Doranidazole and 2’-[14C]-labeled doranidazole ([14C]-doranidazole) were supplied by POLA PHARMA INC. (Tokyo, Japan). The Hypoxyprobe™-1 Kit was obtained from Hypoxyprobe Inc. (Burlington, MA, USA). A BD Matrigel™ reagent was purchased from BD Biosciences (Billerica, MA, USA). Ultrapure N2 gas (99.999%) was obtained from Air Water Technical Supply (Ishikari, Japan). Other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) unless otherwise stated.
Rat glioma cell line C6 was obtained from the Health Science Research Resources Bank (Osaka, Japan). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS: Filtron, Brooklyn, Australia) at 37°C in 5% CO2/95% air.
Cell incubation, X-irradiation and drug treatment in vitro
Tumor cells attached to a 6-cm plastic dish were treated with 10 mM doranidazole before hypoxic incubation. The hypoxic condition (oxygen concentration ≤ 10 mmHg [1.3%]; unpublished data) for tumor cells in the dish was achieved by placing it in a gas-exchangeable chamber
 and continuously passing ultrapure N2 gas for 25 minutes on ice. The cells were then exposed to 20 Gy of X-rays while maintaining the gas flow. X-irradiation was performed with a Shimadzu PANTAK HF-350 X-ray generator (1.0 mm Al filter; 200 kVp; 20 mA; Shimadzu, Kyoto, Japan).
Clonogenic survival assay
After X-irradiation under hypoxia or normoxia, C6 cells were collected by trypsinization and washed with PBS. The proper number (200–30000) of cells were seeded on a 6-cm plastic dish containing fresh medium with 10% fetal bovine serum, followed by incubation at 37°C for 8 days. The cells were then fixed with methanol, stained with Giemsa solution and scored under a microscope. Only colonies containing more than 50 cells were scored as surviving cells. The surviving fraction at each dose was calculated with respect to the plating efficiency of the nonirradiated control.
WKAH/Hkm rats aged 9 weeks were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments in this study were conducted according to the guidelines of the Law for The Care and Welfare of Animals in Japan and approved by the Animal Experiment Committee of the Graduate School of Veterinary Medicine, Hokkaido University.
Intracranial tumor model
The C6 intracranial tumor model was established according to the method detailed in our previous study
. Anesthetized rats were placed on a stereotaxic device (Narishige Scientific Instrument Lab., Tokyo, Japan). A 1-mm hole was drilled through the skull 2 mm anterior and 2 mm lateral to the bregma on the right-hand side of the head. One million of C6 cells in a mixture of 5 μL FBS(−) culture media and 5 μL Matrigel were injected into the cortex at a 3-mm depth at a rate of 2 μL/min. A waiting time of 2 minutes was implemented following injection and the hole was closed using bone wax. The incision was sutured and covered with surgical glue.
Evaluation of the BBB disruption in C6-bearing rats
Vascular permeability in C6-bearing brain was evaluated by perfusing it with Evans blue dye according to the method described previously
. In brief, Evans blue dye solution (2%) was intravenously administered to rats at a dose of 3 ml/kg and allowed to circulate for 60 minutes. To remove intravascular dye, rats were transcardially perfused with saline for 20 minutes. Brains were removed and sectioned at a thickness of 2 mm.
Treatment with doranidazole and X-irradiation
Doranidazole administration and X-irradiation were performed when the tumor reached a size of 50–100 mm3. Animals were randomized into four groups: (1) no treatment; (2) X-irradiation (6 Gy) alone; (3) doranidazole administration alone; and (4) doranidazole administration at 30 minutes before X-irradiation (6 Gy). Doranidazole at a dose of 200 mg/kg was intravenously (i.v.) injected into rats. For irradiation of intracranial tumors, rats were shielded with lead panels, except for the tumor-bearing cranium. X-irradiation was performed with a Shimadzu PANTAK HF-350 X-ray generator at a dose rate of 1.2 Gy/min.
MRI was carried out using a 7.05 T superconducting magnet (Oxford Instruments, Oxford, UK) equipped with a Unity/Inova 300/183 spectrometer (Varian, Palo Alto, CA, USA). Rats were placed in the center of a 35 mm diameter quadrature RF coil. After rapid assessment of the tumor position using a multislice spin-echo (MSE) sequence, T2-weighted images (T2WIs) were also obtained using a MSE sequence with TR/TE = 2000 ms/60 ms, FOV = 80 × 80 and 60 × 60 mm (for sagittal and coronal images, respectively), image matrix = 128 × 128 and slice thickness = 1 mm. Using lengths of tumors measured in three orthogonal dimensions, tumor volume (V) was calculated as: V (mm3) = π(a × b × c)/6, where a, b and c represent width, height and thickness, respectively.
To measure leakage from the BBB, a gadolinium-chelate (Gd-[DTPA]) contrast material (Magnevist®, gadopentetate dimeglumine: Bayer Healthcare Pharmaceuticals, Montville, NJ, USA) was i.v. injected at a concentration of 0.1 mmol/kg body weight. Contrast-enhanced MRI (CE-MRI) images were obtained using multislice T1-weighted images (T1WIs) with spin-echo sequences. The parameters of the CE-MRI were TR/TE = 500 ms/16 ms, slice thickness = 1 mm, FOV = 51.2 × 51.2 mm, and image matrix = 256 × 256. The quantification of the signal enhancement due to Gd-[DTPA] uptake to glioma was performed using Image J software (National Institutes of Health, Bethesda, MD, USA) by calculating the ratio of signal intensity in tumor region to that in normal brain region.
To examine the distribution of doranidazole in the rat brain, we performed autoradiographic analysis using [14C]-doranidazole. Tumor-bearing rats were i.v. injected with 500 μL of [14C]-doranidazole (4.9 MBq/head). At 90 minutes after drug administration, rats were decapitated without prior perfusion with saline. Their brains were immediately removed and frozen. Frozen sections that were 20-μm thick were exposed to a radiosensitive imaging plate (BAS-SR2040: Fuji Film Co. Ltd., Tokyo, Japan) for 4 days with a radioactive standard slide (ARC-146: American Radiolabeled Chemicals Inc., St Louis, MO, USA). The image acquisition was performed using a BAS-2500 Bioimage Analyzer system (Fuji Film Co. Ltd. Tokyo, Japan). After the acquisition of autoradiographic images, parts of sections were fixed with 4% buffered formaldehyde and stained with hematoxylin/eosin (H/E).
At 1 day after treatment with doranidazole and/or X-irradiation tumor-bearing rats were i.v. injected with pimonidazole (Hypoxyprobe™-1 Kit; 60 mg/kg). At 90 minutes after drug administration, rats were perfused with saline and subsequently 4% buffered formaldehyde. Removed brain tissues were fixed, embedded in paraffin and sectioned at 5-μm thickness. The immunostaining procedure for pimonidazole was carried out in accordance with the manufacturer’s instructions. Serial sections were also stained with H/E. The stained images of each section were acquired using a fluorescence microscope (BZ-9000: Keyence, Osaka, Japan).
All results were expressed as the mean ± S.E. The variance ratio was estimated using the F-test and differences in means of groups were determined using Student’s t-test or Welch’s t-test. The minimum level of significance was set at P < 0.05.