Antitumor effects of radionuclide treatment using α-emitting meta-211At-astato-benzylguanidine in a PC12 pheochromocytoma model

Purpose Therapeutic options for patients with malignant pheochromocytoma are currently limited, and therefore new treatment approaches are being sought. Targeted radionuclide therapy provides tumor-specific systemic treatments. The β-emitting radiopharmaceutical meta-131I-iodo-benzylguanidine (131I-MIBG) provides limited survival benefits and has adverse effects. A new generation of radionuclides for therapy using α-particles including meta-211At-astato-benzylguanidine (211At-MABG) are expected to have strong therapeutic effects with minimal side effects. However, this possibility has not been evaluated in an animal model of pheochromocytoma. We aimed to evaluate the therapeutic effects of the α-emitter 211At-MABG in a pheochromocytoma model. Methods We evaluated tumor volume-reducing effects of 211At-MABG using rat pheochromocytoma cell line PC12 tumor-bearing mice. PC12 tumor-bearing mice received intravenous injections of 211At-MABG (0.28, 0.56, 1.11, 1.85, 3.70 and 5.55 MBq; five mice per group). Tumor volumes were evaluated for 8 weeks after 211At-MABG administration. The control group of ten mice received phosphate-buffered saline. Results The 211At-MABG-treated mice showed significantly lower relative tumor growth during the first 38 days than the control mice. The relative tumor volumes on day 21 were 509.2% ± 169.1% in the control mice and 9.6% ± 5.5% in the mice receiving 0.56 MBq (p < 0.01). In addition, the mice treated with 0.28, 0.56 and 1.11 MBq of 211At-MABG showed only a temporary weight reduction, with recovery in weight by day 10. Conclusion 211At-MABG exhibited a strong tumor volume-reducing effect in a mouse model of pheochromocytoma without weight reduction. Therefore, 211At-MABG might be an effective therapeutic agent for the treatment of malignant pheochromocytoma. Electronic supplementary material The online version of this article (10.1007/s00259-017-3919-6) contains supplementary material, which is available to authorized users.

Targeted radionuclide therapy (TRT) is a target-specific systemic therapy with a simple cytotoxic mechanism that directly targets cells such as those with DNA damage from radiation [9]. 131 I-MIBG is a false analog of norepinephrine and is therefore taken into the pheochromocytoma cell via the uptake-1 mechanism [10,11]. 131 I-MIBG, because of the cytotoxic effects of β-radiation, can improve survival in patients with malignant pheochromocytoma [12][13][14]. However, even with high doses of 131 I-MIBG, survival is still limited and 131 I-MIBG is associated with radiation-induced side effects such as bone marrow suppression and lung injury [15]. Therefore, new therapeutic approaches are required to treat malignant pheochromocytoma.
A new generation of TRT involves the use of α-particles. The α-particle is exclusively cytotoxic and not affected by many of the limitations associated with conventional chemotherapy and radionuclide therapy. The α-particle has high mean energy deposition (linear energy transfer, LET) and a limited range in tissue, resulting in strong therapeutic effects with minimal side effects [16]. Theoretically, 211 At-MABG should be more effective and have fewer side effects.
The therapeutic applications of α-emitters have mainly focused on 211 At, 233 Ra, 213 Bi and 225 Ac [16,17]. For our purposes, we required an α-emitter-labeled ligand of the norepinephrine transporter. To maintain the affinity of a benzylguanidine analog for the norepinephrine transporter, we had to use an α-emitter which has similar characteristics to 131 I. Concerning the therapeutic applications of αemitters, 211 At is a halogen and has similar characteristics to 131 I [18]. Therefore, 211 At is suitable for labeling of a benzylguanidine analog with an α-emitter, and 211 At-MABG will have characteristics similar to those of 131 I-MIBG [19]. A previous study showed in vitro cytotoxicity in neuroblastoma cells [20]. However, to date there have been no studies looking at the therapeutic effect of 211 At-MABG in neuroblastoma and pheochromocytoma in vivo in animal models. The purpose of the present study was to investigate the therapeutic effects of 211 At-MABG in a pheochromocytoma model both in vitro and in vivo.

Materials and methods
Production of 211 At and radiosynthesis of 211 At-MABG 211 At was produced and recovered as described previously [19,21] (Supplementary material). The emitted radioactivity of 211 At was 22.1-93.2 MBq at the end of bombardment, and the radiochemical purity of 211 At was more than 99.9% at the end of recovery. Benzylguanidine analog was labeled with 211 At according to a previously published method [19] with slight modification (see a detailed description in the Supplementary material). A high-purity germanium detector was used to measure radioactivity. Radiochemical purity of 211 At-MABG was estimated using reverse-phase radio-high-performance liquid chromatography (radio-HPLC).
Cell culture PC12 rat pheochromocytoma cells (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) that have high norepinephrine transporter expression [22] were cultured in RPMI-1640 (Wako Pure Chemical Industries, Osaka, Japan) containing 10% horse serum (Thermo Fisher Scientific, Waltham, MA) and 5% fetal bovine serum (Serum Source International, Charlotte, NC). The cells were cultured at 37°C in humidified air containing 5% CO 2 .
Cell survival assay PC12 cells (1 × 10 6 cells) were incubated with 0, 0.2, 0.6, 2.0, 6.0 and 20.0 kBq/mL of 211 At-MABG for 24 h. The cells were then washed with phosphate-buffered saline (PBS), suspended in growth medium, and seeded at 400 cells/well in a 96-well plate. After incubation for 14 days, the cells were incubated with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37°C. Absorbance at 590 nm was measured using a plate reader (VMax; Molecular Devices, Sunnyvale, CA). Rates of cell survival were normalized to the absorbance of control cultures treated with 0 kBq/mL. DNA double-strand break assay PC12 cells (1 × 10 6 cells) were incubated with 0, 0.6, 2.0 and 6.0 kBq/mL of 211 At-MABG for 24 h. The neutral comet assay was used to detect DNA double-strand breaks (DSB) using a CometAssay kit (Trevigen, Gaithersburg, MD) according to the manufacturer's instructions. Comet tails were stained with SYBR Green and analyzed using a fluorescent microscope.

Pheochromocytoma mouse model
The animal experimental protocol was approved by the Animal Care and Use Committees of our institutions, and all animal experiments were conducted in accordance with the institutional guidelines regarding animal care and handling. PC12 cells (3 × 10 6 ) were subcutaneously inoculated into the right hind limb of female BALB/c-nu/nu mice at 5 weeks of age (CLEA Japan, Tokyo, Japan) under isoflurane anesthesia.

Biodistribution study
PC12 tumor-bearing mice (five mice per time point) were injected with 100 kBq of 211 At-MABG in 100 μL of PBS into a tail vein. The mice were killed at 1, 3, 6, 12 and 24 h after 211 At-MABG administration. Blood, tumor, and organs of interest were dissected and weighed, and radioactivity was measured using a γ-counter (ARC-7001; Aloka, Tokyo, Japan). The radioactivity of organs and tissues except the thyroid is presented as the percentage injected radioactivity dose per gram (% ID/g), and that of the thyroid as percentage injected radioactivity dose (% ID) [23].

Dosimetry
The mean doses per unit of injected activity (grays per megabecquerel) of α-disintegrations from 211 At absorbed by each organ and tumor were estimated according to the standard method using the Medical Internal Radiation Dose formula [24,25] (Supplementary material).

Monitoring tumor volume and body weight after 211 At-MABG treatment
When the PC12 tumor volumes had reached approximately 50 mm 3 , the mice (body weight 20.89 ± 1.30 g) were injected intravenously with 211 At-MABG (0.28, 0.56, 1.11, 1.85, 3.70 and 5.55 MBq; five mice per dose) or PBS (ten mice). Tumor size and body weight were measured at least twice a week for 8 weeks after 211 At-MABG administration. Tumor size was measured using a digital caliper, and tumor volume was calculated using the formula: tumor volume (mm 3 ) = (length × width 2 )/2.
Body weight loss is considered to be one of the major radiation-related side effects of radiopharmaceuticals. Therefore, we evaluated the change in body weight of the mice as a marker of radiation-related side effects [26].

Primary endpoint after 211 At-MABG treatment
When body weight loss was more than 20% compared with that at baseline (day 0), signs of a moribund state were observed, or the tumor volume had reached more than 800 mm 3 , the mouse was killed humanely by isoflurane inhalation, and the tumor was resected for histological analysis. For Kaplan-Meier survival analysis, a tumor volume of 500 mm 3 was considered the endpoint in addition to a body weight loss of more than 20%.
Pathological analysis: hematoxylin and eosin staining and immunohistochemical staining For analysis of temporal histological change in tumors, subcutaneous PC12 tumors were resected from the mice on days 1, 3 and 7 after administration of 1.11 MBq of 211 At-MABG (three mice per time point). For analysis of dose-dependent tumor volume reduction, PC12 tumors from mice killed on day 3 or 4 after injection of 1.85, 3.70 and 5.55 MBq of 211 At-MABG were evaluated as described above.
The resected tumors were fixed in 10% neutral-buffered formalin and embedded in paraffin. The tumor sections (1 μm thick) were deparaffinized and stained with hematoxylin and eosin (H&E). Immunohistochemical staining for Ki-67 was performed using rabbit anti-Ki-67 (Abcam, Cambridge, MA) and an anti-rabbit HRP/DAB detection kit (Abcam) according to the manufacturer's instructions [27].
To evaluate systemic toxicity of 211 At-MABG, histological changes in the bone marrow, adrenal glands, and heart of the mice were analyzed on H&E-stained sections. These organs were resected from the mice at the same time as the tumors. The organs were fixed and embedded in paraffin as described above. These sections (1 μm thick) were deparaffinized and stained with H&E. Images were obtained using a NanoZoomer S60 virtual slide scanner (Hamamatsu Photonics, Shizuoka, Japan).

Statistical analysis
Continuous measures are presented as means ± standard deviation. Data were analyzed by analysis of variance with Dunnett's multiple comparison test. The survival curves for each treatment group were compared with that for the control group using the log-rank test [28]. A p value <0.05 was considered statistically significant. Statistical calculations were carried out using GraphPad Prism and Statcel 3.

Chemical and biological characterization of 211 At-MABG
The radio-HPLC analysis, cell uptake assay and inhibition assay were performed to confirm that the product was 211 At-MABG. The retention time (t R ) of the product was 19.1 min, which was close to that of nonradioactive MIBG (t R = 18.7 min); Supplementary Fig. 1). The radiochemical yield after HPLC purification was 61.5 ± 14.4% (decay-corrected, n = 4) and the radiochemical purity was over 99.7%. The cell uptake assay showed that the product was rapidly transported into PC12 cells that have high norepinephrine transporter expression ( Supplementary  Fig. 2a). The inhibition assay showed that desipramine (DMI), a selective inhibitor of the norepinephrine transporter, and dl-norepinephrine significantly inhibited cell uptake of the product (p < 0.01, Supplementary Fig. 2b), and uptake was also significantly suppressed by incubation at 4°C.
In vitro tumor cell-damaging effects of 211 At-MABG 211 At-MABG treatment dose-dependently suppressed survival of PC12 cells relative to control cells without 211 At-MABG treatment, as shown using the MTT assay (p < 0.01,vs. control; Fig. 1a). 211 At-MABG treatment induced lactate dehydrogenase (LDH) release (a cell death marker) from PC12 cells (p < 0.01,vs. control; Supplementary Fig. 3). 211 At-MABG treatment dosedependently increased the proportion of cells with DNA DSB and the percentages of cells with DNA DSB treated with 2.0 and 6.0 kBq/mL 211 At-MABG were significantly higher than that in the control group (p < 0.05 for 2.0 kBq/ mL, p < 0.01 for 6.0 kBq/mL; Fig. 1b).

In vivo study
Biodistribution and dosimetry studies Table 1 shows the biodistribution of 211 At-MABG in PC12tumor bearing mice. The uptake of 211 At-MABG in tumors was higher than that in other organs and tissues at all time points (Table 1). 211 At-MABG rapidly accumulated in tumors, and tumor uptake at 1 h after injection reached approximately 30% ID/g. The highest tumor uptake was reached at 3 h, and thereafter uptake decreased gradually. However, accumulation remained high at 24 h. PC12 tumors showed high absorbed doses of 211 At-MABG (10.21 Gy/MBq; Table 2). Compared with other normal organs and tissues, the adrenal gland and heart with high norepinephrine transporter expression showed relatively high uptake (Table 1). The estimated doses absorbed by the adrenal gland and heart were 5.07 and 4.08 Gy/MBq, respectively ( Table 2).

Absorbed radiation dose
The estimated radiation dose absorbed by PC12 tumors was 10.21 Gy/MBq (Table 2), and the calculated dose absorbed by tumors treated with 1.11 MBq of 211 At-MABG was therefore 11.3 Gy. The efficacy of 1.11 MBq of 211 At-MABG was almost equivalent to that of 30 Gy of external X-ray irradiation ( Supplementary Fig. 4).
Weight change after 211 At-MABG treatment 211 At-MABG treatment caused a dose-dependent decrease in body weight soon after administration (Fig. 4). On day 3 after injection, all the mice injected with 0.28 and 0.56 MBq of 211 At-MABG showed a decrease in body weight of less than 5% (p ≤ 0.05 for 0.28 MBq, p ≤ 0.01 for 0.56 MBq). Also, mice injected with 1.11 MBq of 211 At-MABG showed a decrease in body weight of 10-20% (p ≤ 0.01), and a decrease in body weight of 20% was observed in one of the five mice 3 days after injection. However, the body weight decrease in all these mice was temporary, and in these groups body weight gradually recovered. As a result, there were no differences in body weight of the mice treated with 0.28, 0.56 and 1.11 MBq 211 At-MABG compared with that of the control group on day 10 after injection (p = 0.154).
In contrast, all mice treated with 1.85, 3.70 and 5.55 MBq 211 At-MABG showed decreases in body weight of more than 20% on day 3 or 4 after 211 At-MABG administration, and were therefore killed humanely at that time. Their tumors were resected and used for histological analysis. Based on these The values presented are mean ± SD percentage injected radioactivity dose per gram (% ID/g), except the thyroid values which are percentage injected radioactivity dose (% ID)

Histological analysis of PC12 tumors after 211 At-MABG treatment
Since 1.11 MBq of 211 At-MABG was the MTD, we analyzed the temporal histological changes in the PC12 tumors on days 1, 3 and 7 after administration of 1.11 MBq 211 At-MABG.
Tumor sections were stained with H&E and Ki-67 as a proliferation marker. In sections of control tumors (0 MBq), the cells were arranged in a nest pattern and were surrounded by fibrovascular stroma (Fig. 5, left upper panel). In sections of tumors from mice treated with 211 At-MABG, there were no nests of tumor cells, whereas hemorrhage and lymphocyte infiltration were observed (Fig. 5, left panels). The hemorrhage increased in a time-dependent manner, and a small necrotic area was observed on day 3, and the area had expanded by day 7 (Fig. 5, left panels). In Ki-67-stained sections of tumors from mice treated with 211 At-MABG, proliferating (Ki-67-positive) tumor cells tended to decrease in a timedependent manner (Fig. 5, right panels).
Sections of tumors from mice treated with 1.85 MBq 211 At-MABG showed larger hemorrhage and necrotic areas than following treatment with 1.11 MBq 211 At-MABG (Fig. 6  Endpoints were designated as an increase in tumor volume to 500 mm 3 and a decrease in body weight by more than 20% from day 0. Groups treated with 0.56 and 1.11 MBq 211 At-MABG showed significantly better survival than the control group. **p < 0.01, *p < 0.05, vs. 0 MBq panels). In sections of tumors from mice treated with 3.70 MBq 211 At-MABG, partial replacement by fibrous tissue was also observed in addition to hemorrhage and necrosis (Fig. 6, left panels). The area of fibrous tissue was larger following treatment with 5.55 MBq 211 At-MABG (Fig. 6, left panels). In Ki-67-stained sections, proliferating tumor cells tended to decrease in a time-dependent manner (Fig. 6, left panels).

Histological changes in bone marrow, adrenal gland, and heart after 211 At-MABG treatment
In sections of bone marrow from mice treated with 1.11 MBq 211 At-MABG, dilated vascular sinuses filled with erythrocytes were observed on day 1 after 211 At-MABG administration. The number of myeloid cells showed a slight decrease on day 3. However, on day 7 after administration of 1.11 MBq 211 At-MABG, the number of myeloid cells had recovered to the same level as in control mice (Fig. 7a). In the adrenal gland and the heart from mice treated with 1.11 MBq 211 At-MABG, histological changes were not observed on days 1, 3 and 7 (Fig. 7a).
Sections of femur from mice killed 3 or 4 days after administrations of 1.85, 3.70 and 5.55 MBq 211 At-MABG showed decreases in bone marrow cellularity and increases in density of erythrocytes within the expanded vascular sinuses in a dose-dependent manner (Fig. 7b). In particular, administration of 5.55 MBq 211 At-MABG induced severe depletion of cells in the bone marrow, vascular dilation and hemorrhage (Fig. 7b). Although histological changes were not observed in sections of adrenal glands from mice treated with 1.11 MBq and 1.85 MBq 211 At-MABG, some vacuolated medullary cells were observed in the adrenal glands from mice treated with 3.70 and 5.55 MBq 211 At-MABG (Fig. 7b). Sections of heart from mice treated with 1.85, 3.70 and 5.55 MBq 211 At-MABG and mice in the control group showed no differences in morphological features (Fig. 7b).

Discussion
Treatment with 211 At-MABG reduced the tumor volumes in PC12 tumor-bearing mice in a dose-dependent manner. In mice treated with 211 At-MABG, reductions in body weight and in the number of myeloid cells in the bone marrow were not severe. This may indicate that 211 At-MABG has tumorreducing effects without severe radiation-induced side effects.
Histology showed hemorrhage and tumor necrosis soon after 211 At-MABG administration. These histological findings confirm the tumor volume-reducing effects of 211 At-MABG in in vivo studies.

Quality of 211 At-MABG
The radiochemical purity of 211 At-MABG was more than 99.7%. In addition, the selective norepinephrine transporter inhibitor DMI as well as norepinephrine inhibited cell uptake of the radiolabeled product. These results agree with those of previous studies involving neuroblastoma cells [29,30], and indicate that the quality of the 211 At-MABG used in this study was appropriate.

In vitro tumor cell growth suppression effects of 211 At-MABG
In this study, 211 At-MABG reduced the PC12 cell survival ratio in a dose-dependent manner. This effect of 211 At-MABG in reducing cell survival agrees with the findings of previous in vitro studies looking at the toxicity of 211 At-MABG in neuroblastoma cells [20]. This finding further expands the possible role of 211 At-MABG in the treatment of pheochromocytoma. 211 At-MABG induced LDH release as a cell-death marker and dose-dependently increased the number of cells with DNA DSB. These results suggest that decreased cell survival following 211 At-MABG treatment is probably due to cell death induced by DNA DSB. This cell death mechanism, as confirmed in this study, supports the hypothesis of cell injury by α-particles and is most likely the main mechanism by which 211 At-MABG causes cell death in pheochromocytoma [16,31].

Therapeutic effects of 211 At-MABG in PC12 tumor-bearing mice
Similar to the findings of a previous study using a neuroblastoma model [20], biodistribution studies showed that 211 At-MABG accumulated more in the adrenal gland and heart than in other organs. Both the adrenal gland and heart have a rich sympathetic nervous system. Therefore, a norepinephrine analog may accumulate more readily in these organs [32,33]. The current data also show very high 211 At-MABG uptake (about 35% ID/g) in PC12 tumors. A biodistribution study of 211 At-MABG in a neuroblastoma mouse model (SK-N-SH xenograft) showed about 4% ID/g uptake by SK-N-SH tumors [32]. Although we did not directly compare the therapeutic effects of 211 At-MABG in the neuroblastoma model to those in pheochromocytoma models (because we have not yet been able to establish a neuroblastoma mouse model), the current data suggest that 211 At-MABG may be more effective in pheochromocytoma than in neuroblastoma. This possibility should be investigated in future studies.
Some previous studies investigating neuroblastoma and pheochromocytoma cells in vitro have shown the possible therapeutic effects of 211 At-MABG [20]. However, there have been no studies looking at the antitumor therapeutic effects of 211 At-MABG in neuroblastoma and pheochromocytoma animal models in vivo. In this regard, this study showed that 211 At-MABG has therapeutic effects in vivo, and thus provides further insight into the therapeutic potential of 211 At-MABG.
In PC12 tumor-bearing mice, 211 At-MABG treatment at doses of 0.56 MBq and higher reduced tumor volumes compared with those in control mice. The present study once again provides new insights into the therapeutic effects of 211 At-MABG in a pheochromocytoma mouse model. 211 At-MABG administration led to almost complete disappearance of tumor for up to 21 days. The protocol used in this study involved a single administration of 211 At-MABG. Thus, after 21 days, tumor cells began to grow. In the clinical setting, 131 I-MIBG is usually administered at intervals of 6 weeks to 3 months [10,11]. The lack of repeated 211 At-MABG administrations is one of the limitations of this study; future studies should focus on the effects of repeated treatments.

Histological findings on the therapeutic effects of 211 At-MABG
The histological findings of this study confirmed the effectiveness of 211 At-MABG in reducing tumor volumes. Clinical research using 131 I-MIBG has shown that most patients achieve stable disease, and that complete remission was very rare [4,13]. These clinical data suggest that the cytotoxic effects of the β-emitting 131 I-MIBG are limited. In contrast, in this study the PC12 tumors in mice receiving higher doses of 211 At-MABG showed necrosis and fibrosis at earlier time points than tumors in mice receiving lower doses. Although careful dose setting would be necessary, it is possible that 211 At-MABG treatment could lead to complete remission in patients with malignant pheochromocytoma. In contrast, catecholamine released from damaged pheochromocytoma cells can result in catecholamine crisis. This causes hypertension and catecholamine-induced cardiomyopathy [5,34]. Patient management after 211 At-MABG therapy should be considered as the next step prior to clinical trials.
Possible therapeutic effects of 211 At-MABG versus 131

I-MIBG
In this study, we could not directly compare the therapeutic effects of 211 At-MABG with those of 131 I-MIBG. However, in a study by Rutgers et al. using the same pheochromocytoma mouse model, administration of 57 MBq of 131 I-MIBG maximally reduced tumor volume to approximately 30% of the volume on day 0 [35], while in this study tumors treated with 1.11 MBq 211 At-MABG were reduced to approximately 3.3% of the volume on day 0. Therefore, the MTD of 211 At-MABG, while being a fraction of the MTD for 131 I-MIBG, would have a significantly greater tumor-reducing effect (approximately nine times) in pheochromocytoma therapy than 131 I-MIBG.
A systematic review and meta-analysis of the effect of 131 I-MIBG on tumor volume found a complete remission rate after 131 I-MIBG therapy of 3%, a partial remission rate of 27% and a stable disease rate of 52% [13]. In this study, all five mice treated with the MTD of 211 At-MABG (1.11 MBq) showed a reduction in tumor volume, and in two of them the tumor disappeared until day 28 after 211 At-MABG administration. Moreover, based on the percentage tumor volume reduction, in mice receiving 1.11 MBq 211 At-MABG, tumor volumes were reduced by 96.7% compared to the volumes at baseline. Strictly speaking, although the RECIST criteria cannot be applied to our data [36], in mice treated with the MTD of 211 At-MABG (1.11 MBq), 40% achieved complete remission and 60% had partial remission. In terms of side effects of 131 I-MIBG, the most frequently reported side effects were hematologic toxicity with grade 3 or 4 neutropenia that occurred in up to 87% and grade 3 or 4 thrombocytopenia that occurred in up to 83% [13]. Although we did not monitor complete blood cell counts before and after 211 At-MABG administration, bone marrow histological findings revealed rapid recovery of bone marrow cellularity. Therefore, 211 At-MABG may have greater therapeutic effects with fewer side effects than 131 I-MIBG.

Safety aspects of 211 At-MABG therapy
One of the greatest limitations of current TRT is radiationrelated side effects such as bone marrow suppression. Since β-emitting radiotracers have far-reaching effects, surrounding organs are irradiated and damaged [9]. On the other hand, because of the short range of α-particles (<100 μm) radiation-induced side effects are minimized in the clinical setting [37]. In this study, we measured the body weight of the mice to monitor radiation-induced adverse effects and, based on the body weight reduction, decided that the MTD of 211 At-MABG in nude mice was 1.11 MBq. Mice receiving a dose of 211 At-MABG lower than or equal to the MTD showed temporary weight reduction after administration but then a gradual recovery in body weight. Thus, there was no significant weight reduction at 10 days after 211 At-MABG administration compared with the body weight of control mice.
Changes in bone marrow cellularity indicate systemic toxicity from exposure to chemicals or radiation. Therefore, evaluation of bone marrow is important in toxicity and safety assessments [38,39]. We evaluated bone marrow cellularity using H&E-stained femur sections. The treatment with 211 At-MABG at the MTD (1.11 MBq) caused no marked change in bone marrow cellularity compared with the bone marrow of the control group. In contrast, treatment with 1.85, 3.70 and 5.55 MBq of 211 At-MABG induced obvious decreases in the number of nucleated cells and increases in the density of erythrocytes within the dilated vascular spaces. Sections of the adrenal gland and heart that express norepinephrine transporter were also evaluated. There were no marked histological changes in any organs treated with the MTD of 211 At-MABG, whereas some vacuolated medullary cells were detected in adrenal gland treated with 3.70 and 5.55 MBq of 211 At-MABG. These results indicate that the MTD of 211 At-MABG determined in terms of body weight should be safe based on these histological findings.

Study limitations
The protocol used in this study involved a single 211 At-MABG administration because the aim was to clarify the initial therapeutic effects and to evaluate the adverse effects. Based on the current data, we determined the therapeutic MTD of 211 At-MABG. The next step will be to conduct a repeated-treatment study in the near future. We evaluated the therapeutic and adverse effects of 211 At-MABG but did not directly compare these with the effects of 131 I-MIBG. We compared the therapeutic efficacy of 211 At-MABG with that of 131 I-MIBG in previously published studies, and we estimated the effective dose of 211 At-MABG.

211
At-MABG showed a strong tumor volume-reducing effect in a pheochromocytoma mouse model without severe adverse effects such as weight reduction and reductions in the numbers of myeloid cells in the bone marrow. Therefore, 211 At-MABG might be an effective therapeutic agent for the treatment of malignant pheochromocytoma.
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