ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 13, No. 6, 397-400, 1999 

Effect of edetate calcium disodium on yttrium-90 activity in bone of mice 

Naoyuki WATANABE,*,** Noboru ORIUCHI,* Shuji TANADA,* Hajime MURATA,* Tomio INOUE,** E. Edmund KIM,*** Yasuhito SASAKI* and Keigo ENDO** 

*Division of Advanced Technology for Medical Imaging, National Institute of Radiological Sciences **Department of Nuclear Medicine. Gunma University School of Medicine ***Department of Nuclear Medicine, The University of Texas M.D. Anderson Cancer Center, Houston. Texas, USA 

The kinetics of Yttrium-90 (Y-90) in bone of mice was investigated in combination with edetate calcium disodium (CaNa2EDTA). One group of mice were intraperitoneally administered 37.5 mg/ kg CaNa2EDTA or 0.9% NaCl as a control at 1, 22, 34, 46, 58, 70, 82, 94, 154 and 166 h after injection of Y-90 acetate (post-administration), and the biodistribution was studied at 3, 24, 72, 120 and 168 h postinjection of Y-90 acetate. No difference between the post-CaNa2EDTA-treated mice and the control was demonstrated in the radioactivity in the bone. A decrease in radioactivity in the liver and kidneys was accelerated, and the radioactivity was lower than the control at 120 h postinjection. The other group of mice were also given the same dose of chelator at 12 h and 1 h preinjection of Y-90 acetate and at 1, 22, 34, 46, 58, 70, 82, 94, 154 and 166 h after injection of Y-90 acetate (pre- and post-administration), the radioactivity in bone at 3 h postinjection was significantly lower than in the control (24.4+-3.92% ID/g vs. 31.7+-2.26% ID/g, p < 0.05), but the decrease was not sequential. A significant reduction in radioactivity in the blood, kidneys and liver was demonstrated at 3 h, 72 h and 72 h postinjection. In conclusion, the CaNa2EDTA with the administration schedule employed here cannot chelate the Y-90 from bone but the free Y-90 before deposition into bone. 

Key words: edetate calcium disodium, bone uptake 

INTRODUCTION 

YTTRIUM-90 (Y-90) is a candidate radionuclide for radioimmunotherapy because of its high maximum energy of 2.3 MeV, long-range beta-emitting character (maximum tissue penetration of nearly 1 cm with 80% of the energy deposited in the first 4 to 5 mm), and halflife of 64.1 h but Y-90 dissociated from Y-90-labeled-monoclonal antibody (MAb) specifically accumulates in bone.1-6 The bone uptake of Y-90 may cause myelosuppression, and prevent its therapeutic potential from being realized. The use of a therapeutic chelating agent for heavy metal poisoning, edetate calcium disodium 

 Received April 19, 1999, revision accepted August 25, 1999.
 For reprint contact: Naoyuki Watanabe, M.D., Division of Advanced Technology for Medical Imaging, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inageku, Chiba 263-8555, JAPAN.
 E-mail: w_nao@nirs.go.jp 

(CaNa2EDTA), to suppress the undesirable radioactivity in bone, was investigated.7 The administration of CaNa2EDTA reduced the radioactivity in bone and safely allowed the dose of Y-90-labeled-MAb to be increased to a sufficient level for therapy without severe myelosuppression.8,9 We have also confirmed the effect of CaNa2EDTA on the radioactivity in the bone of tumorbearing nude mice with Y-90 labeled MAb10 but it was not clear whether the reduced radioactivity in the bone resulted from chelating of Y-90 from bone or from removal of free Y-90 that did not localize in bone. In this study, the kinetics of Y-90-activity in bone of mice was investigated in combination with CaNa2EDTA, and the mechanism of CaNa2EDTA-mediated reduction in radioactivity in bone is discussed. 
MATERIALS AND METHODS. 

Animals Female normal mice with a Balb/c background were obtained from the Institute of Experimental Animal Research, Gunma University School of Medicine at 4 weeks of age (16.5 g-18.7 g mouse body weight). The mice were allowed free access to commercial chow and distilleddeionized water during the experiment. The ambient temperature during the study was maintained between 21 deg.C and 23deg.C, and the mice were exposed to a 12-h light, 12-h dark cycle. Animal studies were performed in compliance with relevant regulations relating to the conduct of animal experiments issued by Gunma University. 

Effect of CaNa2EDTA on biodistribution in normal mice injected with Y-90 acetate 
One group of mice were slowly, intraperitoneally administered 37.5 mg/kg CaNa2EDTA solution (Bleian Inj.R, Nisshin Pharmacy Co. Ltd., Yamagata, Japan) or an equal volume of 0.9% NaCl solution as a control at 1 , 22, 34, 46, 58, 70, 82, 94, 154 and 166 h after intravenous injection of Y-90 acetate (740 kBq) (post-administration). The Y-90 acetate (925 MBq/mL) was prepared by mixing Y-90-chloride in 50 mM HCI ( 1850 MBq/mL) (Nordion, Kanata, Ontario, Canada) with an equal volume of 1 M sodium acetate buffer, pH 5.8, for 30 min at room temperature. The other group of mice were slowly intraperitoneally administered 37.5 mg/kg CaNa2EDTA solution or an equal volume of 0.9% NaCl solution as a control at 12 h and 1 h before and at 1, 22, 34, 46, 58, 70, 82, 94, 154 and 166 h after intravenous injection of Y-90 acetate (740 kBq) (pre- and post-administration). Every five mice were sacrificed by heart puncture under general anesthesia at 3, 24, 72, 120 and 168 h postinjection of Y-90 acetate. Circulating blood was then replaced by 0.9% NaCl with 0.01 % heparin: the right atrium was cut while 0.9% NaCl with 0.01% heparin was mildly perfused into the left ventricle with a small syringe. The liver, kidneys, stomach, intestines, spleen, lungs, muscle (quadriceps) and bone (femur) were removed and cut into pieces weighing less than 200 mg. The radioactivity of each samples was counted in a NaI well-type gamma counter (Aloka. Tokyo, Japan) with an energy window ranging from 100 keV to infinity. The counting efficiency of the NaI well-type gamma counter in measuring Y-90 preparation was approximately 12%. A sufficient linearity between radioactivity and count rate was obtained when non-dissolved samples were below 200 mg in weight. Results were expressed as a percent injected dose/gram tissue % ID/g) normalized to 20 g mouse. Serum calcium levels from blood of mice pre- and post-administered CaNa2EDTA and 0.9% NaCl were measured at 168 h postinjection by the orthocresolphtalein complexone (OCPC) method to check for possible side effects of the chelator. 

Data analysis 
Statistical comparison of values was made by Student's unpaired t-test. Differences were considered statistically significant when a p value was less than 0.05. 

RESULTS 

In the control mice, the radioactivity in the bone had reached a maximum level of approximately 30% ID/g at 3 h post-Y-90 acetate-injection, and the level showed no change for up to 168 h (Fig. 1). The radioactivity in the other normal tissues and blood decreased with time (Figs. 2-4). In the post-CaNa2EDTA-treated mice, no change in the radioactivity of the bone was demonstrated compared to the control mice (Fig. 1 ). The reduction in Y-90 activity in the kidneys and liver was accelerated, and the radioactivity was significantly lower than the control at 120 h postinjection of Y-90 acetate (Figs. 2 and 3), but not the blood (Fig. 4), spleen, lungs, or muscle (data not shown). In the pre- and post-CaNa2EDTA-treated mice, the radio-activity in the bone at 3 h postinjection was significantly suppressed compared to the control mice (Fig. 1 ) but the bone radioactivity was not decreased further throughout the study (Fig. 1). The reduction in radioactivity in the blood, kidneys and liver was accelerated, and their radio-activity was lower than in the control at 3 h, 72 h and 72 h postinjection of Y-90 acetate (Figs. 2-4). There was no significantly accelerated reduction in the Y-90-activity in the other tissues (data not shown). No significant difference in the concentration of serum calcium was noted between the CaNa2EDTA-pre- and post-treated mice and the control mice (4.59+-0.25 mEq/L vs. 4.60+-0.26 mEq/L, respectively). There was no inflammation of the abdominal wall in any of the CaNa2EDTA-treated mice. No weight loss or other sign of toxicity was observed. No macroscopic change was observed in the kidneys of the mice. 

DISCUSSION 

CaNa2EDTA-treatment has been reported to accelerate the elimination of yttrium-radioactivity in rabbits and to remove yttrium from the skeleton7 but our study did not demonstrate any chelating effect of CaNa2EDTA on the radioactivity in the bone of normal mice. There was a significant difference between the doses of CaNa2EDTA and animal species employed in the two studies; approximately 460 mg/kg of CaNa2EDTA once or twice a day was given to young rabbits after yttrium (1 h after Y-91 and daily for 9 days) in the former study7 whereas 37.5 mg/kg of CaNa2EDTA twice a day was administered to 4-week-old normal mice in the latter (our) study. The dose of 37.5 mg/kg twice a day is the clinically maximum daily dose for children,11 and the CaNa2EDTA-administration schedule in our study has no influence on serum metallic concentrations such as copper and lead in mice (data not published). Moreover, the introduction of pre-administration of CaNa2EDTA effectively suppressed the radio-activity in the bone, but even the pre- and post-CaNa2EDTA treatment could not chelate Y-90 from the bone. There-fore, it is acceptable that the binding of Y-90 to the bone matrix may be too tight to allow chelation by CaNa2EDTA. The pre-administered chelate is speculated to be immediately available to clear free radioactivity. On the other hand, in the post-CaNa2EDTA-treated mice, the radioactivity in the kidneys and liver was reduced. And the pre- and post-administration of CaNa2EDTA was reduced earlier than with post-administration only. Earlier introduction of CaNa2EDTA-administration was speculated to effectively reduce the radioactivity in the kidneys and liver, however, it is not so well understood how Y-90 distributes at the tissues that the exact mechanism of CaNa2EDTA-mediated reduction in radioactivity is also unclear. In conclusion, the CaNa2EDTA with the administration schedule employed here cannot chelate the Y-90 from bone but can chelate the Y-90 before deposition into bone. In the pre- and post-CaNa2EDTA-treated nude mice bearing tumors with Y-90 labeled MAb, the radio-activity in the bone may be successfully decreased by removing free Y-90 before its deposition into bone. 

REFERENCES 

1. Hnatowich DJ, Chinol M, Siebeker DA, Gionet M, Griffin T, Doherty PW, et al. Patient biodistribution of intraperitoneally administered yttrium-90 labeled antibody. J Nucl Med 29: 1428-1434, 1988. 

2. Sharkey R, Kaltovich FA, Shih LB, Fand I, Govelitz G, Goldenberg DM. Radioimmunotherapy of human colonic cancer xenografts with yttrium-90 labeled monoclonal antibodies to carcinoembryonic antigen. Cancer Res 48: 3270-3275, 1988. 

3. MacDonald NS, Nusbaum RE, Alexander GV, Ezmirlian F, Spain P, Rounds DE. The skeletal deposition of yttrium. J Biol Chem 195: 837-841, 1952. 

4. Jowsey J, Sissons HA, Vaughan J. The site of deposition of Y-91 in the bones of rabbits and dogs. J Nucl Energy 2: 168-176, 1956. 

5. Jowsey J, Rowland RE, Marshall JH. The deposition of the rare earths in bone. Rad Research 8: 490-501, 1958. 

6. Durbin PW. Metabolic characteristics within a chemical family. Health Phys 2: 225-238, 1960. 7. Vaughan JM, Tutt ML. Use of ethylene diamine tetraacetic acid (versene) for removing fission products from the skelton. Lancet 2: 856-859, 1953. 

8. Rowlinson-Busza G, Snook D, Epenetos AA. 90Y-labeled antibody uptake by human tumor xenografts and the effect of systemic adntinistration of EDTA. Int J Rediation Oncology Biol Phys 28: 1257-1265, 1994. 

9. Stewart JSW, Hird V, Snook D, Dhokia B, Sivalapenko G, Hooker G, et al. Intraperitoneal yttrium-90-labeled monoclonal antibody in ovarian cancer. J Clin Oncol 8: 1941-1950, 1990. 

1O. Watanabe N, Oriuchi N, Endo K, Inoue T, Kuroki M, Matsuoka Y, et al. Use of CaNa2EDTA for improvement of radioimmunodetection and radioimmunotherapy with indium-111 and yttrium-90-DTPA-anti-CEA MAbs in nude mice bearing human colorectal cancer. J Nucl Med (in press) 

11 . Klaassen CD. Toxicology. In Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman Gilman A, Rall TW, Nies AS, Taylor P (eds.), 8th ed., New York, Pergamon Press, Inc., pp. 1592-1614, 1990.