211At was produced on CYPRIS MP-30 cyclotron (Sumitomo Heavy Industries, Ltd., Tokyo, Japan) in the Advanced Clinical Research Center at Fukushima Medical University [21]. [125I]Sodium iodide (629 GBq/mg) was purchased from PerkinElmer (Waltham, MA, USA). [67Ga]Ga-citrate was purchased from Nihon Medi-Physics Co., Ltd. (Tokyo, Japan), and converted [67Ga]GaCl3 by using Sep-Pak® Silica Plus Light Cartridge (Waters Co., Ltd., Milford, MA, USA) according to a previous report [22]. 4-(4-Iodophenyl)butyric acid was purchased from Ambeed (Arlington Heights, IL, USA). (2RS)-2-[4-(2-Methylpropyl)phenyl]propionic acid (ibuprofen) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). U-87 MG glioblastoma cells were purchased from DS Pharma Bio-medical (Osaka, Japan). Other reagents were of reagent grade and used as received.

Ga-DOTA-K([211At]APBA)-c(RGDfK) ([211At]1), [67Ga]Ga-DOTA-K(IPBA)-c(RGDfK) ([67Ga]2), and Ga-DOTA-K([125I]IPBA)-c(RGDfK) ([125I]2) were synthesized according to our previous report [16].

Experiments with animals were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University. The experimental protocols were approved by the Committee on Animal Experimentation of Kanazawa University. The animals were housed with free access to food and water at 23 °C with a 12-h alternating light/dark schedule. Normal mice were used as 6-week-old male ddY mice (29–36 g, Japan SLC, Inc., Hamamatsu, Japan).

To prepare tumor-bearing mice, 5 × 106 of U-87 MG cells were subcutaneously inoculated into the right shoulder of 4-week-old female BALB/c nude mice (13–15 g, Japan SLC, Inc.) as previously reported [23]. At approximately 10-day postinoculation of U-87 MG cells, they were used for each experiment after tumor size reached 0.3–0.5 cm3.

To evaluate the effects of albumin-binding inhibitors in normal mice, [211At]1 (37 kBq) and [125I]2 (37 kBq) were intravenously coadministered. At 1-h postinjection of radiotracers, vehicle (saline), ibuprofen at 10 molar equivalent of blood albumin (2.5 mg, 11 µmol), or IPBA at 2 (680 µg, 2.2 µmol), 5 (1.7 mg, 5.5 µmol), or 10 (3.4 mg, 11 µmol) molar equivalent of blood albumin was administered. Ibuprofen and IPBA were administered as sodium salts. The total blood albumin (1.1 µmol) in normal mice was calculated from the albumin concentration (3.0 g/dL ≈ 455 µM) in blood, which was informed from Japan SLC, Inc., total blood volume (2.5 mL) as 8% of body weight, and the molecular weight of albumin as 66,000 g/mol. Mice were sacrificed at 4-h postinjection of radiotracers.

In the case of tumor-bearing mice, [211At]1 (37 kBq) and [125I]2 (37 kBq) were intravenously coadministered. At 1-h postinjection of [211At]1 (37 kBq) and [125I]2 (37 kBq), IPBA (1.9 mg, 6.1 µmol) was administered at 10 molar equivalent of blood albumin. Mice were sacrificed at 65-, 70-min, 2-, 4-, 12-, and 24-h postinjection of [211At]1 and [125I]2. Tissues of interest were removed and weighed, and radioactivity counts of 125I and 211At were determined with an auto well gamma counter (ARC-7010; Hitachi Medical, Ltd., Tokyo, Japan) and corrected for background radiation [21]. To determine the radioactivity excreted from the body for 24 h, mice were housed in metabolic cages (Metabolica, Sugiyama-Gen Co., Ltd., Tokyo, Japan).

For estimation of the radiation dose absorbed, the blood, bone, and muscle mass of mice were assumed to be 8%, 5%, and 48% of body weight, respectively [24]. The stomach was assumed to be distributed only in the wall, the heart, small intestine, and colon equally in the content and wall, and the colon was further assumed to be equally distributed in the left, right, and rectosigmoid. According to the International Commission on Radiological Protection, an equal distribution of radionuclide to trabecular and cortical bones was assumed [25]. The non-decay-corrected activity from each source organ was converted into a percentage of the injected dose. The area under each organ’s activity curve from time 0 to infinity was calculated by extrapolation of biodistribution data. To correct for the different ratios of organ to total body weights in mouse and in human, we used the following organ correction factor (CR):

$$\mathrm\;=\;\;\mathrm/\mathrm\;\mathrm\;\mathrm\right)}_}/\;\mathrm/\mathrm\;\mathrm\;\mathrm\right)}_}$$

Human organ weights and total body weights used data for adult males from previous reports [26]. Tumor volume was converted from mouse-to-human total body weight ratio. According to the values, the radiation doses were calculated for an adult male patient using MIRDcalc v1.21 software (Society of Nuclear Medicine and Molecular Imaging) [27]. In the case of without administrating IPBA, calculation was performed using the data of a biodistribution study in our previous study [16].

SPECT/CT imaging of [67Ga]2 with or without administration of IPBA in above-mentioned U-87 MG tumor-bearing mice was performed using a small animal SPECT system (VECTor/CT, MIlabs, Houten, the Netherlands). At 1-h postinjection of [67Ga]2 (7.4 MBq), IPBA was administered at 10 molar equivalent of blood albumin. SPECT scanning was performed for 1 h from 3-h postinjection of [67Ga]1. The mice were sacrificed at 3-h postinjection because of long time acquisition of SPECT scanning.

Data were acquired in list mode, and photopeak windows were set after the acquisition. The energy windows of 80–110, 165–200, 265–320, and 350–410 keV were employed. Data were reconstructed using pixel-based order-subsets expectation maximization, with correction for attenuation on computed tomography, in 16 subsets and 6 iterations. Data were filtered with the 1-mm size Gaussian filter as the post filter. The voxel size was 0.8 × 0.8 × 0.8 mm. The obtained SPECT/CT images were analyzed using an image-processing application (AMIDE Imaging software, version 1.0.4, Slashdot Media, LCC., San Diego, CA, USA).

U-87 MG tumor-bearing mice were divided into three groups, [211At]1 (1.85 MBq) (n = 6) injected group, only [211At]2 (1.85 MBq) and IPBA (n = 6) injected group, and vehicle and IPBA (n = 3) injected group as a control group. In IPBA injected group, it was administered at 10 molar equivalent of blood albumin at 1-h postinjection of [211At]1 or vehicle. Tumor volume and body weight of mice were monitored 5 or 6 times weekly. Tumor size was measured with a slide caliper, and tumor volume was calculated using a formula: volume = 4/3 π (1/2 length × 1/2 width × 1/2 height). Tumor volume and body weight compared to the values on the day of treatment (relative tumor volume). The mice were euthanized humanely when body weight was less than 80% at baseline (day 0) or when the tumor weight reached more than 10% of body weight as the endpoint.

In biodistribution experiments, the difference between injected tracers with a double tracer method was determined by paired Student’s t test, and the difference among types and doses of inhibitors was determined by one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc test. Therapeutic experiments were analyzed by unpaired Student’s t test.