I. INTRODUCTION
Chronic pain syndrome is an important complications of bone metastases. It is well acknowledged that the first radio-nuclide therapy of bone metastases was done in 1960 by administration of 32P-phosphate [1]. Since then, a variety of β-radioisotopes have been used to treat bone Metastases [2, 3]. Among them, 89Sr (Metastron) is used the most frequently worldwide, despite the inconvenience in obtaining it, hence the expensive price. It is only recently available in Australia. In mid 1990s, it was found that 186Re hydroxyethylidene diphosphonate (HEDP) is effective for pain palliation in patients with osseous metastases from prostate cancer [4, 5, 6, 7]. Radiochromatographic method was developed later for quality control and stability test of [186Re]-HEDP [8, 9]. However, the ideal agent for bone pain palliation has not yet been identified. 188Re, emitting electrons in maximum energy of 2.1 MeV and 155 keV γ photons (15%) with T1/2 = 16.9 h, is an attractive candidate for bone tumor therapy. It can be obtained in no-carrier-added form of 188W/188Re generator. Palmedo et al. found in 2003 that repeated 188Re-HEDP therapy was good for patients with prostate cancer patients with bone metastases [10]. Recently, Biersack et al. found that repeated administrations of 188Re-HEDP reduced the pain and improved survival rates of prostate cancer patients with bone metastases [11]. The 188Re-HEDP for bone pain palliation has become an effective method for treating bone metastasis pain, and has been a radiopharmaceutical used clinically. The preparation of 188Re-HEDP has been reported by many researchers [12, 13]. However, the synthesis process is still complicated, and is not conducive to rapid production.
In this paper, we report a system labeling study of 188Re-HEDP lyophilized kit with 188Re obtained from 188W/188Re generator using radiometric methods. The 188Re-HEDP’s obtained by lyophilized kit is of high radiochemical purity (RCP) and radiochemical yields (RY). The relationship is investigated, too, between the bone uptake and precise rhenium mass levels. The biodistribution of mice and SPECT imaging of rabbit are performed. All these efforts are made towards routine clinical application in bone tumor therapy with the188Re-HEDP lyophilized kit.
II. EXPERIMENTAL
A. General
Hydroxyethylidene diphosphonate (HEDP), sodium acetate (NaOAc), phosphate-buffered saline (PBS), sodium bicar-bonate (NaHCO3), glacial acetic acid, phosphorus trichloride (PCl3), 2,5-dihydroxybenzoic acid (DHB), and acetyl chloride were purchased from Sigma-Aldrich (St. Louis, MO). 188ReO4- was eluted from 188W/188Re generator (Shanghai Institute of Applied Physics, CAS) using 188W produced in the High Flux Isotope Reactor. Other commercial chemicals were purchased from VWR International (San Diego, CA). BABLC/SPF mice (about 30 g) and New Zealand rabbit were kept under pathogen-free conditions and were handled according to Institutional Animal Care and Use Committee guidelines. Thighbone and focile uptakes were analyzed using two-tailed, unpaired Student t-tests, with p < 0.05 being considered as statistically significant. All statistical computations were performed using Excel.
B. Analytical methods
1H NMR spectra were recorded on a Varian XL-300 spectrometer (Varian, Inc., Palo Alto, CA) operating at 300 MHz with tetramethyl silane (TMS) as internal standard. The samples were analyzed by a radioactivity thin-layer scanner (Bioscan, IAR-2000, USA) and an automated gamma scintillation counter (Perkin Elmer, 1470-002, USA). High performance liquid chromatography (HPLC) analysis was performed using a Dionex P680 system equipped with a tunable absorption detector and a PDA-100 photodiode-array detector, using a Hypersil BDS C-18 reversed phase column (5 μm, 250 mm×4.6 mm). The HPLC solvents were 0.1% TFA in H2O (solvent A) and 0.1% TFA in acetonitrile (solvent B). Conditions: Gradient B: 0%–10%, 0 min–5 min; 10%–50%, 5 min–8 min; 50%–80%, 8 min–10 min; 80%–10%, 10 min–12 min.
C. Radio-synthesis of 188Re-HEDP by conventional methods
The synthesis and radiolabeling of 188Re-HEDP is shown in Fig. 1. The precursor HEDP was synthesized by a previous procedure with some modifications [14]. Briefly, 30 g glacial acetic acid was dissolved in 15 mL water under N2 atmosphere in a three neck bottle. PCl3 was added dropwise in 60 min at 50 ℃. The mixtures were stirred for four more hours at 150 ℃. The acetyl diphosphonate mixtures were obtained. Then, the mixtures were further distilled at 150 ℃ till the acetic acid evaporated (about 4 h). The HEDP was obtained by crystallization from glacial acetic acid/H2O/ethanol (1/3/1) as white crystal in 90% total yield. Formation of HEDP was analyzed by electrospray ionization mass spectrometry using the Agilent LC/MSD TOF mass spectrometer (Santa Clara, CA, USA) equipped with a Vydac C-18 column (4.6 mm×250 mm, 7 μm particle sizes, 300 Å pore size, Anaheim, CA, USA).
188Re was obtained from 370 MBq (10 mCi) alumina-based 188W/188Re generator. The specific volume of above eluates was 10 mL (saline), and then was concentrated to 1.5 mL. For preparation of the 188Re-HEDP, 5.8 μL (0.22 μmol) of NH4ReO4 (10 mg/mL) in saline was added to the concentrated 188Re solution. This carrier-added 188ReO4- was used for the labeling reaction through a 0.22-mm sterile filter to a kit vial containing 8.3 mg (0.04 mmol) of HEDP, 3.0 mg (0.02 mmol) of DHB, and 3.9 mg (0.02 mmol) of SnCl2. The vials were kept for 20 min at 90 ℃, and the reaction was monitored by radio-TLC (eluted A: Acetone; eluted B: 5% saline buffer). Quality control of carrier-added 188Re-HEDP was performed using instant Xin-Hua 1 test paper [11].
D. Kit formulation and labeling with 188Re
Effects of reductant amount (SnCl2) and carrier content (NH4ReO4) in labeling of 188Re-HEDP were investigated. The radio-labeling yields were determined with Radio-TLC. Each set of reaction conditions was run 2–3 times.
The kit formulation was prepared under aseptic conditions, with Vial A containing 1 mL 5% NaHCO3 buffer and 5.0 mg DHB in final pH 6.0, while Vial B containing HEDP and SnCl2. Each vial was transferred to the freeze-dryer and the process continued. The vials were closed under dry sterile nitrogen gas and stored at 2 ℃–8 ℃.
The freeze-dried Vial B was added with 2 mL of generator-eluted 188ReO4- (370 MBq), extracted with injector, added to Vial A and incubated for a while to form 188Re-HEDP.
E. Radiochemical analysis of 188Re-HEDP lyophilized kit
Dependence of the 188Re-HEDP labeling yields upon the reaction temperature and times were investigated. Radio-labeling yields were determined with Radio-TLC. Each set of reaction conditions was run 2–3 times.
F. Quality control of radioactive 188Re-HEDP lyophilized kit
The in vitro stability of 188Re-HEDP in saline and with vitamin C solution were evaluated by measuring RCP with Radio-TLC at each time point after incubation at 37 ℃. Radiolabeling yield of the kits was tested for three months.
G. Biodistribution study in normal mice
Radiolabling yield of 188Re-HEDP kit was over 95%. It was used without further purification in the following biodistribution and SPECT imaging studies.
Relationship between bone uptake and precise rhenium mass levels was studied. Each amount of NH4ReO4 was added to 188ReO4- with finial concentration of 0.05 to 0.3 mg/5 mg HEDP. About 0.37 MBq (100 μL) of 188Re-HEDP with each amount of NH4ReO4 was injected through the tail vein of mice (n= 4). Three hours after injection, the mice were sacrificed, and the tissues and organs of interest were collected, wet weighed and counted in a γ-counter. The percentage of injected dose per gram (%ID/g) for each sample was calculated by comparing its activity with appropriate standards of the injected dose (ID), and the values are expressed as mean±SD. The contrast study was performed to acquire the relation between bone uptake and precise rhenium mass levels. The results were expressed as %ID/g. Averages and standard deviations were calculated. The T/NT and F/NT (thighbone or focile to normal tissues) values were calculated.
Biodistribution of optimal NH4ReO4 added 188Re-HEDP were investigated. The mice were injected intra-venously with 188Re-HEDP (0.37 MBq), and sacrificed at 0.5, 1, 4 and 6 h after injection.
H. SPECT imaging of 188Re-HEDP
About 74 MBq of 188Re-HEDP (contain 0.15 mg NH4ReO4/5 mg HEDP) in 0.2 mL of PBS solution was injected in marginal ear vein of a New Zealand rabbit. It was placed near the center of the field of view (FOV) of a Siemens SPECT scanner, where the highest image resolution and sensitivity are available. The rabbit was deeply anesthetized by intravenous injection of a mixture of ketamine (25 mg/kg) and diazepam (1.1 mg/kg). Anesthesia was supplemented as needed. Computer acquisition of the gamma camera data was initiated after 4 hours administration of the 188Re-HEDP.
III. RESULTS AND DISCUSSION
A. Chemistry and radiolabeling produce
The reaction procedure is described in Fig. 1. HEDP was prepared by recrystallization from three phase system as white crystal in 90% total yield. It was identified by elemental analysis, mass spectrum, and 1H-NMR, and the results agree well with the expected chemical structures. HEDP contains phosphoric acid and alcohol hydroxyl groups, we performed electrospray ionization mass spectrometry. ESI-MS m/z = 205.0082 for [M-H]-, calculated for C2H7O7P2-: 204.9667. 1H NMR (D2O, 300 MHz) δ: 2.0–2.2 (s, 5H, -OH), 1.1 (s, 3H, -CH3). Anal. calcd for C2H8O7P2 (%): C, 11.66; H, 3.91; Found (%): C, 11.58; H, 4.13.
HEDP was labeled with 188Re obtained from 188W/188Re generator by reduction with SnCl2 in the conventional method. For Radio-TLC analysis, with the 5% saline system, the Rf values of 188Re-HEDP, and free 188ReO4- were about front (Rf 0.9–1.0), while 188Re-colloidal impurities remain at original. With the acetone system, the 188Re-colloidal impurities and 188Re-HEDP remains at the origin and free 188ReO4- moves with the front (Rf 0.9–1.0). For each radiolabeled complex, the single peak in the HPLC-chromatogram clearly showed the formation of only one complex and excluded the possibility of residual 188ReO4- or other component. This means that the chelation of rhenium with the bisphosphonates moiety is complete.
B. Kit formulation and radiolabeling procedure
The composition of the kit was similar to conventional methods (ligand-HEDP, antioxidant-DHB, reducing agent-SnCl2, and carrier-NH4ReO4). It was found that over 95% yields of complex were at pH 4–6 and the labeling yield did not depend on the DHB concentration. We concerned with the influence of reductant amount (SnCl2), and carrier content (NH4ReO4) obtained the optimal condition. The labeling yield of HEDP increased with the SnCl2 content ([HEDP] =10 mg/mL, Fig. 2(a)). However, as shown in Fig. 3(a), the absence of carrier decreased the labeling yield to 90%. At a concentration of 0.01 mg–1 mg NH4ReO4 carrier/5 mg HEPD the radiolabling yields were >95%.
A lyophilized kit was prepared under optimal conditions: 188Re-labeled HEDP in 5% NaHCO3 buffer with the final pH 6.0. Dispensed cold kits were tested according to Chinese Pharmacopoeia to be both sterile and pyrogen free.
Figure 3 shows the labeling yield as a function of temperature and reaction time. The temperature effect was investigated in the synthesis of 188Re-HEDP (Fig. 3(a)), and the desired compound was prepared in good yield at 90 ℃. As shown in Fig. 3(b), 15 min is suitable for this reaction. At 90 ℃, the reaction was rapid and efficient. After 15 min, the highest radiochemical yield was (96±2)% (n = 3) with 188Re-HEDP kit.
So, to the above freeze-dried vial, 2 mL of generator-eluted 188ReO4- (370 MBq) was added to Vial B, which was extracted with injector and added to Vial A and incubated at 90 ℃ for 15 min to give 188Re-HEDP.
In vitro stability of the complex was evaluated by measuring the RCP with Radio-TLC at different hours after preparation (Fig. 4(a)). The RCP were still over 95% at 5 h, indicating that 188Re-HEDP was stable in vitro at least for 4 h at room temperature. The stability was improved by adding 5 mg/mL Vitamin c (Vc) under the same condition. As shown in Fig. 4(b), long term deposit of kit did not affect the radiolabeling yields in three months, and the kit stored at 2 ℃–8 ℃ for three months had more than 95% radiolabling yield in optimal conditions.
C. Biodistribution
Many authors focus on 188Re radiolabling of phosphonate compounds for bone metastases therapy [15-17], but evidence shows that coordination compound [188Re] Re-HEDP is a successful alternative for palliation of metastatic bone pain. This stimulates studies on 188Re-HEDP kit formulation and its effect on biological activity and therapeutic use.
Tissue distribution of 188Re-HEDP in BABLC/SPF mice was studied. The biodistributions of 0.37 MBq 188Re-HEDP co-injected with 0.05 mg–0.3 mg NH4ReO4/5 mg HEDP at 3 h after injection were given in Table 1.
| Organa | 0.05 mg Re | 0.10 mg Re | 0.15 mg Re | 0.20 mg Re | 0.30 mg Re |
|---|---|---|---|---|---|
| Blood | 0.13±0.03 | 0.23±0.09 | 0.19±0.06 | 0.11±0.01 | 0.11±0.02 |
| Heart | 0.05±0.01 | 0.06±0.01 | 0.06±0.0 | 0.04±0.01 | 0.05±0.01 |
| Liver | 0.13±0.07 | 0.18±0.12 | 0.13±0.01 | 0.10±0.01 | 0.12±0.06 |
| Spleen | 0.07±0.01 | 0.08±0.01 | 0.09±0.02 | 0.07±0.02 | 0.07±0.02 |
| Kidney | 0.68±0.08 | 0.83±0.03 | 1.13±0.26 | 0.66±0.08 | 0.69±0.10 |
| Lung | 0.15±0.03 | 0.13±0.02 | 0.17±0.02 | 0.09±0.02 | 0.11±0.02 |
| Stomach | 0.35±0.16 | 0.58±0.21 | 0.40±0.03 | 0.61±0.61 | 0.36±0.15 |
| Intestine | 0.04±0.01 | 0.07±0.02 | 0.07±0.02 | 0.05±0.01 | 0.07±0.01 |
| Muscle | 0.03±0.01 | 0.04±0.01 | 0.04±0.03 | 0.03±0.01 | 0.02±0.01 |
| Thigh | 21.16±1.09 | 20.58±0.57 | 26.66±1.02 | 20.04±3.06 | 21.06±2.88 |
| Focile | 21.06±1.86 | 23.82±2.47 | 30.55±5.02 | 21.62±1.29 | 18.58±1.79 |
It is well known that the presence of macroscopic amounts of stable rhenium in Re-HEDP preparations is decisive with respect to the form of phosphonate chemical species [18]. In Re carrier-added preparations, these species consist of rhenium-rhenium bonds that cannot be formed using carrier-free 188Re. As shown in Fig. 3(a), the carrier-added HEDP showed higher RY.
From Table 1, the biodistribution and bone uptake of each amount of carrier-added 188Re-HEDP differ from each other. The radioactivity in the blood was 0.13±0.03, 0.23±0.09, 0.19±0.06, 0.11±0.01 and 0.11±0.02 %ID/g for uptake of 0.05, 0.10, 0.15, 0.20 and 0.30 mg Re per 5 mg HEDP, respectively, indicating that the188Re-HEDP can be eliminated quickly from the blood. The uptakes of 188Re-HEDP in liver (< 0.18 %ID/g) and spleen (0.07–0.09 %ID/g) were especially low. No significant uptake was seen in any other organ except bone. The biodistribution also showed that skeletal uptake of 0.15 mg Re were the highest (26.66±1.02% and 30.55±5.02% in thighbone and focile, respectively). There was no statistical significance in bone uptake between thighbone and focile (P = 0.16, unpaired two tail test).
The T/NT and F/NT values were calculated, as shown in Fig. 5. Due to high radioactivity level in bones, the uptakes in non-target organs decreased, such as muscle, blood, spleen and liver (T/NT > 200). 0.10 mg Re/5 mg HEDP carrier added 188Re-HEDP showed the lowest T/NT value. F/NT showed same tendency compared with T/NT. For all this, both thighbone and focile showed excellent uptake of 188Re-HEDP. Among then, 0.15 mg Re/5 mg HEDP carrier added tracer showed the highest T/NT and F/NT value.
Biodistributions of 0.15 mg NH4ReO4 per 5 mg HEDP in BABLC/SPF mice are given in Table 2. No sign of toxicity through the study period was observed. This is consistent with the general observation that Re-HEDP has low toxicity and can be used therapeutically at a high dose. 188Re-HEDP cleared from the blood rapidly, being 1.74±0.06, 0.43±0.06, 0.268±0.100 and 0.17±0.07 %ID/g at 0.5, 1.0, 4.0 and 6.0 h post injection. 188Re-HEDP showed predominant kidney uptake. As described above, the re-uptake of radio-metabolites may be the main reason for high kidney uptake.
| Organ | 0.5 h | 1.0 h | 4.0 h | 6.0 h |
|---|---|---|---|---|
| Blood | 1.74±0.06 | 0.43±0.06 | 0.27±0.10 | 0.17±0.07 |
| Heart | 0.85±0.03 | 0.16±0.03 | 0.08±0.02 | 0.04±0.01 |
| Liver | 0.54±0.05 | 0.23±0.02 | 0.27±0.02 | 0.21±0.04 |
| Spleen | 0.30±0.005 | 0.14±0.03 | 0.16±0.06 | 0.11±0.01 |
| Kidney | 2.08±0.42 | 1.72±0.41 | 1.33±0.26 | 1.45±0.18 |
| Lung | 0.79±0.06 | 0.24±0.03 | 0.22±0.08 | 0.15±0.04 |
| Stomach | 0.62±0.04 | 0.44±0.19 | 0.50±0.10 | 0.66±0.22 |
| Intestine | 0.32±0.06 | 0.18±0.08 | 0.26±0.18 | 0.15±0.07 |
| Muscle | 0.27±0.03 | 0.21±0.08 | 0.11±0.04 | 0.06±0.02 |
| Thighbone | 24.97±0.70 | 25.96±3.11 | 26.50±1.72 | 27.72±1.40 |
| Focile | 24.42±4.23 | 28.42±10.14 | 30.03±11.01 | 26.94±1.22 |
| Blood | 1.74±0.06 | 0.43±0.06 | 0.27±0.10 | 0.17±0.07 |
These results show that 188Re-HEDP lyophilized kit has high selective uptake in the skeletal system and low background uptake in soft tissues, and is of great potential for instant clinical assessment of bone tumor therapy.
D. SPECT imaging
A typical SPECT image is shown in Fig. 6. High bone activity accumulation was observed at 4 h after injection. From this image, it can be seen that 188Re-HEDP complexes has highly selective skeletal uptake in the rabbit after administration of intravenous injection. The SPECT results agree well with the biodistribution studies in mice, indicating that 188Re-HEDP kit possesses excellent characteristics for instant clinical assessment of bone tumor therapy.
IV. CONCLUSION
In this paper, we focus on the development of 188Re-HEDP kit for instant clinical bone tumor therapy. The kit preparation process was simple and fast. The sterile and pyrogen-free HEDP kit was developed to produce instant preparation of 188Re-HEDP suitable for clinical bone tumor therapy. The amounts of stable rhenium in 188Re-HEDP preparations and biodistribution are determined. Both the biodistribution experiments and SPECT imaging studies demonstrate bone targeting. The development of the lyophilized HEDP kit affords the new opportunity for routine clinical assessment of bone tumor therapy.
