spectra (ESI–MS) were determined using a Waters Platform ZMD4000 LC/MS (Waters, USA). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-500 spectrometer (Bruker, Germany), and the chemical shift values were referenced to the internal tetramethylsilane (TMS). The IR spectra were recorded on a Bruker TENSOR-27 spectrometer (Bruker, Germany) using KBr pellet in the region of 4,000–400 cm−1. The Xinhua chromatography paper (Shanghai, China) was used for thin layer chromatography (TLC). Analytic high performance liquid chromatography (HPLC) were accomplished on Waters 1525 Binary HPLC pump, a Waters 2487 dual λ absorbance detector, and a Perkin Elmer Radiomatic 610TR radioactivity detector, which were operated by Breeze and proFSA software. A Packard-multi-prias γ Counter (Perkins Elmer, USA) were used for the radioactivity counting. Philips SKY Light emission computed tomography (ECT) (Philips, USA) was used for the bone scanning of the rabbit.

Institute of Cancer Research (ICR) mice (18–22 g) and New Zealand rabbit (1.5–1.7 kg) were supplied by Shanghai SLAC Laboratory Animal Company. Animals are bred according to the standards set forth in the Guide for the Care and Use of Laboratory Animals (published by the National Academy of Science, National Academy Press, Washington, DC). All procedures and animal protocols were approved by our Institutional Animal Experimentation Committee.

Synthesis and radiolabeling

BIDP was synthesized according to the similar method as reported in the previous literatures (Lin et al., 2011; Widler et al., 2002). The synthesis route has been summarized in Scheme 2. In brief, BIDP (5 mg, dissolved in 0.1 mL 0.2 M NaOH solution), SnCl2·2H2O solution (100 μL, freshly prepared, 10 mg SnCl2·2H2O dissolved in 10.0 mL 0.5 M HCl solution), and Na99mTcO4 (92.50 MBq freshly eluted) were added into a penicillin vial in turn. The pH of the mixture was adjusted to pH = 6.0 by adding 0.2 M phosphate buffer solution (PBS). The final volume of the solution was adjusted to 2.0 mL by water. The reaction mixture was heated at 70 °C for 30 min.

Scheme 2
scheme 2

Synthesis of BIDP and 99mTc-BIDP: i BrCH2COOEt, KOH, K2CO3, tetrabutyl ammonium bromide (TBAB), CH2Cl2, reflux, 7 h; ii HCl, H2O, reflux, 7 h; iii PCl3, H3PO4 (85 %), PhCl, 120 °C, 10 h; iv HCl, H2O, reflux, 5 h; v SnCl2, Na99mTcO4, PBS (pH 6.0), 100 °C, 20 min

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Quality control

The radiolabeling yield (RLY) and radiochemical purity (RCP) of 99mTc-BIDP were determined by TLC method. Strips of Xinhua No. 1 paper chromatography of 13 cm long and 0.5 cm wide were marked at 1.5 cm from the bottom and lined into sections 1 cm each, up to 10 cm. About 3 μL 99mTc-BIDP solution was applied with a syringe at 1.5 cm from the bottom of paper strips, and then the strips were developed in distilled water and acetone. After complete development, the chromatographic paper strips were cut into pieces of 1 cm and the radioactivity of these pieces was counted by a γ counter to determine the RLY and RCP.

At the same time, the RLY and RCP of 99mTc-BIDP were also measured by HPLC method. The column was eluted with isocratic solvents of 70 % water and 30 % acetonitrile, and the flow rate was 1.0 mL min−1. Radioanalysis of the labeled compound was performed using a Cd (Te) detector.

In vitro stability

The in vitro stability of 99mTc-BIDP was studied in mouse serum (MS) and human serum (HS) for 1–6 h. In brief, 200 μL (about 3.7 MBq) of 99mTc-BIDP was added into 200 μL of MS and HS, respectively. After incubation at 37 °C for 1–6 h, an aliquot of HS and MS solution were added to 100 μL of 50 % trifluoroacetic acid (TFA). After centrifugation, the upper solution was taken for TLC analysis.

Octanol–water partition coefficient

The octanol–water partition coefficient (log P) was determined for 99mTc-BIDP at pH 7.4 by measuring the radioactivity of the radiolabeled compound in octanol and PBS at equilibrium, respectively. First, 99mTc-BIDP solution was diluted with PBS (100 + 900 μL). Then, the solution was further mixed with 1.0 mL octanol, vortexed for 5 min, and centrifuged at 4,000 rpm for 5 min to ensure complete separation of layers. An aliquot (100 μL) of organic and aqueous phases were collected and the radioactivity was measured with a γ counter. The log P was calculated using the formula log P = log (radioactivity in octanol layer/radioactivity in PBS layer). The reported value is the average obtained from three independent experiments.

Plasma protein binding assay

99mTc-BIDP (100 μL, 37 KBq) was mixed with the human plasma (100 μL) in the centrifuge tube. After the mixture was incubated at 37 °C for 2 h, the plasma protein was precipitated by adding 1.0 mL trichloroacetic acid (TCA, 250 g L−1) to the mixture. The supernatant and precipitate were separated by centrifugation at 4,000 rpm for 5 min. The radioactivities of both phases were measured separately. The above procedure was repeated thrice. The percentage of protein binding was determined by the following equation: \( {\text{Plasma binding}} \,\% = \left[ {{\text{Precipitate CPM}}/\left( {{\text{Precipitate CPM}} + {\text{Supernatant CPM}}} \right)} \right]\;* 100\,\% \)

Pharmacokinetics

For pharmacokinetic analysis, six ICR mice were intravenously injected with 7.4 MBq of 99mTc-BIDP. Blood samples were drawn from each rat through the tail vein by microliter pipette (20 μL) at several time points between 5 min and 6 h after injection. The radioactivity of each blood sample was measured for by the γ counter and expressed as a percentage of the injected dose per gram of blood (% ID g−1). Pharmacokinetic parameters were calculated using 3P97 software (edited by Chinese Mathematical Pharmacology, 1997, Beijing, China) (Xue et al., 1997). In the present work, the pharmacokinetic of 99mTc-BIDP can be described by the following biexponential equation: C = Ae−αt + Be−βt, where A and B are the extrapolated zero-time intercepts of the distribution and elimination phases, respectively; C is the plasma level of the tracer at any given time t; and α and β are rate constants of distribution and elimination phases, respectively.

Biodistribution studies

99mTc-BIDP (7.4 MBq, 0.2 mL) was administered to ICR mice via the tail vein injection. The mice were sacrificed by decapitation at 5, 10, 15, 30, 60, 120, and 240 min post injection. Interested organs were collected and weighed, and 200 μL of blood were taken from carotid artery. Their radioactivities were measured by a γ counter and expressed as % ID g−1 to determine the percentage of radioactivity absorbed by the organ or tissue. Then, the distribution in different organs was determined and the uptake rations of bone to other soft tissues were also obtained from the % ID g−1 values. The experiment at each time point was repeated five times. The data were expressed as mean values with standard deviations.

Bone imaging of rabbit

In order to comprehensively investigate the whole body localization of 99mTc-BIDP, SKY Light ECT was used to study the imaging of the rabbit. Prior to scanning, two New Zealand rabbits were anesthetized with ethyl carbamate solution (8.0 mL, 25 %) and fixed on the board. Pediatric urine bag was used to absorb the urine. Then 99mTc-BIDP (1.5 mL, 92.5 MBq) was injected intravenously into the rabbit through the marginal ear vein and bone scanning was carried out subsequently with ECT. The whole body image was observed and collected for 4 h. During the first hour, scanning images were collected every 5 min under the conditions of low energy, high resolution, and 128 × 128 matrix. Regions of interest (ROIs) were directly drawn on the SPECT composite image including bone, muscle, heart, liver, kidney, and stomach. Shapes and sizes of ROIs were kept constant for all subsequent images. The uptakes of femur, muscle close to femur, and other soft tissues were obtained, and the uptake ratios of bone to soft tissues were calculated from the same ROIs. Then, a series of static bone scanning images were collected at 1, 2, 3, and 4 h, respectively.

Results and discussion

Chemistry and radiolabeling

Compound 2 was prepared through the N-alkylation reaction and phase transfer catalytic (PTC) reaction, as illustrated in Scheme 3. To accelerate the reaction rate and achieve a satisfactory yield, TBAB was added to the reaction system as a phase transfer catalyst (Hui et al., 1999). Then, BIDP was obtained through the phosphine acidification reaction of the carboxylic acid. Noteworthy is that the speed of adding PCl3 has critical effect on the yield of the reaction. If the addition is fast, the reaction may be hard to control and the yield is also low. Otherwise, the reaction time may be too long. A moderate speed (about 0.4 mL min−1) should be selected to control the reaction temperature and increase the yield. The structure of BIDP and intermediate were confirmed by melting point, mass spectra, IR, 1H, and 13C NMR spectra.

Scheme 3
scheme 3

Mechanism of phase transfer reaction

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2-(2-Butyl-1H-imidazole-1-yl)acetic acid: yield: 26.6 %. mp: 138–141 °C; IR (KBr)/cm−1: 3,395 (ν O–H), 1,726 (ν C=O), 1,398 (ν C–N); MS(ESI): m/z = 182 (found: 181); 1H-NMR (400 MHz, DMSO): δ7.63 (d, 1H, CH-ring), 7.57 (d, 1H, CH-ring), 5.14 (s, 2H, COOHCH 2), 2.90 (t, 2H, CH3CH2CH2 CH 2), 1.61 (m, 2H, CH3CH2 CH 2), 1.25 (m, 2H, CH3 CH 2), 0.85 (t, 3H, CH2 CH 3); 13C-NMR (100 MHz, D2O): δ170.02 (s, COOH), 148.70 (s, N=C–N), 122.85 (s, C-ring), 118.02 (s, C-ring), 48.27 (s, N–CH 2), 27.97 (s, CH3CH2 CH 2), 23.76 (s, ring-CH 2), 21.38 (s, CH 2CH3), 12.78 (s, CH3).

1-Hydroxy-2-(2-butyl-1H-imidazole-1-yl) ethylidene-1,1-diphosphonic acid (BIDP): yield: 17.1 %. mp: 54–56 °C; IR (KBr)/cm−1: 3,410(ν O–H), 7,55 (ν P–C), 1,605 (ν C=N); MS(ESI): m/z = 328 (found: 329); 1H-NMR (400 MHz, NaOH/D2O): δ7.38 (d, 1H, CH-ring), 7.13 (d, 1H, CH-ring), 4.50 (s, 2H, NHCH 2), 2.98 (t, 2H, CH3CH2CH2 CH 2), 1.60 (m, 2H, CH3CH2 CH 2), 1.25 (m, 2H, CH3 CH 2), 0.79 (t, 3H, CH2 CH 3); 13C-NMR (100 MHz, D2O): δ151.56 (s, N=C–N), 123.94 (s, C-ring), 123.15 (s, C-ring), 75.57 (t, P–C–P), 50.29 (s, N–CH 2), 29.40 (s, CH3CH2 CH 2), 25.88 (s, ring-CH 2), 21.78 (s, CH 2CH3), 13.27 (s, CH3).

BIDP was further radiolabeled by Na99mTcO4 with deoxidation of stannous chloride. Also noteworthy is that the pH value is very important for the radiolabeling of BIDP. When the acidity of the reaction system is high (pH 1–3), the RLY will be less than 90 % due to the fact that 99mTcO4 − is not completely reduced in a strong acidic condition. On the contrary, when the pH is larger than 6, Na99mTcO4 will be easily changed into the colloid of 99mTc by SnCl2 under the alkaline condition, and therefore, the RLY will decrease significantly. So, the best pH is 4–6 where the RLY can be more than 95 %. The effect of other factors on the labeling yield, such as the amount of SnCl2•2H2O, DP, and 99mTcO4 −, has also been investigated according to the previous studies (Motaleb et al., 2011; Qiu et al., 2012). This labeling method also meets the clinical requirement for the production of other 99mTc-labeled diphosphonates, such as 99mTc-MDP.

Quality control

The radio-TLC analysis was used to monitor the progress of the radiolabeling reaction and check the purity of the radiolabeled compound. All of the chemical species involved in the radiolabeling reaction were separated by TLC method (Fig. 1a). In the system of distilled water, 99mTcO2·nH2O remained at the origin (R f = 0–0.1), while Na99mTcO4 and 99mTc-BIDP both migrate with the solvent front (R f = 0.8–1.0). In the system of acetone, 99mTcO2 · nH2O and 99mTc-BIDP remain at the origin (R f = 0–0.1), while Na99mTcO4 migrated with the solvent front (R f = 0.9–1.0). Under optimal radiolabeling condition, TLC analysis showed that Na99mTcO4 was completely reduced and 99mTc-colloidal amount was less than 2 %.

Fig. 1
figure 1

a RCP of 99mTc-BIDP in the acetone and water estimated by TLC. b RP-HPLC radioanalysis of 99mTc-BIDP (A) and Na99mTcO4 (B). Conditions: C18 reversed-phase column (10 μm, 250 × 4.6 mm), flow rate of 1.0 mL min−1 with CH3CN (30 %) and H2O (70 %). 99mTc-BIDP: Rt = 2.8 min, Na99mTcO4: Rt = 8.9 min

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The HPLC analyses of 99mTc-BIDP and Na99mTcO4 were shown in Fig. 1b. It was observed that the retention time of 99mTc-BIDP was 2.8 min, while that of Na99mTcO4 was 8.9 min. The single peak suggested that only one product (99mTc-BIDP) was formed. The specific activity of the radiotracer at the end of synthesis was estimated to be about 32 GBq/mmol. This value is comparable to that of other technetium-99 m labeled small molecule drug (Nayak et al., 2008). Since the radiotracer was used immediately after preparation for both in vitro and in vivo studies, the RCP was identical to the RLY. Therefore, the RCP of 99mTc-BIDP was also larger than 95 %.

In vitro stability

The stability was assessed by measuring the RCP based on the TLC analysis. As shown in Fig. 2, more than 95 % of 99mTc-BIDP remained intact in the MS and HS after 6 h of incubation. This suggested that the radiolabeling efficiency was high and 99mTc-BIDP was stable enough to allow further biological and imaging studies.

Fig. 2
figure 2

RCP of 99mTc-BIBDP in mouse serum and human serum determined by TLC analysis after incubation for 1, 2, 4, and 6 h, respectively

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Log P and plasma protein binding

The octanol–water partition coefficient (log P) of 99mTc-BIDP was determined to be −1.55, indicating that 99mTc-BIDP has high hydrophilic property. This may be attributed to the fact that the structure contains many hydroxyl groups and an imidazole ring. Compared with previous studies, we have found that the lipophilicity of 99mTc-BIDP was higher than 99mTc-MIDP, 99mTc-EIDP, and 99mTc-PIDP (Luo et al., 2005; Niu et al., 2008; Chen et al., 2009). Since log P is an important parameter for assessing the biological distribution of these complexes (Valko, 2004), higher lipid molecules have more positive effect on the bone binding model (Zhang et al., 2009). Thus, it is speculated that 99mTc-BIDP has more positive effect on the bone uptake than the previous 99mTc-DPs.

The percentage of protein binding was 58.93 % for 99mTc-BIDP. In general, the plasma protein binding efficiency also has significant influence on the bone uptake (Kroesbergen et al., 1988). Compared with 99mTc-MIDP, 99mTc-EIDP, and 99mTc-PIDP (Luo et al., 2005; Niu et al., 2008; Chen et al., 2009), 99mTc-BIDP has higher plasma protein binding. It turned out that with the extension alkyl chain in the ring, the plasma protein binding increases and the blood clearance will decrease. The blood clearance will be discussed in detail in the pharmacokinetic studies.

Pharmacokinetics of 99mTc-BIDP

In Fig. 3, a correlation between the blood radioactivity and time has been plotted for 99mTc-BIDP, which shows that the complex can be washed out quickly from the blood. And the blood clearance of 99mTc-BIDP accords well with a double exponential equation, C = 5.891e−0.0224t + 14.27e−0.420t + 0.3063. The distribution half-life (t 1/2α ) and elimination half-life (t 1/2β ) of 99mTc-BIDP were 1.65 and 30.91 min, respectively. This indicates that 99mTc-BIDP can be quickly absorbed by the blood and also eliminated rapidly from the blood (Liu et al., 2009).

Fig. 3
figure 3

Pharmacokinetic curve of 99mTc-BIDP in mice (n = 5, mean ± SD). t 1/2α : distribution half-life, t 1/2β : elimination half-life

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Biodistribution

Although ZL has been known to possess low toxicity and been used therapeutically at a high dose (Berenson et al., 2005), the toxicity profile of its derivatives is uncertain. In the period of biodistribution study, the mice were watched carefully for any sign of adverse reaction after the radiotracer 99mTc-BIDP was given via intravenous injection, respectively. Through the study period of 4 h, the mice showed no sign of toxicity. The biodistribution data of 99mTc-BIDP in normal mice have been listed in Table 1. It was observed that 99mTc-BIDP can reach the skeleton within 5 min after injecting, especially quickly for the joint. The amount of 99mTc-BIDP deposited in the bone began to increase continuously to a maximum of 24.60 %ID g−1 at 120 min. It has been proved that 99mTc-MIDP, 99mTc-EIDP, and 99mTc-PIDP have excellent bone binding efficiency. In order to better evaluate the bone uptake efficiency, the bone uptake of 99mTc-BIDP will be compared with them, as shown in Fig. 4. It was observed that the bone uptake of 99mTc-BIDP was higher than 99mTc-MIDP, 99mTc-EIDP, and 99mTc-PIDP at 15 min and 120 min. The results clearly show that an increase in the lipophilicity of the DP ligand through extending the alkyl chain in the imidazolyl group leads to the tendency of targeting and accumulation of 99mTc-DPs to the bone. It was also noteworthy that compared with those of the clinically widely used bone imaging agent 99mTc-MDP, the bone uptake of 99mTc-BIDP were all higher than those of 99mTc-MDP (Liu et al., 2004).

Table 1 Biodistribution of 99mTc-BIDP in normal mice (mean ± SD, n = 5)
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Fig. 4
figure 4

Comparison of 99mTc-MIDP, 99mTc-EIDP, 99mTc-PIDP, and 99mTc-BIDP in mice

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Moreover, the uptake of 99mTc-BIDP in blood and muscle were also higher than those of 99mTc-MIDP, 99mTc-EIDP, and 99mTc-PIDP, which can be observed intuitively from Fig. 4. This may be attributed to the fact that 99mTc-BIDP has a higher Log P value and plasma protein binding. However, at 60–120 min post injection, the uptakes in the blood and the muscle among these 99mTc-DPs are similar. In addition, the bone uptake of 99mTc-BIDP increased to a maximum of 24.60 % ID g−1 at 120 min. These results indicate that 99mTc-BIDP has larger uptake ratios of bone to blood and muscle among 99mTc-MIDP, 99mTc-EIDP, 99mTc-PIDP, and 99mTc-BIDP. Among all the non-target organs, the kidney uptake of 99mTc-BIDP was the highest but the concentration decreased quickly. It decreased from 12.7 % ID g−1 at 5 min to 7.07 % ID g−1 at 60 min. Therefore, a conclusion was drawn that 99mTc-BIDP can be excreted rapidly through urine. As a whole, the radiotracer 99mTc-BIDP has higher bone uptake and lower background at 60 min post injection, compared to 99mTc-MIDP, 99mTc-EIDP, and 99mTc-PIDP. In comparison with its analogs of 99mTc-labeled disubstituted zoledronic acids [99mTc-DMIDP (Qiu et al., 2012) and 99mTc-EMIDP (Lin et al., 2010)], the radiotracer 99mTc-BIDP also displays significantly higher bone to muscle uptake ratios from 60 min post injection. For instance, at 60 min the bone to muscle uptake ratio of 99mTc-BIDP (37.37) was larger than those of 99mTc-DMIDP (30.67) and 99mTc-EMIDP (29.79). At 120 min post injection, the bone to muscle uptake ratio of 99mTc-BIDP was much larger than those of 99mTc-DMIDP and 99mTc-EMIDP (98.40 vs. 60.25 and 33.04). The higher target-to-background ratio will help to obtain a clearer skeletal image. This indicates that 99mTc-BIDP is excellent and it holds great potential as a promising diagnostic radiopharmaceutical.

Bone imaging

Dynamic images

In order to better evaluate the bone imaging efficiency and biodistribution as well as clearance of 99mTc-BIDP in vivo, bone scanning images of the normal rabbit after intravenous administration of 99mTc-BIDP were collected dynamically and compared with those of 99mTc-ZL, which have been presented in Fig. 5. It was observed that these two compounds exhibited similar behavior in vivo, which mainly accumulated in the skeleton, kidney, and urinary bladder. It also demonstrated that 99mTc-BIDP was metabolized through the kidney of the rabbit, which is consistent with the biodistribution result of the mouse. Also noteworthy is that a clear image of the rabbit skeleton can be obtained at 1 h after injecting 99mTc-BIDP. This reveals that 99mTc-BIDP has highly selective bone uptake and rapid clearance from soft tissues in the rabbit after the first hour, which accords well with the biodistribution studies in mice.

Fig. 5
figure 5

Dynamic images of rabbit bone scanning in 1 h post injection of 99mTc-ZL (a) and 99mTc-BIDP (b)

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Static images

For comparison, a series of static images of the rabbit bone scanning after intravenous administration of 99mTc-BIDP and 99mTc-ZL, respectively, were also collected in Fig. 6. From these images, one can see that two radiolabeled complexes are all excreted from the kidney, and 99mTc-BIDP has highly selective skeletal uptake in the rabbit at different time intervals after intravenous injection. However, it is notable that 99mTc-BIDP is eliminated faster from the liver than 99mTc-ZL, and more satisfactory images can be obtained from 99mTc-BIDP. The rabbit SPECT imaging results agree well with the biodistribution studies in mice and suggest that 99mTc-BIDP possesses excellent characteristics for the potential application as a bone scanning agent. However, utilizing a bone pathology model to compare the bone lesion with the normal bone uptake will be necessary to more fully assess the clinical utility of 99mTc-BIDP. This will be taken into account in the subsequent research.

Fig. 6
figure 6

Static images of the rabbit bone scanning from 1 to 4 h post injection of 99mTc-ZL (a) and 99mTc-BIDP (b)

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Conclusion

In summary, a novel radiotracer 99mTc-BIDP was prepared with high radiolabeling yield, radiochemical purity, and stability. Kinetics of blood clearance showed that 99mTc-BIDP can be quickly absorbed by the blood and also eliminated from the blood rapidly. In the biodistribution studies, from 99mTc-MIDP to 99mTc-BIDP with the lipid solubility increasing, 99mTc-BIDP exhibits significant advantages on the bone uptake. At 1 h post injection, excellent images of the rabbit skeleton can be obtained from99mTc-BIDP, which was faster than 99mTc-ZL. So, 99mTc-BIDP displays attractive features as a promising bone scanning agent, and it is worth of further investigation as a novel SPECT imaging agent in larger animals or animals with bone metastases.