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Experimental Therapeutics |
-Particle-emitting 223Ra Demonstrated in an Experimental Skeletal Metastases Model1
Department of Chemistry, University of Oslo, Oslo, Norway [G. H., R. H. L.]; Anticancer Therapeutic Inventions AS, N-0884 Oslo [G. H., R. H. L.]; Department of Tumor Biology, [K. B., Ø. F.]; and Department of Oncology, The Norwegian Radium Hospital, N-0310 Oslo, [Ø. S. B.] Norway
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ABSTRACT |
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-particle-emitting radionuclide 223Ra (t1/2 = 11.4 days) in the treatment against experimental skeletal metastases in rats was addressed. Biodistribution studies, involving measurement of 223Ra in bone marrow samples, were performed in rats after i.v. injection. To study the therapeutic effect of 223Ra, an experimental skeletal metastases model in nude rats was used. Animals that had received 106 MT-1 human breast cancer cells were treated with 223Ra doses in the range of 6–30 kBq after 7 days. The biodistribution experiment demonstrated that 223Ra was selectively concentrated in bone as compared with soft tissues. The femur content of 223Ra was 800 ± 56% of injected dose per gram tissue times gram body weight (b.w.; mean ± SD) 1 day after the injection and 413 ± 23% of injected dose per gram tissue times gram b.w. at 14 days. The femur:kidney ratio increased from (5.9 ± 2.0)·102 at 1 day to (7.2 ± 3.0)·102 at 14 days, whereas the femur:liver ratio increased from (6.2 ± 0.2)·102 to (9.1 ± 6.6)·102. Femur:spleen ratio increased from (8.1 ± 0.3)·102 at 1 day to (6.4 2.2)·103 at 14 days. The femoral bone:marrow ratio was 6.5 ± 2.1 after day 1 and larger than 15 at day 14. All of the tumor-bearing control animals had to be sacrificed because of tumor-induced paralysis 20–30 days after injection with tumor cells, whereas the rats treated with 
10 kBq of 223Ra had a significantly increased symptom-free survival (P < 0.05). Also 36% (5 of 14) of rats treated with 11 kBq and 40% (2 of 5) of rats treated with 10 kBq were alive beyond the 67-day follow-up period. No signs of bone marrow toxicity or b.w. loss were observed in the groups of treated animals. The significant antitumor effect of 223Ra at doses that are tolerated by the bone marrow is most likely linked to the intense and highly localized radiation dose from -particles at the bone surfaces. The results of this study indicate that 223Ra should be additionally studied as a potential bone marrow-sparing treatment of cancers involving the skeleton. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bone-targeting radiopharmaceuticals have been studied clinically as a treatment of cancer that has metastasized to the skeleton (4, 5, 6, 7) . The compounds used were based on ß-particle emitters (8) and lately also a conversion electron emitter (9) . 89Sr (Metastron) and 153Sm-EDTMP3 (Quadramet) have been made commercially available for palliation of pain because of skeletal metastases (7 , 10 , 11) , whereas several others are under clinical investigation (10 , 11) . Because of the relatively long radiation range, a significant bone marrow exposure is associated with the use of ß-emitters. This has restricted bone treatment with ß-emitters to pain palliation (10 , 11) .
A possible alternative could be radionuclides emitting -particles, which are characterized by short range (<0.1 mm) and highly energetic radiation compared with low LET radiation like ß-particles and 
-rays. Thus, -particles are classified as high-LET radiation, which is generally more lethal to cells (12)
. -Emitters could potentially be used as sources in metabolically concentrated radionuclide therapy against skeletal metastases, because the short range of the -particles could affect a reduction in bone marrow exposure. In a recent study, -emitting 211At and ß-emitting 131I linked to bone-seeking bisphosphonates (13)
were compared as bone seeking agents. Dosimetry estimates indicated that the bone surface:bone marrow dose ratio could be increased 
3-fold by substituting the ß-emitter with the -emitter (13)
.
Other than 211At, only a few -particle-emitting radioisotopes are considered useful for biomedical applications (14)
. Among these is 212Bi, which has been evaluated as a bone-seeking agent (15)
. However, the short physical half-life of 212Bi (t1/2 = 60 min) and because the time required for the bismuth phosphonate complex to localize in the target tissue is substantial, the use of this compound results in significant normal tissue exposure during the uptake and elimination phases (15)
. The ß-emitter 212Pb (t1/2 = 10.6 h) was evaluated as an in vivo generator for 212Bi. However, a substantial redistribution of 212Pb and 212Bi was observed (15)
rendering this strategy less useful.
The -emitting radionuclide 223Ra (t1/2 = 11.43 days) has been suggested for use in targeted radiotherapy (16)
. Because of the bone-seeking properties of alkaline earth elements (Ca, Sr, Ba, and Ra), Ra2+ may be useful for metabolically concentrated radionuclide irradiation of osseous sites e.g., bone surfaces and skeletal metastases growth zones. These elements are probably concentrated because of inclusion in the bone mineral calcium hydroxy apatite, where radium and strontium could substitute calcium during mineral formation. In a recent study with 223Ra in mice, biodistribution was measured at 1 h, 6 h, 24 h, 3 days, and 14 days after injection. A rapid uptake and prolonged retention was demonstrated in the skeleton, whereas soft tissue radioactivity cleared relatively rapidly. It was also demonstrated, by measuring 211Bi and 223Ra in bone samples immediately after sacrificing the animals, that only very small amounts (<1%) of daughter radionuclides redistributed from the site of 223Ra decay in bone at day 3 after i.v. injection of 223Ra.4
This high retention of daughters is probably because of the rapid decay of the radon-219 daughter (t1/2 = 3.96 s) preventing translocation, because animal experiments with 224Ra, which radon-220 daughter has a t1/2 = 56 s, showed that a large fraction of the daughter products escaped from bone (17
, 18)
.
223Ra has several favorable features, which could be exploited in radionuclide therapy. Firstly, it can be produced relatively inexpensively, readily, and in large amounts: sources of 227Ac (t1/2 = 21.7 years) could potentially be used as a long-term operating generator for 223Ra (19)
.227Ac can in turn be produced by neutron irradiation of relatively commonly available 226Ra. Secondly, 223Ra has a physical half-life of 11.4 days, which provides sufficient time for preparation, distribution, including long distance shipment, and administration of the -emitting radionuclide. Thirdly, 223Ra decays through a chain of progeny (Fig. 1)
with the emission of approximately 28 MeV of energy. The fraction of energy borne by particulate radiation emitted as -particles is 96%.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The eluate containing 223Ra was evaporated to dryness, and thereafter 5 mM sodium citrate (pH 7.4) was added followed by filtration through sterile 0.22-µm nylon filters (Nalgene, Rochester, NY). The final solution used for injection had a 223Ra activity concentration of 25–150 kBq/ml in 5 mM sodium citrate.
Biodistribution Experiment.
Nude rats (Han rnu/rnu: Rowett) with a b.w. of 120–150 g (n = 4) were used in the biodistribution experiment. This was slightly heavier than the animals used for therapy. 223Ra was administered by injection into the tail vein using 200 µl of a 5 mM sodium citrate solution containing 10 kBq of 223Ra. Animals were sacrificed and dissected either at 24 h or at 14 days after injection.
The tissue uptake of 223Ra was measured with its progeny at radioactive equilibrium. Hence, samples were stored for a time interval corresponding to at least five half-lives of 211Pb before the measurement was performed. Two different procedures were used: (a) tissue samples were measured by using a NaI(Tl) well-type detector (Harshaw Chemie BV, De Meern, Holland) combined with a Scaler Timer ST7 digital unit (NE Technology Ltd., Reading, United Kingdom); and (b) to improve counting statistics for samples with lower quantities of 223Ra, some tissues were recounted using the more sensitive but also more laborious and waste producing liquid scintillation counting method. For this procedure, soft tissue samples were dissolved by adding 1–3 ml of Soluene 350 (Packard, BioScience BV, Groningen, Holland) per 100 mg tissue, and bone samples were dissolved in HClO4:H2O2 1:2 (v/v). All of the tissue samples were kept at 50°C until they were completely dissolved. When required, nontransparent soft tissue samples were bleached by adding H2O2. Finally, Instagel Plus II scintillation mixture (Packard) was added, and the samples were then stored in the dark to allow decay of luminescence.
Samples of 223Ra in equilibrium with its daughter nuclides were used as counting references.
Detection validation studies with the two methods showed a good agreement between the two radioactivity counting procedures.
Activity ratio between two tissues was calculated as mean ± SD by using the ratios for each individual animal as the input values.
Therapy Study.
Tumors were established in nude rats (Han: rnu/rnu Rowett) by intraventricular injection of 1 x 106 MT-1 breast cancer cells into the left side of the heart (20)
.
The effect of 223Ra in prolonging symptom-free survival of rats bearing experimental breast cancer skeletal metastases was studied for two treatment regimens. In treatment regimen A, animals were treated 7 days after tumor cell inoculation. At this time animal b.w.s varied between 80 and 110 grams, and animals were randomized in groups so the average weight within a group was 100 g at the time of treatment. Each animal in the treatment groups received either 6 or 11 kBq of 223Ra administered by tail vein injection, whereas animals in the control group were injected with an equivalent volume of 5 mM sodium citrate solution.
For treatment regimen B, in an effort to inhibit bone erosion, which could possible remove 223Ra from the tumor-affected skeletal area, the bone resorption inhibitor, 3-amino-1-hydroxypropylidene bisphosphonate, di-sodium salt (Aredia, Novartis AG, Switzerland), a bisphosphonate used against skeletal complications of cancer (21)
, was added as a supplemental treatment to 223Ra. Animals in the treatment groups were treated on day 7 after tumor cell inoculation, which is in accordance with previous studies using this model (Table 1)
, by i.v. injection of 5, 10, or 30 kBq of 223Ra. The following day, 1.5 mg/kg b.w. of Aredia, which is the standard dose for humans, was administered by i.v. injection. Animals in the control group received 5 mM sodium citrate solution on day 7 and 1.5 mg/kg b.w. of Aredia on day 8.
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Toxicity Study.
In a separate experiment, conventional Balb/c mice were injected with 1 MBq/kg b.w. of 223Ra and observed for short and longer term (90 days) to investigate if possible life-threatening bone marrow toxicity would occur and to demonstrate that the use of -emitter could spare the bone marrow even for high average skeletal doses and extremely high bone surface doses.4
All of the procedures and experiments involving animals were approved by the National Animal Research Authority and carried out according to the European Convention for the Protection of Vertebrates Used for Scientific Purposes.
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RESULTS |
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. The bone uptake was as follows: femur, 6.2 ± 0.2 and 3.0 ± 0.3; spine, 3.5 ± 0.1 and 2.6 ± 0.1; rib, 2.5 ± 1.1 and 1.7 ± 0.3% ID/g at the 24-h and 14-day point, respectively. For soft tissues the following was found: blood, (2.2 ± 0.5) x 10-3 and (6.1 ± 1.6) x 10-3; kidney, (1.1 ± 0.3) x 10-2 and (4.5 ± 0.2) x 10-3; liver, (1.0 ± 0.3) x 10-3 and (4.1 ± 0.3) x 10-3; spleen, (1.3 ± 0.7) x 10-2 and (1.3 ± 1.5) x 10-3; large intestine, (2.9 ± 0.1) x 10-2 and (4.9 ± 1.1) x 10-3 % ID/g at the 24-h and 14-day time points, respectively.
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Normalized for b.w. the femur content of 223Ra was 800 ± 56% ID·g/g (mean ± SD) 1 day after the injection and 413 ± 23% ID·g/g at 14 days. The femur:kidney ratio increased from (5.9 ± 2.0) x 102 at 1 day to (7.2 ± 3.0) x 102 at 14 days, whereas the femur:liver ratio increased from (6.2 ± 0.2) x 102 to (9.1 ± 6.6) x 102. Femur:spleen ratio increased from (8.1 ± 0.3) x 102 to (6.4 ± 2.2) x 103. The femoral bone:marrow ratio was 6.5 ± 2.1 after day 1 and >15 at day 14.
Therapy Study.
Dose survival plots for rats treated with 223Ra under therapy regimen A are presented in Fig. 3
. The increase in survival for animals injected with 11 kBq relative to control was significant at the 1% level. Also, when taken together, the increase in survival of the animals in the 6 and 11 kBq groups was significant compared with the control group ( P < 0.05). Furthermore, the 11 kBq group had 36% (5 of 14) symptom-free survivors beyond 50 days, whereas the animals injected with 6 kBq had 20% (1 of 5) symptom-free survivors, but survival improvement was not significant, because paralysis occurrences were not statistical significantly delayed compared with the control group.
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the survival of groups treated under regimen B are presented. The control group receiving Aredia had no survivors beyond day 21 and, hence, showed no therapeutic response at the dose level administered. The 10 and the 30 kBq groups each had 40% (2 of 5) survivors beyond 50 days. The 10 kBq and 30 kBq groups had a survival that was significantly improved (P < 0.05) compared with the control. However, at the 5 kBq dose level the survival was not significantly different from the control group. To compare larger cohorts of animals an additional graph was made by combining the animals receiving 223Ra (with or without Aredia) in one cohort, and these were compared with a cohort made of all of the control animals (with or without Aredia treatment) in Fig. 5
. The data indicated a significant therapeutic response (P < 0.005) in the 223Ra cohort compared with the controls.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-particles, on the other hand, could possibly have a future role in the treatment of metastatic cancers because their short range in tissues match well to treatment requirements of multiple small tumor foci. Because their maximum range is typically <100 µm, crossfire into bone marrow from sources located on the bone surfaces should be significantly reduced compared with ß- and conversion electron-emitting bone seekers. Also, because of the high LET quality of -particles, their cytotoxicity is nearly independent of dose rate, cell cycle phase, and oxygen concentration (12)
. -Particles could strongly inhibit tumor development if they can be successfully targeted to tumors.
To compare the bone-seeking properties of 223Ra2+ with those of other bone-seeking radioactive compounds, data from uptake in rodents were compiled and normalized so that animals of different size could be compared (Table 2)
. As can be seen 223Ra2+ was among the bone seekers with the highest uptake and had a considerably higher bone uptake than the commercially available bone seekers 89Sr2+ and 153Sm-EDTMP. It should be noticed that the magnitude of bone uptake of 223Ra2+ in rats and mice was almost identical when b.w. normalization was performed.
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10 kBq of 223Ra had significantly increased symptom-free survival compared with control, indicating that metabolically concentrated -particle emitters irradiating the bone surfaces and calcified tumor sites could slow down progression of skeletal metastases.
For comparison, the results of the current study and previous studies exploring various therapeutic modalities in the MT-1 model are presented in Table 1
. The data indicate that 223Ra has a significant therapeutic potential for use against metastatic bone disease compared with chemotherapeutic agents, immunotoxins and bisphosphonate labeled with the therapeutic ß-emitter 131I evaluated in the MT-1 model. Because 223Ra is concentrated in osseous sites including the skeletal surfaces, the antitumor effect of 223Ra may be expected to be independent of the cell characteristics, which may impede the effect of chemotherapeutic agents and immunotoxins. In contrast to immunotoxins, the antitumor effect of 223Ra is not dependent on a high and uniform expression of the antigen on all of the tumor cells and a relatively low cross-reactivity with normal cells. In addition, because 223Ra is a high LET radiation -emitter, a significant antitumor effect should also be expected on nonproliferating (resting) tumor cells adjacent to the bone surface.
The highest activity of 223Ra that was administered in the tumor treatment study corresponded to 0.3 MBq/kg b.w. or 30 kBq/animal. The therapeutic gain of increasing the dosage beyond the 10–11 kBq/animal level was not significant, indicating that a therapeutic plateau was reached with 223Ra. There could be several reasons for this. One reason could be that the complex growth pattern of the tumor model used. Because the uptake of radioactive bone seekers, like radium and strontium, would be in the growth zones of skeletal metastases, i.e., the mineralized interface between the bone and the tumor, and not in the tumor cell itself, the tumor could in some instances be out of reach for the short ranging -particles, e.g., metastases could develop from inside of marrow cavity instead of growing adjacent to the bone surface.
The bone metabolism is a complex process involving osteoblasts and osteoclasts, which are cells affecting bone formation and bone resorption. Irradiation could affect the activity of these cells in bone, and if osteoclast-mediated demineralization of bone was the effect, this could cause resorption of radionuclide from the bone. Therefore, additional treatment with the osteoclast inhibitor Aredia was included as a substudy in this work. However, the treatment did not seem to improve therapeutic response at the 30-kBq versus the 10-kBq dose level.
In general the therapeutically effective activity of 223Ra seemed to be well tolerated, because no signs of bone marrow toxicity or b.w. loss could be seen in the groups of treated animals. In the experiment with mice, an activity level corresponding to 1 MBq/kg was administered without signs of severe bone marrow toxicity or b.w. loss within 3 months after the injection of 223Ra. This indicates that therapeutically relevant doses of 223Ra could be tolerated.
223Ra decays via a multistep chain releasing four -particles. Another radium isotope with multistep decay chain, i.e., 224Ra, has been used medically in the treatment of noncancerous bone diseases, e.g., ankylosing spondylitis (22
, 23)
. A new study of 224Ra in patients with ankylosing spondylitis was reported recently (24)
indicating that significant pain relief could be obtained in the majority of patients. Compared with 223Ra the half lives of 224Ra and the nuclides in the decay chain after 224Ra transformation could be less favorable for the purpose of bone targeting. As has been demonstrated in animal experiments, a significant fraction of the daughter isotopes of 224Ra escape from bone (17
, 18)
, most probably because of the noble gas daughter radionuclide, 220Rn (t1/2 = 55.6 s), which diffused away from the site of 224Ra decay. 223Ra, on the other hand, has a half life of 11.43 days, which is about three times that of 224Ra. This allows a deeper incorporation into the bone matrix before decay occurs, and less radiation to soft tissues during the uptake and elimination phase because a lower fraction of the injected radium atoms would decay in the first few days. Also, perhaps even more important, the radon daughter 219Rn has a short half-life (3.9 s), which is likely to diminish translocation of the radon daughter. Data from rodent indicate a high retention of 223Ra daughter nuclides in bone as measured by comparing the activity levels of 211Bi with 223Ra in bone samples. On the basis of the favorable properties demonstrated in preclinical studies, 223Ra has been included recently in a clinical trial concerning patients suffering from skeletal metastases from breast and prostate cancer.
In conclusion, the present work shows that i.v. injection of 223Ra may slow down tumor progression in the skeleton. The antitumor effect is most likely linked to the delivery of an intense and highly localized radiation zone from -particles targeting the bony surfaces. This may eliminate early skeletal metastases, whereas bone marrow cells distant from the bone surfaces are spared. A clinical trial has been initiated recently to evaluate 223Ra treatment in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Jeanette and Søren Bothner Legacy. 
2 To whom requests for reprints should be addressed, at Anticancer Therapeutic Inventions AS, Kjelsåsveien 172 A, N-0884, Oslo, Norway.
3 The abbreviations used are: EDTMP, ethylene diamine N,N'-tetra(methylene) phosphonic acid; b.w., body weight; % ID/g, percentage of injected dose per gram tissue; % ID·g/g, percentage of injected dose per gram tissue x gram body weight; LET, linear energy transfer.
4 G. Henriksen, D. R. Fisher, J. C. Roeske, Ø. S. Bruland, and R. H. Larsen. Targeting of osseous sites with emitting 223Ra: comparison with the ß emitter 89Sr in mice, submitted for publication.
Received 9/19/01. Accepted 3/26/02.
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