A study on the preparation, evaluation of biological characteristics, and preliminary imaging of [188Re]Re-ibandronate

Bone is a common site of metastasis of malignant tumor. Several radiopharmaceuticals are available to relieve bone pain in patients with cancer. However, there is still a need to to investigate easily accessible and high bone anity radiopharmaceuticals. Radionuclide 188 Re has an advantage in this regard because of its commercial extraction from 188 W/ 188 Re generators. It can be used on demand and is cost-effective. The rst-generation bisphosphonate hydroxyethylidene diphosphonate (HEDP) is the commonly used bisphosphonate for 188 Re-labelling. And the third-generation bisphosphonates ibandronate (IBA) has higher bone anity and ability to inhibit bone resorption than HEDP. However, there have been no reports on 188 Re-labelling with IBA. We used IBA and 188 Re for radiolabeling to develop and evaluate a novel type of bone-seeking radiopharmaceutical.


Introduction
Bone is a common site of metastasis of malignant tumor. Up to 80% and 50% of the patients with prostate cancer and breast cancer, respectively, have bone metastasis [1,2]. The risk of bone metastasis in patients with lung, thyroid, and kidney cancer is about 30-40% [3,4]. Patients with bone metastasis usually experience severe and refractory pain. Furthermore, it may be accompanied by pathological fractures, spinal cord compression, hypercalcemia, and other complications, thus seriously affecting the qualitya of life [5]. Therefore, a 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, radiotherapy, bisphosphonate therapy, hormone therapy, and the use of painkillers [5]. However, the aforementioned methods have some limitations [6]. The use of bone-seeking radiopharmaceuticals is another effective method to relieve bone pain. It has the advantages of simultaneous treatment of multiple metastases, repeatability, and combination with other treatments [7]. Moreover, it can reduce or postpone the incidence of skeletal-related events [8]. There are several radionuclides available for bone-targeted radionuclide therapy, including 89 Sr, 153 Sm, 186 Re, 188 Re, 177 Lu, and 223 Ra [6,9]. 89 Sr and 223 Ra are calcium mimetic radionuclides. They have natural tropism for the bone, which enables the deposition of hydroxyapatite in the bones [10]. While 89 Sr releases β − rays, 223 Ra treats bone lesions by releasing α particles. Both are primarily used in the form of chloride ([ 89 Sr]SrCl 2 and [ 223 Ra]RaCl 2 ). In contrast, 153 Sm, 186 Re, 188 Re, and 177 Lu do not have natural tropism for the bone. This necessitates their radiolabeling with bisphosphonates. The major forms of 153 Sm, 186 Re, 188  Despite the diversity of options, commercially available radiopharmaceuticals are limited. Most of the radionuclides used for the treatment are produced through reactors, thus are extremely expensive. The radionuclide 188 Re has an advantage owing to its availability from commercial 188 W/ 188 Re generators. It can be used on demand and is cost-effective. 188 Re has a physical half-life of 16.9 h and can produce β − rays with a maximum energy of 2.1 MeV for treatment [11]. 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 [11]. [ 188 Re]Re-HEDP is one of the most widely used bisphosphonate radiopharmaceuticals in clinical nuclear medicine that relieves bone pain caused by prostate cancer, breast cancer, or other tumors [12]. However, there is a need to identify bisphosphonates with stronger bone-targeting 188 Re-compound. Bisphosphonates are analogues of endogenous pyrophosphates, characterized by P-C-P bonds. They comprise a hydroxyl group in one position of the carbon, which has a high a nity for calcium phosphate, the primary mineral of the bone [13]. Moreover, it comprises a side chain structure that inhibits bone resorption in the other position of the carbon [13]. The side chain structure of the rst-generation bisphosphonate does not contain nitrogen. HEDP is one of its representative drugs. In contrast, the side-chain structure of the second and third-generation bisphosphonates contains nitrogen. Furthermore, the third-generation bisphosphonate side chain also contains a heterocyclic structure and its ability to inhibit bone resorption is signi cantly stronger than that of the rst-and second-generation bisphosphonates. However, nitrogen-containing bisphosphonates may have signi cant 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 of patients with renal insu ciency. Ibandronic acid and zoledronic acid are the most powerful and widely used third-generation bisphosphonates. Studies [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 is equivalent to 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]mentioned that the pain relief rate and improvement in the quality of life in patients with bone tumor were higher in the ibandronic acid group than the zoledronic acid group (P < 0.05).
Nonetheless, the rate of adverse reaction was lower in the ibandronic acid group than in the zoledronic acid group (P < 0.05). There have been no reports on 188 Re-labelling with IBA. Therefore, we selected IBA and 188 Re for radiolabeling to develop and evaluate a new type of radiopharmaceutical with potential bone-seeking properties and low toxicity, which may contribute to individualized treatment in the era of precision medicine. We will shed light on the preparation, optimization of conditions, biological evaluation, and preliminary imaging studies of [ 188 Re]Re-IBA in detail.

Materials
[ 188 Re]NaReO 4 was eluted from the alumina based 188 W/ 188 Re generator (OncoBeta, Germany) with saline solution (0.9% NaCl). We purchased IBA from Twbio Technology Co., Ltd., Beijing, China. Ascorbic acid, potassium perrhenate (KRe04), and stannous chloride (SnCl 2 ) were purchased from Macklin Biochemical Co., Ltd., Shanghai, China. All the aforementioned reagents can be directly used without further puri cation. We used Xinhua No. 1 chromatography paper (Xinhua Paper Industry Co., Ltd., Hangzhou, China) for paper chromatography (PC). Moreover, the distribution of radioactivity on the PC strips was measured by a thin-layer chromatographic (TLC) scanner (Bioscan Inc, Washington, DC, USA). We used a dose calibrator (CRC-25R; Hengyide Technology Co., Ltd., Beijing, China) and a gamma counter (SN-695B; Hesuo Rihuan Photoelectric Instrument Co, Shanghai, China) to measure the radioactivity of the samples. respectively. We then added the fresh eluted [ 188 Re]ReO 4 solution. Subsequently, we adjusted the pH value to 0.5-9 with 1 N sodium acetate solution and 1 N hydrochloric acid. The reaction occurred at temperatures of room temperature (25±2℃), 60℃, and 95℃ for 10-60 min, respectively. After the reaction was completed, it was cooled to room temperature (25±2℃). The pH value of each tube was adjusted to 6-7. We used an aseptic lter membrane of 0.22 μm for sterilization and ltration.

Quality control
The RCP of [ 188 Re]Re-IBA was determined by TLC. The method of radioactivity quanti cation involved cutting the chromatography paper into 2 cm wide and 15 cm long PC strips. We used a pencil to draw a straight line, 2 cm away from one end of the strips to mark the origin. We eventually added 3-5 μL of the nal solution at the origin of the strips. We used acetone and saline as the solvents. A TLC scanner was used to measure the distribution of radioactivity The result is expressed as mean ±standard deviation (x±s).

Lipids and water distribution coe cient
Three 5 ml test tubes were numbered A1, A2, and A3. We added freshly prepared 1.85 MBq [ 188 Re]Re-IBA to each tube under optimal labeling conditions. Each test tube was shaken for 20 min with a vortex mixer, followed by centrifugation at 2000 r/min for 5 min. The upper liquid (organic phase) 0.1 ml was collected into three test tubes (numbered B1, B2, and B3). Moreover, the lower liquid (water phase) 0.1 ml was collected into three test tubes (numbered C1, C2, and C3). The radioactivity counts of organic phase and water phase were measured by a γ counter. Furthermore, the lipid-water partition coe cient (lgp) was calculated by using the formula as follows: The results are expressed as mean±standard deviation (x±s). Nonetheless, the overall stability was good. The lgP of the marker was evaluated by the above-mentioned method.  This in turn was signi cantly higher than that in other organs and tissues. In addition, the radioactivity ratio of the bone to the heart, liver, blood, and muscles was highest at 48 h, which were 327.902, 111.183, 326.053, and 291.551, respectively. The highest uptake occurred in the kidneys except the bone, which is related to the kidney as the primary excretory organ. We could observe the distribution of radioactivity in the stomach within 8 h. However, the gastric uptake gradually decreased with time.  There was an obvious accumulation in both kidneys, bladder, and bones 20 min after the injection. As time goes on, the accumulation in the kidneys gradually faded and got excreted with urine; furthermore, the soft tissue accumulation gradually faded and disappeared; the whole-body bone imaging was clear with a high contrast between the bone and the background, which consistent with the in vivo distribution in mice.

Discussion
Numerous radiopharmaceuticals have been used to target and relieve bone pain since the introduction of radionuclides to treat bone pain in the 1940s [19].  [6,9]. However, each radiopharmaceutical has its own advantages and disadvantages. The choice is extremely dependent on the patient's status, such as the renal function and bone marrow reserve, cancer extent (extraskeletal lesions and the bulk of the tumor), and physical properties of radionuclides [19]. As the commercial availability of radiopharmaceuticals is limited, the availability of each radiopharmaceutical also needs to be considered. Moreover, most of the radionuclides used for treatment are produced through reactors, thus are extremely expensive. Therefore, it is important to choose an easily accessible and cost-effective radiopharmaceutical. Radionuclide 188 Re has an advantage in this regard because of its commercial extraction from 188 W/ 188 Re generators, which can be used on demand. Moreover, it is cost-effective. The rst-generation bisphosphonate HEDP is the commonly used bisphosphonate for 188 Re-labelling. It has lower bone a nity and ability to inhibit bone resorption than the third-generation bisphosphonates IBA and is zoledronic acid. In addition, the rate of adverse reactions, including nephrotoxicity of IBA is lower than that of zoledronic acid [15,16,18]. We selected IBA and 188 Re for radiolabeling was based on the unique advantages of 188 Re in palliative treatment of metastatic bone pain and the low toxicity of IBA.
Previous studies [20] have identi ed ascorbic acid as the best antioxidant in [ 188 Re]Re-bisphosphonate, which is better than gentian acid and citric acid. Therefore, we use ascorbic acid as the antioxidant. In addition, we chose

Conclusion
This study encompassed the successful preparation of [ 188 Re]Re-IBA, a novel bisphosphonate radiopharmaceutical. The aforementioned radiopharmaceutical has the advantages of a simple preparation method, high stability, plasma protein binding rate, and good hydrophilicity. The in vivo biological distribution in mice and imaging of New Zealand rabbits con rmed the following: rapid blood clearance, high a nity to the bone, long retention time in the bone, high T/NT ratio, and low uptake in the liver, spleen, and soft tissue. Therefore,