Preparation, Characterization, and Preliminary Imaging Study of [<sup>188</sup>Re]Re-Ibandronate as a Novel Theranostic Radiopharmaceutical for Bone Metastasis

Xu, Tingting; Wang, Yingwei; Chen, Zan; Liu, Hanxiang; Yang, Songsong; Liu, Guangfu; Zhao, Yan; Fu, Wenhui; Liu, Lin; Xiang, Ke; Peng, Dengsai; Chen, Yue

doi:https://doi.org/10.1155/2022/7684076

Contrast Media & Molecular Imaging

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Research Article | Open Access

Volume 2022 | Article ID 7684076 | https://doi.org/10.1155/2022/7684076

Preparation, Characterization, and Preliminary Imaging Study of [¹⁸⁸Re]Re-Ibandronate as a Novel Theranostic Radiopharmaceutical for Bone Metastasis

Tingting Xu,^1,2,3Yingwei Wang,^1,2,3Zan Chen,⁴Hanxiang Liu,^1,2,3Songsong Yang,^1,2,3Guangfu Liu,^1,2,3Yan Zhao,^1,2,3Wenhui Fu,^1,2,3Lin Liu,^1,2,3Ke Xiang,^1,2,3Dengsai Peng,^1,2,3and Yue Chen^1,2,3

Academic Editor: Alexey P. Kostikov

Received08 Oct 2021

Revised21 Dec 2021

Accepted01 Feb 2022

Published25 Feb 2022

Abstract

Background. Bone is a common site of metastasis from a malignant tumor. Several radiopharmaceuticals are available to relieve bone pain in patients with cancer. However, every radiopharmaceutical has its own disadvantages, and there is still a need to investigate easily accessible and high bone affinity radiopharmaceuticals. Ibandronate (IBA) and ¹⁸⁸Re were used for radiolabeling to develop and evaluate a novel type of bone-seeking radiopharmaceutical. Methods. The preparation conditions of [¹⁸⁸Re]Re-IBA were investigated, and thin-layer chromatography was used to analyze radiochemical purity. The stability, plasma protein binding rate, lipid-water distribution coefficient, safety and biodistribution in normal mice, and bone imaging of [¹⁸⁸Re]Re-IBA in New Zealand rabbits were studied. In addition, the nude mice model of bone metastasis was established, and biodistribution and imaging characteristics of [¹⁸⁸Re]Re-IBA in these nude mice were studied. Results. [¹⁸⁸Re]Re-IBA was successfully prepared with radiochemical purity >95%. The optimum preparation conditions were as follows: IBA, 0.8–1.4 mg; ascorbic acid, 0.2–0.5 mg; stannous chloride, 0.14–0.18 mg; potassium perrhenate, 0.005 mg; and [¹⁸⁸Re]ReO₄^- activity, 18.5–296 MBq, reacted for 30 min at 95°C with pH = 2. [¹⁸⁸Re]Re-IBA demonstrated good stability, high plasma protein binding rate, good hydrophilicity, and low toxicity. The biodistribution and bone imaging in normal animals showed rapid blood clearance, high bone uptake, low uptake in the solid organs and soft tissue, and high contrast during imaging. The biodistribution and imaging of bone metastasis in nude mice showed that [¹⁸⁸Re]Re-IBA has higher uptake in bone metastasis lesions than normal bone. Conclusions. Our study encompassed the successful preparation of [¹⁸⁸Re]Re-IBA, a novel bone-seeking radiopharmaceutical, and confirmed it has potential in the treatment of bone metastasis and monitoring through imaging.

1. Introduction

Bone is a common site of metastasis from a malignant tumor. Patients with bone metastasis usually experience severe and refractory pain. In addition, it may be accompanied by pathological fractures, spinal cord compression, hypercalcemia, and other complications, thus seriously affecting the quality of life [1]. Therefore, timely and effective treatment is needed to alleviate the symptoms and improve the quality of life.

The currently available treatments for bone metastasis are chemotherapy, external beam radiotherapy, surgery, bisphosphonate therapy, hormone therapy, and the use of painkillers [1]. In addition, the use of bone-seeking radiopharmaceuticals is also an effective method of relieving bone pain. It has the advantages of simultaneous treatment of multiple metastases, repeatability, and combination with other treatments [2]. Moreover, it can reduce or delay the incidence of skeletal-related events [3]. Numerous radiopharmaceuticals are available to relieve bone pain in patients with cancer, including [⁸⁹Sr]SrCl₂, [¹⁵³Sm]Sm-ethylenediamine tetramethylene phosphonate (EDTMP), [¹⁸⁶Re]Re-bisphosphonate hydroxyethylidene diphosphonate (HEDP), [¹⁸⁸Re]Re-HEDP, [¹⁷⁷Lu]Lu-EDTMP, and [²²³Ra]RaCl₂ [4, 5]. However, every radiopharmaceutical has its advantages and disadvantages. The choice is extremely dependent on the status of the patient, such as the renal function, bone marrow reserve, cancer extent, and physical properties of the radionuclides [6]. As the commercial availability of radiopharmaceuticals is limited, the availability of each radiopharmaceutical also needs to be considered. Moreover, most radionuclides used for treatment are produced through reactors; thus, they are extremely expensive. Therefore, it is important to choose an easily accessible and cost-effective radiopharmaceutical. Radionuclide ¹⁸⁸Re has an advantage in this regard because of its commercial extraction from ¹⁸⁸W/¹⁸⁸Re generators, which can be used on-demand and are cost-effective.

¹⁸⁸Re has a physical half-life of 16.9 h and can produce β⁻ rays with a maximum energy of 2.1 MeV which can be used for treatment [7]. It also emits γ rays with an energy of 155 keV for imaging, which facilitates visualizing the distribution of radioactive tracers in the body during treatment [7]. [¹⁸⁸Re]Re-HEDP is one of the most widely studied bisphosphonate radiopharmaceuticals in nuclear medicine which can relieve bone pain caused by prostate cancer, breast cancer, or other tumors [8]. In addition, a variety of new ¹⁸⁸Re labeled bisphosphates have been synthesized, such as [¹⁸⁸Re]Re-diethylenetriamine-N,N,N′,N″,N″-pentakis acid [9], [¹⁸⁸Re]Re-nitrilotris acid [10], [¹⁸⁸Re]Re-ethylenediamine–N,N,N,N-tetrakis acid [11], [¹⁸⁸Re]Re-pamidronate (PMA) [6], [¹⁸⁸Re]Re-zoledronate (ZNA) [12], and [¹⁸⁸Re]Re-risedronate [13]. These drugs show potential for application in preclinical studies. It is still necessary to identify bisphosphonates with a stronger ability to target bones for ¹⁸⁸Re labeling. Bisphosphonates are analogues of endogenous pyrophosphates, characterized by P-C-P bonds. There is a hydroxyl group attached to one side of the carbon atom, which has a high affinity for calcium phosphate, the primary mineral of the bone [6]. Moreover, there is a side-chain structure attached to the other side of the carbon atom that inhibits bone resorption [6]. The side-chain structure of the first-generation bisphosphonate does not contain nitrogen, and HEDP is one of its representative drugs. The second- and third-generation bisphosphonates contain nitrogen, and their ability to inhibit bone resorption is significantly stronger. Furthermore, third-generation bisphosphonates also contain a heterocyclic structure, and their bone affinity and ability to inhibit bone resorption are significantly stronger. However, nitrogen-containing bisphosphonates may have significant side effects, including renal failure, hypocalcemia, and osteonecrosis of the jaw [14]. The choice of drugs with lower toxicity is an important factor in determining the treatment for patients with renal insufficiency. Ibandronic acid and zoledronic acid are the most powerful and widely used third-generation bisphosphonates. The studies in [15, 16] on the effects of the aforementioned bisphosphonates on renal safety have reported on the possible occurrence of nephrotoxicity while using zoledronic acid. Nonetheless, the nephrotoxicity of ibandronic acid is extremely low and equivalent to that of the placebo. Another study [17] conducted on 44 patients treated with ibandronate (IBA) reported no impairment of renal function during an average follow-up of 18.5 months. In addition, Han et al. [18] noted that the pain relief rate and improvement in the quality of life in patients with bone tumors were higher in the ibandronic acid group than in the zoledronic acid group ( < 0.05). Nonetheless, the rate of adverse reactions was lower in the ibandronic acid group than in the zoledronic acid group ( < 0.05). There have been no reports on ¹⁸⁸Re labeled with IBA. Therefore, IBA and ¹⁸⁸Re were selected for radiolabeling to develop and evaluate a new radiopharmaceutical with potential bone-seeking properties and low toxicity, which may contribute to individualized treatment in the era of precision medicine. Light will be shed on the preparation, optimization of conditions, biological and safety evaluation, and imaging studies of [¹⁸⁸Re]Re-IBA.

2. Materials and Methods

2.1. Materials

[¹⁸⁸Re]NaReO₄ was eluted from the alumina based ¹⁸⁸W/¹⁸⁸Re generator (OncoBeta, Germany) with saline solution (0.9% NaCl). IBA was purchased from Twbio Technology Co., Ltd., Beijing, China. Ascorbic acid, potassium perrhenate (KReO₄), and stannous chloride (SnCl₂) were purchased from Macklin Biochemical Co., Ltd., Shanghai, China. Xinhua No. 1 chromatography paper (Xinhua Paper Industry Co., Ltd., Hangzhou, China) was used for paper chromatography (PC). Other equipment, chemicals, and animals used in the experiment were provided by the Nuclear Medicine and Molecular Imaging Key Laboratory of Sichuan Province. All studies were approved by the Ethics Committee of Southwest Medical University.

2.2. Methods

2.2.1. Radiolabeling and Quality Control

Various reaction parameters were studied by using the control variables method (changing one parameter at a time) to determine the effects of IBA (Figure 1), the antioxidant (ascorbic acid), carrier (KReO₄), reducing agent (SnCl₂), [¹⁸⁸Re]ReO₄^- activity, pH, temperature, and reaction time on the radiochemical purity (RCP) of [¹⁸⁸Re]Re-IBA. First, 0.1–1.8 mg, 0–0.5 mg, 0.02–0.4 mg, and 0–0.019 mg of IBA, ascorbic acid, SnCl₂, and KReO₄ were mixed sequentially. Then, the fresh eluted [¹⁸⁸Re]ReO₄^- solution 18.5–444 MBq was added. Subsequently, the pH value was adjusted to 0.5–4 with 1 N sodium acetate solution and 1 N hydrochloric acid. The reaction occurred at room temperature (25 ± 2°C), 60°C, or 95°C for 10–60 min, respectively. After the reaction was completed, the solution was cooled to room temperature (25 ± 2°C). The pH value of each tube was adjusted to 6–7. An aseptic filter membrane of 0.22 μm was used for sterilization and filtration. In this study, the specific fixed values of all parameters were as follows: IBA, 1.0 mg; ascorbic acid, 0.3 mg; SnCl₂, 0.14 mg; KReO₄, 0.005 mg; and [¹⁸⁸Re]ReO₄^- activity, 37 MBq, reacted for 30 min at 95°C and pH = 2.

The RCP of [¹⁸⁸Re]Re-IBA was determined by PC. Acetone and 0.9% NaCl were used as the eluents. A TLC scanner was used to measure the distribution of radioactivity on the PC strips. The RCP of [¹⁸⁸Re]Re-IBA was calculated from the peak area measurements as follows:

RCP = 100% − (% [¹⁸⁸Re]ReO₄^- + % [¹⁸⁸Re]Re-colloid).

2.2.2. In Vitro Stability

Freshly prepared [¹⁸⁸Re]Re-IBA was incubated in 0.9% NaCl and fresh human serum at 37°C. The RCP of the tubes was determined by PC at 1 h, 3 h, 6 h, 12 h, and 24 h. The experiment was repeated thrice. The results are expressed as mean ± standard deviation ( ± s).

2.2.3. Plasma Protein Binding Rate

0.1 mL fresh human plasma and freshly prepared 1.85 MBq [¹⁸⁸Re]Re-IBA were added in a tube labeled A and incubated at 37°C for 2 h. Then, 25% trichloroacetic acid solution (1 mL) was added to tube A and centrifuged under a centrifugal force of 587 g for 5 min. The supernatant was collected into another tube labeled B. Centrifugation was repeated, and the supernatant was collected thrice. A γ counter was used to measure the radioactivity counts of tubes A and B. After repeating the experiment thrice, the plasma protein binding (PPB) rate was calculated as follows: PPB = [(A − background)/(A + B − background × 2)] × 100%. The results are expressed as mean ± standard deviation ( ± s).

2.2.4. Lipids and Water Distribution Coefficient

Freshly prepared 1.85 MBq [¹⁸⁸Re]Re-IBA was added to a tube labeled A and it was shaken for 20 min with a vortex mixer, followed by centrifugation at 587 g for 5 min. The 0.1 mL upper liquid (organic phase) was collected into a tube labeled B. The 0.1 mL lower liquid (water phase) was collected into a tube labeled C. The radioactivity counts of the organic phase and water phase were measured by a γ counter. After repeating the experiment thrice, the lipid-water partition coefficient (logP) was calculated by using the following formula: logP = log[(B-background)/(C-background)]. The results are expressed as mean ± standard deviation ( ± s).

2.2.5. Toxicity Test of Mice

Sixteen Kunming mice were randomly divided into four groups (equal number of males and females): normal control group and low-, middle-, and high-dose [¹⁸⁸Re]Re-IBA groups. The control group was injected with 0.9% NaCl, and the experimental groups were injected with [¹⁸⁸Re]Re-IBA solution of 3.7 MBq, 18.5 MBq, and 37 MBq, respectively. The body weight and general condition of the mice in each group were measured. After 4 weeks, the blood of the mice in each group was drawn for routine blood examination and liver and kidney function. A pathological examination of important tissue and organs was performed.

2.2.6. The Biodistribution of [¹⁸⁸Re]Re-IBA and [¹⁸⁸Re]ReO₄^- in Mice

Twenty Kunming mice (originated from Swiss albino mice and introduced into Kunming, China.) were randomly divided into five groups (equal number of males and females). Each group was injected with 3.7 MBq [¹⁸⁸Re]Re-IBA. Then, the mice were sacrificed by CO₂ asphyxiation at 1 h, 3 h, 6 h, 24 h, and 48 h. The blood and important tissue and organ samples were collected to measure the radioactivity count by γ counter. Following the time attenuation correction, the percentage injection dose rate per gram of tissue (%ID/g) was calculated at each time point. The result is expressed as mean ± standard deviation ( ± s). The in vivo distribution of [¹⁸⁸Re]ReO₄^- was studied using the aforementioned method.

2.2.7. Imaging of New Zealand Rabbits with [¹⁸⁸Re]Re-IBA

The New Zealand rabbit was anesthetized by intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg). Then, 100 MBq [¹⁸⁸Re]Re-IBA was injected. Bone imaging was performed at different time points (acquisition equipment: American GE Infinia T4 double probe single-photon emission computed tomography, high energy collimator; scanning parameters: energy peak: 155 keV, posture: supine position, matrix: 128 × 128, and window width: ±10%). Following image acquisition, the images were processed by using the software of the postprocessing workstation.

2.2.8. Biodistribution and Imaging of Bone Metastasis Nude Mice

The nude mice of the bone metastasis model were established by tibial bone marrow injection. The specific methods were as follows: 25 μL of prostate cancer PC-3 and breast cancer MDA-MB-231 cell culture medium were injected into the left tibial bone marrow cavity of healthy male and female nude mice, respectively. At approximately 3-4 weeks after inoculation, they were scanned by microcomputed tomography (CT, SIEMENS InveonTM, Munich, Germany) to determine the condition of the model. If CT showed bone destruction in the left tibia (osteolytic, osteogenic, or mixed types); it suggested that the model was successful. Bone metastases were initially evaluated by CT and confirmed by pathology after the completion of related studies.

Ten PC-3 and ten MDA-MB-231 nude mice with bone metastasis were randomly divided into five groups. The mice were sacrificed by CO₂ asphyxiation at 1 h, 3 h, 6 h, 24 h, and 48 h. The important tissue and organ samples were collected for the biodistribution study.

The nude mice were injected with [¹⁸F]F-sodium fluoride (NaF), [^99mTc]Tc-methylene diphosphonate (MDP), and [¹⁸⁸Re]Re-IBA at the first, second, and fifth days, respectively, for bone imaging. Photon emission tomography/computed tomography (PET/CT) acquisition equipment was SIEMENS InveonTM. Single-photon emission computed tomography/computed tomography (SPECT/CT) acquisition equipment was GE SPECT, pinhole collimator.

2.2.9. Statistical Analysis

SPSS 26.0 was used for statistical analysis, and the quantitative data were expressed as mean ± standard deviation ( ± s). In the toxicity test, repeated-measures analysis of variance was used to compare the body weight of mice in each group. < 0.05 was considered statistically significant.

3. Results

3.1. Radiolabeling and Quality Control

A third-generation bisphosphonate derivative targeting bone metastasis [¹⁸⁸Re]Re-IBA was prepared. The proposed structure of [¹⁸⁸Re]Re-IBA is represented in Figure 2. The optimum preparation conditions in our study were as follows (see Figure 3): IBA, 0.8–1.4 mg; ascorbic acid, 0.2–0.5 mg; SnCl₂, 0.14–0.18 mg; [¹⁸⁸Re]ReO₄^- activity, 18.5–296 MBq; and KReO₄, 0.005 mg, reacted for 30 min at 95°C and pH = 2. Typical radioactivity distribution in PC is shown in Figure 4. Upon using acetone as an eluent, the colloidal impurities [¹⁸⁸Re]ReO₂ and product [¹⁸⁸Re]Re-IBA remained at the origin of the PC strip; however, [¹⁸⁸Re]ReO₄^- moves with the eluent front (Figure 4(a)). In contrast, upon using saline as the eluent, the colloidal impurities [¹⁸⁸Re]ReO₂ remained at the origin, while [¹⁸⁸Re]ReO₄^- and the product [¹⁸⁸Re]Re-IBA move with the eluent front (Figure 4(b)). [¹⁸⁸Re]Re-IBA could be consistently prepared in > 95% RCP under the best preparation conditions. Moreover, it did not require further purification.

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3.2. In Vitro Stability

Table 1 summarizes the results of in vitro stability. The results show that RCP is 91.44% and 93.06% in 0.9% NaCl and serum at 24 h, respectively, showing that [¹⁸⁸Re]Re-IBA has good stability.

3.3. Plasma Protein Binding Rate and Lipids and Water Distribution Coefficient

The PPB of [¹⁸⁸Re]Re-IBA incubated in plasma for 2 h was 79.8 ± 0.71%. logP of [¹⁸⁸Re]Re-IBA was - 2.33 ± 0.02, which indicated its high hydrophilicity.

3.4. Toxicity Test of Mice

There were no adverse reactions in all mice within 4 weeks, and the weight of each group increased with the increase of time (see Figure 5). The mean values of routine blood and liver and kidney function examination of the four groups were within the normal reference range (see Figure 6). Compared with the normal control group (see Figure 7), there were no abnormal pathological changes in important tissues and organs among the low-dose group (3.7 MBq), medium-dose group (18.5 MBq), and high-dose group (see Figure 8). In addition, there was no significant difference in weight among the low-dose group (3.7 MBq), medium-dose group (18.5 MBq), and control group. However, there were significant differences between the high-dose group (37 MBq) and the other three groups, and the average difference between the high-dose group and the other three groups was negative, which suggested that high-dose [¹⁸⁸Re]Re-IBA inhibited the weight gain of mice to a certain extent. However, the dose of 37 MBq was too high for mice, and the body weight of this group still showed an increasing trend, and the pathological results were not abnormal, suggesting that [¹⁸⁸Re]Re-IBA is safe and has low toxicity.

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3.5. The Biodistribution of [¹⁸⁸Re]Re-IBA and [¹⁸⁸Re]ReO₄^- in Mice and Imaging of New Zealand Rabbits with [¹⁸⁸Re]Re-IBA

Table 2 summarizes the biodistribution of [¹⁸⁸Re]Re-IBA in mice. A rapid blood clearance and low uptake of [¹⁸⁸Re]Re-IBA in the soft tissue, brain, liver, lung, and spleen were observed. The bone uptake of [¹⁸⁸Re]Re-IBA is relatively high, and the %ID/g reaches the maximum at 6 h (7.724 ± 2.292%ID/g), and %ID/g remains 5.239 ± 2.029 at 48 h. In addition, the radioactivity ratio of the bone to heart, liver, and muscles is rather high at 48 h. In addition to the bone, the highest uptake of [¹⁸⁸Re]Re-IBA occurred in the kidneys, which is related to the kidney as the primary excretory organ. Table 3 outlines the distribution of [¹⁸⁸Re]ReO₄^- in mice. The highest uptake of [¹⁸⁸Re]ReO₄^- occurs in the stomach and thyroid gland. However, the uptake in the bone is negligibly low. Furthermore, the radioactivity ratio of bone to heart, liver, and muscle tissue is low, and the highest ratio is as low as 2.372 (bone/muscle, 3 h).

Figure 9 depicts the bone imaging of the New Zealand rabbits in different time points. There was an obvious accumulation in bones and mild tracer uptake in soft tissue at 20 min after the injection. Over time, the soft tissue accumulation gradually faded and disappeared, and the bone imaging was clear with a high contrast between the bone and the background. In addition, the elimination of most of the [¹⁸⁸Re]Re-IBA activity was through the kidneys, which was consistent with the biodistribution in mice.

3.6. Biodistribution and Imaging of Bone Metastasis Nude Mice

Table 4 summarizes the biodistribution of [¹⁸⁸Re]Re-IBA in bone metastasis nude mice. A rapid blood clearance and low uptake in soft tissue, brain, liver, lung, and spleen of [¹⁸⁸Re]Re-IBA were observed, which is consistent with the biodistribution in mice. The comparison between the model bone and the contralateral bone shows that the uptake of the model bone is lower than that of the contralateral bone at 1–3 h but higher than that of the contralateral bone from 6 to 48 h.

[¹⁸⁸Re]Re-IBA bone imaging of nude mice with bone metastasis showed that, at 1–3 h after injection, the uptake in the model side was higher than that of the control side, but the concentration was mainly distributed in the surrounding soft tissue; at 6 h, 16 h, and 32 h, the bone uptake of the model bone was higher than that of the contralateral bone. Table 5 shows the ROI ratio of model bone/contralateral bone in nude mice of bone metastasis model at 6 h, 16 h, and 32 h.

4. Discussion

The amount of ligand IBA is an important evaluation parameter in the labeling process. A sufficient number of ligands must be provided in the formula to achieve high RCP complex formation. However, using excessive quantities of bisphosphonate ligand should be avoided to achieve high specific activity labeling and avoid the formation of undesired side product, which increases the impurity and inversely affects the labeling yield [6, 12]. Our study found that the highest RCP complex formation can be obtained when the number of ligands in the formula is 0.8–1.4 mg. With further increase of the dosage, the RCP gradually decreased, which may be due to the nonlabeled ligand which interfered with the labeling process or the presence of too many ligands making the reaction solution supersaturated and thus affecting the labeling. In this study, ascorbic acid was used as the antioxidant and stannous ion stabilizer. The results showed that when the quantity of ascorbic acid in the formula was 0.15–0.5 mg, [¹⁸⁸Re]Re-IBA could be prepared in >95% RCP. An inorganic compound of tin, SnCl₂, has been widely used in nuclear medicine as a reducing agent for pharmaceutical products radiolabeled with ^99mTc or ¹⁸⁸Re [19]. In our study, SnCl₂ was chosen as the reducing agent to reduce [¹⁸⁸Re]ReO₄^- to a lower oxidation state and facilitate its reaction with IBA. Despite its widespread use, there are several side effects associated with this ion and its derivatives described in the literature, for example, irritation of oral mucosa in rats [20], deterioration in semen quality of male rabbits [21], free radical induction and damage to liver and kidney function in rabbits [22], and marked hazardous alterations in some enzymatic activities and biochemical parameters in rabbits [23]. Furthermore, genotoxicity, mainly related to SnCl₂, has been reported [24], which could mediate single-strand breaks in plasmid DNA through reactive oxygen species (ROS) formation [19]. Our research showed that the RCP is high enough when the amount of SnCl₂ in the formula reaches 0.14–0.18 mg. An increase in the amount of SnCl₂ beyond this dosage range is not beneficial to the labeling yield and might lead to the formation of colloidal impurities in the product. However, an insufficient amount of SnCl₂ will not reduce all [¹⁸⁸Re]ReO₄^-. Therefore, it is necessary to ensure an optimum ratio of SnCl₂ with IBA and [¹⁸⁸Re]ReO₄^-, and the excess SnCl₂ should be removed as much as possible. In addition, avoiding the potential toxicity of SnCl₂ in the formula is also a problem worth discussing. Several studies showed that ascorbic acid, a well-known antioxidant and water-soluble ROS scavenger, is capable of detoxifying the hazardous effects of SnCl₂, including alleviation of reproductive toxicity [21] and the toxicity to some enzyme activities and oxidative damage [22], as well as increase in the activities of antioxidant enzymes [23]. Therefore, the ascorbic acid used in our formula may also act as a protectant against the toxicity of SnCl₂. The ¹⁸⁸Re obtained in the generator was carrier-free. In previous studies [9, 25–32], [¹⁸⁸Re]Re-bisphosphonate, without cold carrier rhenium, had a poor stability and little bone uptake. Hence, nonradioactive KReO₄ was added as a cold perrhenate carrier to ensure the stability and bone uptake.

In addition, [¹⁸⁸Re]Re-IBA, like [¹⁸⁸Re]Re-HEDP [33], [¹⁸⁸Re]Re-ZNA [12], and [¹⁸⁸Re]Re-ABP [34], may be an anionic six-coordinated complex with one metal atom bound to two IBA ligand molecules. Figure 2 shows the proposed structure of [¹⁸⁸Re]Re-IBA. However, the structure we provide is only a basic coordination structure. In fact, [¹⁸⁸Re] Re-IBA is likely to exist in a form with a complex spatial structure. Elder et al. [35] carefully studied the structure of Re-HEDP and found that medically effective species of Re-HEDP with Re-Re bonds may exist in two forms of complex mixture: as a linear tetramer of rhenium atoms bridged by HEDP ligands, HEDP ligands which also bind an equivalent number of tin atoms with additional HEDP ligands, or as a triangular cluster of rhenium atoms bicapped by two HEDP ligands and bridged to three tin atoms by HEDP to form a complex. As a similar compound of Re-HEDP, we speculate that [¹⁸⁸Re]Re-IBA may also exist in a similar structural form. Therefore, its structure may contain Re-Re bonds and tin atoms, bridged by HEDP ligands, forming a complex spatial structure. However, at present, little is known about the specific structure of this radiopharmaceutical. To determine the exact structure of [¹⁸⁸Re]Re-IBA, further research should be pursued.

The stability of radiopharmaceuticals is extremely important for therapy. Our study showed that the [¹⁸⁸Re]Re-IBA has good stability, which is beneficial for further research. The differences in stability between [¹⁸⁸Re]Re-IBA and other drugs were also compared, and the results showed that there was no significant difference in stability between [¹⁸⁸Re]Re-IBA and [¹⁸⁸Re]Re-HEDP (91.44 ± 0.8% versus 91.2% at 24 h) in 0.9% NaCl [32], but its stability improved compared to [¹⁸⁸Re]Re-risedronate (80.3% at 24 h) and [¹⁸⁸Re]Re-PMA (81.9% at 24 h) [6, 13]. In addition, the stability of [¹⁸⁸Re]Re-IBA (93.06 ± 1.6% at 24 h) in human serum was improved compared to those of [¹⁸⁸Re]Re-ZNA (91.53 ± 1.39% at 24 h)) and [¹⁸⁸Re]Re-risedronate (73 ± 1.21% at 24 h) [12, 13].

Radiopharmaceuticals should achieve ideal therapeutic effects and minimize the related toxicity to gain efficacy as therapeutic drugs [4]. Toxicity test of mice showed that [¹⁸⁸Re]Re-IBA is safe and has low toxicity; thus it can be used for later imaging and therapeutic research. The biodistribution and imaging of normal animals indicated that [¹⁸⁸Re]Re-IBA highly targets the bone and can remain in the bone for a long time. As expected, the in vivo distribution of [¹⁸⁸Re]ReO₄^- revealed that the free [¹⁸⁸Re]ReO₄^- was poorly targeted to the bone, thus confirming the success and bone-targeting capability of the labeled [¹⁸⁸Re]Re-IBA. In addition, the imaging results of the New Zealand rabbits revealed fast soft tissue clearance and low nontarget tissue uptake, and the overall image quality was good. Hence, [¹⁸⁸Re]Re-IBA has potential for the treatment of bone pain and can also be used to monitor therapeutic imaging.

The bone uptake of [¹⁸⁸Re]Re-IBA in normal mice was also compared with those of other ¹⁸⁸Re labeled bisphosphonates [6, 12, 36–38]. Although the mouse strain used for evaluation of [¹⁸⁸Re]Re-IBA (Kunming mice) is not completely the same as those used for evaluation of other ¹⁸⁸Re labeled bisphosphonates (Swiss mice or Kunming mice), we believe that these data can be compared considering that Kunming mice also originate from the Swiss mice strain. We noted that, among the ¹⁸⁸Re labeled third-generation bisphosphonates (i.e., IBA and ZNA), [¹⁸⁸Re]Re-IBA had the highest bone uptake. The highest femur bone uptake value of [¹⁸⁸Re]Re-IBA in Kunming mice was 7.724 ± 2.292%ID/g (6 h), and the minimum value was 5.239 ± 2.029%ID/g (48 h). The bone uptake of [¹⁸⁸Re]Re-ZNA in Swiss mice reached the maximum at 1 h (1.08 ± 0.14%ID/g); however, with the extension of time, the bone uptake decreased gradually and reached the lowest value of 0.36 ± 0.06%ID/g at 24 h [12]. In addition, the highest and lowest bone uptakes of ¹⁸⁸Re labeled second-generation bisphosphonate PMA in Swiss mice were 0.81 ± 0.25%ID/g (4 h) and 0.40 ± 0.05%ID/g (1 h), respectively [6]. Therefore, according to the existing research results, their bone activity uptake is categorized as [¹⁸⁸Re]Re-IBA > [¹⁸⁸Re]Re-ZNA > [¹⁸⁸Re]Re-PMA. In addition, the comparison results in bone uptake of [¹⁸⁸Re]Re-HEDP (Kunming mice or Swiss mice) and [¹⁸⁸Re]Re-IBA (Kunming mice) showed different conclusions. In two studies [37, 38], it was found that the bone uptake of [¹⁸⁸Re]Re-HEDP in Kunming mice (22.36 ± 4.11%ID/g at 24 h and 11.08 ± 4.25%ID/g at 24 h, respectively) was significantly higher than that of [¹⁸⁸Re]Re-IBA (6.177 ± 2.163%ID/g at 24 h). However, in another study [36], the maximum bone uptake of [¹⁸⁸Re]Re-HEDP (1.0 ± 0.1%ID/g at 4 h) in Swiss mice was significantly lower than that of [¹⁸⁸Re]Re-IBA (7.724 ± 2.292%ID/g at 6 h). The differences in these results may be related to the differences in the drugs obtained by different labeling methods or the differences in experimental operation methods. The differences of osteophilic properties between [¹⁸⁸Re]Re-IBA and other ¹⁸⁸Re labeled bisphosphonates, especially [¹⁸⁸Re]Re-HEDP, should be directly compared in future studies.

In addition, we noticed that there was a significant uptake of [¹⁸⁸Re]Re-IBA in the joints of the New Zealand rabbits upon bone imaging. El-Mabhouh and Mercer [39] explained that the joints are areas of increased osteoblastic activity in young animals due to bone growth; in addition, the distribution of bisphosphonates in the bone is not uniform but will rather be concentrated in the joints where bone growth and consequently osteoblastic activity are more pronounced. This may explain the significant imaging agent activity in the joint area of the New Zealand rabbits. The joint bone surface will provide an animal model for the activity present at sites of osteoblastic bone metastases [39].

Biodistribution and imaging of bone metastasis in nude mice showed that the uptake of [¹⁸⁸Re]Re-IBA in the model bone is higher than that in the normal bone, suggesting that [¹⁸⁸Re]Re-IBA is more targeted in bone metastasis. However, there was only a slightly increased uptake of [¹⁸⁸Re]Re-IBA in the model bone compared to that in the contralateral bone, which may be due to almost all model bone being osteolytic lesions, which may be related to the way of modelling. In addition, we speculate that [¹⁸⁸Re]Re-IBA may have better bone targeting in bone metastasis than the conventional bone imaging using [^99mTc]Tc-MDP. However, the current research is limited by the detection performance of our SPECT/CT equipment for nude mice and the lack of an osteogenic metastasis model, which will be further improved in future research.

5. Conclusions

This study encompassed the successful preparation of [¹⁸⁸Re]Re-IBA, a novel bisphosphonate radiopharmaceutical. The aforementioned radiopharmaceutical has the advantages of a simple preparation method, high stability and PPB, good hydrophilicity, and low toxicity. The in vivo biological distribution in mice and imaging of New Zealand rabbits confirmed the following: rapid blood clearance, high affinity to the bone, long retention time in the bone, low uptake in the solid organs and soft tissue, and high contrast of imaging. The biodistribution and imaging of bone metastasis nude mice confirmed that [¹⁸⁸Re]Re-IBA has a higher bone affinity to bone metastasis lesion than normal bone. Therefore, [¹⁸⁸Re]Re-IBA is a suitable candidate for the treatment of bone metastasis and monitoring the therapeutic effects. However, the method of modelling should be further improved to obtain an osteogenic metastasis model that is more suitable for research. In addition, the efficacy of [¹⁸⁸Re]Re-IBA in the treatment of animal models of bone metastasis and comparisons with other radiopharmaceuticals should be explored to completely clarify its value. This is the goal of our future research.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure

An earlier version of the manuscript has previously been presented in the form of a preprint [40].

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

TX, YW, and ZC contributed to the study design and TX wrote the manuscript. HL, SY, GL, YZ, WF, LL, KX, and DP are responsible for the integrity of the data and the accuracy of the data analysis. YC was responsible for revising of important intellectual content. All authors read and approved the final manuscript. TX, YW, and ZC contributed equally to this work.

Acknowledgments

This paper was supported by National Natural Science Foundation of China (Project 81671721). The authors would like to thank Editage (http://www.editage.com) for English language editing.

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Copyright © 2022 Tingting Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Contrast Media & Molecular Imaging

Preparation, Characterization, and Preliminary Imaging Study of [188Re]Re-Ibandronate as a Novel Theranostic Radiopharmaceutical for Bone Metastasis

Abstract

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Methods

2.2.1. Radiolabeling and Quality Control

2.2.2. In Vitro Stability

2.2.3. Plasma Protein Binding Rate

2.2.4. Lipids and Water Distribution Coefficient

2.2.5. Toxicity Test of Mice

2.2.6. The Biodistribution of [188Re]Re-IBA and [188Re]ReO4- in Mice

2.2.7. Imaging of New Zealand Rabbits with [188Re]Re-IBA

2.2.8. Biodistribution and Imaging of Bone Metastasis Nude Mice

2.2.9. Statistical Analysis

3. Results

3.1. Radiolabeling and Quality Control

3.2. In Vitro Stability

3.3. Plasma Protein Binding Rate and Lipids and Water Distribution Coefficient

3.4. Toxicity Test of Mice

3.5. The Biodistribution of [188Re]Re-IBA and [188Re]ReO4- in Mice and Imaging of New Zealand Rabbits with [188Re]Re-IBA

3.6. Biodistribution and Imaging of Bone Metastasis Nude Mice

4. Discussion

5. Conclusions

Data Availability

Disclosure

Conflicts of Interest

Authors’ Contributions

Acknowledgments

References

Copyright

Preparation, Characterization, and Preliminary Imaging Study of [¹⁸⁸Re]Re-Ibandronate as a Novel Theranostic Radiopharmaceutical for Bone Metastasis

2.2.6. The Biodistribution of [¹⁸⁸Re]Re-IBA and [¹⁸⁸Re]ReO₄^- in Mice

2.2.7. Imaging of New Zealand Rabbits with [¹⁸⁸Re]Re-IBA

3.5. The Biodistribution of [¹⁸⁸Re]Re-IBA and [¹⁸⁸Re]ReO₄^- in Mice and Imaging of New Zealand Rabbits with [¹⁸⁸Re]Re-IBA