Metastatic bone disease develops as a result of many interactions between bone cells and tumor cells such as carcinomas of unknown primary site, like lung, breast, kidney, prostate, and thyroid that often occur during the final stages of cancer and can lead to bone pain [1-3].
Methods of palliative treatment of painful bone metastases normally are nonsteroidal analgesics to opioids and chemotherapy or hormonal therapy, and radiation treatment using external-beam, sealed or unsealed sources . However, many of these treatments are limited in their efficacy or duration and have significant side effects . Bone-seeking radiopharmaceuticals labeled with beta emitters to relieve intense bone pain resulting from metastases have been shown to be clinically useful . Substantial advantages include the ability to simultaneously treat multiple sites of disease with a more probable therapeutic effect in earlier phases of metastatic disease, the ease of administration, the repeatability, and the potential integration with other treatments .
Radiopharmaceuticals developed for bone pain palliation use the following radionuclides: 32P, 89Sr, 186Re, 188Re, 153Sm, and 177Lu . The major challenge in developing effective agents for palliative treatment of bone pain arising from skeletal metastases is to ensure the delivery of adequate doses of ionizing radiation, at the site of the skeletal lesions, with minimum radiation-induced bone marrow suppression . When lanthanides are administrated intravenously as salts, the main part of the dose (around 60-80%) is accumulated in the skeleton and the liver, and when lanthanides are chelated with organic ligands these molecular complexes distribute more homogeneously in the body and are essentially excreted by the kidneys in a few h .
175Yb is one of the potential lanthanide that has suitable radionuclidic properties for developing various radiotherapy agents. 175Yb decay by emission of β-particles with 470 keV maximum energy (86.5%) to stable 175Lu with a convenient half-life of 4.2 days. 175Yb also emits photons of 113 keV (1.9%), 282 keV (3.1%) and 396 keV (6.5%) which are appropriate for studying the biolocalization . 175Yb can be produced by thermal neutron bombardment of natural ytterbium target. The simplified production scheme is:
174Yb (n, γ) 175Yb → 175Lu (Stable) σ= 69 barn
Reactions leading to the formation of radionuclidic impurities upon thermal neutron bombardment of natural ytterbium target include:
168Yb (n, γ) 169Yb → 169Tm (Stable) σ= 2300 barn
176Yb (n, γ) 177Yb → 177Lu→177Hf (Stable) σ= 2.4 barn 
By attention to the presence of low amounts of 169Yb in natural ytterbium target (0.13%) should not cause any serious problem in the in vivo application of 175Yb. On the other hand, the presence of 169Yb will be useful in extended studies of the pharmaco- logical characteristics of the 175Yb labeled radiopharmaceuticals in biological systems . Also, 177Lu is another radionuclidic impurity and is itself a potential therapeutic radionuclide already under investigation. The radionuclidic characteristics and chemical properties of 177Lu are very similar to 175Yb. Hence, the presence of 177Lu in very small quantities in the 175Yb produced should not restrict the use of the latter in the in vivo therapy .
Multidentate aminomethylenephosphonic acids form stable complexes with different radionuclide’s, and they have already proven to be very effective for palliation of bone pain . The choice of using cyclic chelator is also based on the more pronounced thermodynamic stability and kinetic inertness of their lanthanide complexes when compared to that of their acyclic analog . In this direction, cyclic tetraphosphonate, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaminome thylene-phosphonate as DOTMP has been labeled with 175YbCl3. The paper describes the successful radio labeling of this ligand with 175Yb (Figure 1).
Figure 1. Chemical structure of DOTMP.
Figure 2. The HPGe spectrum for Yb-175.
2. Materials and methods
The natural ytterbium oxide was purchased from Isotec Inc, USA and 175Yb was produced in the Tehran Research Reactor (TRR).Chemical components were obtained from Sigma-Aldrich Chemical Co. U.K. All radioactivities counting related to paper chromatography were carried out using a NaI (Tl) scintillation counter on adjustment of the base line at 396 keV. The activity as well as the radionuclidic purity of the 175Yb produced was ascertained by gamma spectroscopy on the base of 396 keV peak by using the HPGe detector and betaspectroscopy was carried out by the Wallac 1220 Quantulus liquid scintillation spectrometer. Animal studies were performed in accordance with the United Kingdom biological council's guidelines on the use of living animals in scientific investigations. All the values were expressed as mean±standard deviation (Mean±SD).
2.1. Production and quality control of 175YbCl3 solution
Ytterbium-175 was produced by neutron irradiation of 1 mg of natural Yb2O3 at the neutron flux of 3×1013 n/cm2/s. Irradiation was carried out for 7 d. The irradiated target was dissolved in 0.1 M HCl and the resultant solution was evaporated until shrivels and was reconstituted in double distilled water. The radionuclidic purity of the solution was checked using high purity germanium (HPGe) spectroscopy for the detection of various interfering gamma emitting radionuclides.The radiochemical purity of the 175YbCl3 was checked using one solvent system of ITLC (NH4OH: MeOH: H2O (1:10:20)).
2.2. Labeling of DOTMP with 175YbCl3
DOTMP solution was prepared by dissolving 10 mg of ligand in 1 ml of NaHCO3 (0.5 M) at the pH 9. Then 0.3 ml of this solution was added to 100 μl 175YbCl3 (100MBq). The reaction mixtures were remained with stirring at room temperature for one h. Sterility, apyrogenicity and toxicity were ascertained by routine methods.
2.3. Quality control techniques
2.3.1. Paper chromatography
For determination of the stability of complex (175Yb-DOTMP), it was applied to Whatman no. 3 chromatography paper in NH4OH: MeOH: H2O (1:10:20) system.
2.3.2. In vitro stability studies
The stability of the complex stored at room temperature (22 ºC ), fridge (4 ºC) and in the presence of freshly prepared human serum (at37 ºC) was checked at different time points by paper chromatography in NH4OH:MeOH: final 175Yb-Cl3 solution with 160-180 μCi was injected intravenously to rats, too. The animals were sacrificed post-anesthesia at 2,4 h and 2, 4 d post-injection. The tissues and the organs were excised and the activity associated with each organ was measured in a NaI(Tl) scintillation counter. The distributed activity in different organs was determined by calculation as the percentage of the injected activity (based on area under the curve of 396 keV peak) per gram of the organ. The institutional and international guide for the care and use of laboratory animals were followed.
3. Results and discussion
3.1. Production of 175Yb
Around 1.3-1.5 GBq/g (35-40 Ci/g) of 175Yb activity was obtained after 7 days H2O (1:10:20) system to determine the irradiation at a flux of 3×1013 n/cm2/s using radiochemical purity of the radiolabeled complex.
2.3.3. Biodistribution studies
Biodistribution studies of 175Yb complex were carried out in Wistar rats. Rat’s weight was 170-220 g and two rats were sacrificed for each time point. The complex solutions (0.15-0.2 ml; 160-180 μCi) were injected through the tail vein of the rats. For comparison, free Yb3+ cation buffer solution was also administered. Briefly, 0.15-0.2 ml of natural Yb2O3 target. Other result of this irradiation is also 169Yb and 177Lu as radionuclidic impurities. The gamma-ray spectrum of irradiated target after chemical processing is shown in Figure 2. The observed gamma-photo peaks correspond to the gamma-photo peaks of 175Yb (113, 144, 286 and 396 keV), 169Yb (63, 110, 130, 177, 198, 261 and 307 KeV) and 177Lu (208 and 250 keV). By analyzing the gamma-ray spectra, the radionuclidic purity of 175Yb was found to be 96.2% with the presence of 2.1% 169Yb and 1.7% 177Lu as radionuclidic impurities.
3.2. Labeling optimization studies
To obtain the highest labeling yield, quantitative studies were performed. In this study, different amounts of the ligand for a specific amount of radioactivity (2.8 mCi of 175YbCl3 for instance) was used in a suitable
temperature (25 °C). The labeling yield of 45-88% was obtained at room temperature using different amounts of the ligand within 24 h (Figure 3).
Figure 3. Radiochemical yield (RCY) of 175Yb-DOTMP in radio labeling at 25°C in 24 h.
3.3. Stability of 175Yb-EDTMP in final formulation
By attention to optimized reaction conditions the stability of the 175Yb-DOTMP complex was studied and was observed that the complex has excellent stability when stored at room temperature. The complex remained stable to the extent of 88% up for 96 h, whereas stability of this compound was shown 90% for 72 h in refrigerator. The free ytterbium cation in 175Yb3+ form remains at the origin (Rf= 0.0) and the 175Yb-DOTMP complex migrates to higher Rf (Rf>0.88).
3.4. Biodistribution of 175Yb cation and 175Yb- DOTMP in rats
For substantiating the significant accumulation of 175Yb-DOTMP in bone, it is necessary to perform a comparison between 175Yb-DOTMP and free ytterbium cation biodistribution data. Thus the biodistribution of the cation was checked in various vital organs after injection of 6-7 MBq of the 175YbCl3 pre-formulated by the normal saline (pH= 8) to each rat. On the other hand, a volume (0.2 ml) of 175Yb-DOTMP solution with 0.16-0.18 mCi activity and pH=8-9 was injected intravenously to each rat through tail vein. The animals were sacrificed at the exact time intervals (2, 4, 48 h and 4 d), and specific activity of different organs was calculated as percentage of injected dose per gram using NaI(Tl) detector (Figures 4 and 5).
Figure 4. Percentage of injected dose per gram (ID/g%) of 175YbCl3 in wild-type rat tissues at 2, 4 h and 2, 4 d post injection
Figure 5. Percentage of injected dose per gram (ID/g%) of 175Yb-DOTMP in wild-type rat tissues at 2, 4 h and 2, 4 d post injection.
The Liver uptake of the free 175Yb cation is relatively high. About 2.3% of the activity accumulates in the liver after 2 days. Beside the kidney was one of the major accumulation sites of the radiolabeled DOTMP. Therefore, it can be concluded that free 175Yb is extracted from the liver due to free cation release through the biliary tract, while in case of 175Yb-DOTMP the uptake reaches its maximum at 2 h followed by excretion. Lung, muscle and also skin do not demonstrate significant uptake which is in accordance with other cations accumulation.
For demonstration of ligand effect in organs uptake can be compared of 175Yb- DOTMP and 175YbCl3 behavior in wild-type rat tissues. By attention to Figure 6 it can be realized for 175Yb-DOTMP the blood content is low at all time intervals and this shows the rapid removal of activity in the circulation. Toward 175YbCl3 the activity in blood is in the highest value at first two hours and with different mechanism was washed out from the circulation after 4 days. A 2.12% bone uptake is observed for the cation in 2 days after injection and then decreased (Figure 7). On the other hand, 175Yb-EDTMP complex was rapidly taken up in the bone in 2 h after injection (ID/g%=3.92) and remained almost constant after 4 days (ID/g%=3.91) 175Yb cation is accumulated in the liver in the 2 days post injection, and it can be assumed that later the activity is excreted from liver. But liver uptake for 175Yb-DOTMP is negligible (Figure 8).
Figure 6. Comparative blood activity for 175Yb-DOTMP and 175YbCl3 in wild-type rats.
Figure 7. Comparative bone activity for 175Yb-DOTMP and 175YbCl3 in wild-type rats.
Figure 8. Comparative liver activity for 175Yb-DOTMP and 175YbCl3 in wild-type rats.
It was observed from the animal tests and quality control data of 175Yb-DOTMP that is shows good features to be used as bone pain palliation again. Quality control and animal tests data of 175Yb-DOTMP show good features to be used as bone pain palliation agent.175Yb-DOTMP complex was prepared and was carried out quality control using optimized condition. For 175Yb-DOTMP, radiochemical purity was higher than 98%, also radionuclidic purity was acceptable. The labeling and quality control took one hour and radiolabeled complex was stable in human serum for least 2 days. The biodistribution data on normal rats showed at least 4% uptake of 175Yb-DOTMP is in gram of the bone tissues. The produced 175Yb-DOTMP properties such as relatively long half-life, appropriate beta and gamma energy, low cost and easy production suggest good potential for efficient use of this radiopharmaceutical for bone pain palliation of skeletal metastases.
The authors would like to thank Dr. Majid Shahriari for his guidance and supervision when he was with us. God bless his soul.