Boron compounds
Maleimide-functionalized closo-dodecaborate (MID), which has been successfully synthesized, was mixed with albumin from human serum lyophilized powder (Sigma-Aldrich, Tokyo, Japan) at a ratio of 1:10. The conjugate was subsequently used in several experiments (Maleimide-functionalized closo-dodecaborate albumin conjugate, MID-AC) [14]. BPA (L-isomer) was kindly provided by Stellar Chemifa (Osaka, Japan) and converted into a fructose complex [15]. All boron compounds used in this study were boron-10 concentrates.
Cell Culture
In this study, we used F98 rat glioma cells because they can infiltrate and invade the normal brain parenchyma of a Fischer rat and are reportedly refractory to various treatments, including radiotherapy. Moreover, F98 rat glioma models progress histologically to undifferentiated or anaplastic gliomas in in vivo pathological histology [16]. They often serve as brain tumor models for evaluating the BNCT therapeutic effects [17–20]. F98 rat glioma cells were kindly provided by Dr. Rolf Barth (The Ohio State University, Columbus, Ohio, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 10% fetal bovine serum and penicillin, streptomycin, and amphotericin B at 37 ℃ in an atmosphere of 5% CO2. All materials were purchased from Gibco Invitrogen (Grand Island, NY, USA).
F98 rat glioma model
Each 10-week-old male Fischer rat used in this study weighed between 200 and 250 g. The rats were anesthetized by intraperitoneal injection of a mixed anesthetics containing medetomidine (ZENOAQ, Fukushima, Japan) (0.4 mg/kg), midazolam (SANDOZ, Yamagata, Japan) (2.0 mg/kg), and butorphanol (Meiji Seika, Tokyo, Japan) (5.0 mg/kg). Each head was fixed with a stereotactic frame (Model 900; David Kopf Instruments, Tujunga, CA, USA), and F98 rat glioma cells were implanted into each rat brain. 103 F98 rat glioma cells diluted in a 10 µL solution of DMEM containing 1.4% agarose (Wako Pure Chemical Industries, Osaka, Japan) for therapeutic experiments or 105 F98 rat glioma cells for biodistribution experiments were injected at a rate of 20 µL/min by an automated infusion pump, respectively. Our research groups use these surgical procedures routinely [17–20].
Biodistribution of boron compounds in F98 rat glioma models
Approximately 12 days post-implantation of 105 F98 rat glioma cells, when the tumor was expected to have grown sufficiently, each boron compound was administered at the following bodyweight (b.w.) doses: 12 mg boron (B)/kg b.w. for BPA and 20 mg B/kg b.w. for MID-AC, respectively. All rats were euthanized at a fixed time, and the tumor, brain, blood, heart, lung, liver, kidney, spleen, skin, and muscle were removed. Consequently, each organ was weighed and digested with 1N nitric acid solution. After each organ dissolved sufficiently, the boron concentration was measured by using inductively coupled plasma atomic emission spectroscopy (ICP-AES; iCAP6300 emission spectrometer, Hitachi, Tokyo, Japan). Results were normalized as µg B/g.
Survival analysis of a neutron irradiation experiment for F98 rat glioma models
This study aimed to evaluate the therapeutic effects of MID-AC on brain tumors. A neutron irradiation experiment was conducted at the nuclear reactor (Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Osaka, Japan) for 14 days post-implantation of 103 F98 rat glioma cells into the cells the brains of Fischer rats. Thirty-five F98 rat glioma models were randomly divided into the following five groups: untreated control group (Untreated), neutron-irradiated control group (Irradiation only), neutron irradiation following 2.5 hours after termination of intravenous administration (i.v.) of BPA (BNCT using BPA 2.5 h), and neutron irradiation following 2.5 or 24 hours after termination of i.v. of MID-AC (BNCT using MID-AC 2.5 h or 24 h). Regarding MID-AC, this neutron irradiation experiment was conducted not only following 2.5 h after termination of i.v. of MID-AC, but also at 24 h, in order to evaluate the effect of the long retention of boron in the tumor. In other words, it was confirmed whether the administration of a boron compound with a long retention time in the tumor would have sufficient BNCT effect even after a long time after administration.
All rats were anesthetized by intraperitoneal injection of a mix of anesthetics described above, and the study groups received BPA or MID-AC. Excluding their heads, all rat bodies were attached on a plate lined with 6LiF ceramic tiles to shield and reduce neutron irradiation, and then neutron irradiation was performed. F98 rat glioma models were irradiated at a reactor power of 5 MW with the Heavy Water Irradiation Facility for 20 minutes. After neutron irradiation, all rats remained under the same experimental conditions as the control groups, and observations continued until their time of death or euthanasia. Finally, the therapeutic effects of BNCT were evaluated by Kaplan–Meier survival curves, and the percent increased life span (%ILS) was determined based on the median survival times (MST). %ILS was calculated by the equation (MST of each BNCT group - MST of Untreated group) × 100 / (MST of Untreated group).
Determining the physical dose, estimated photon-equivalent doses, and the compound biological effectiveness based on the in vivo neutron irradiation experiment
The physical dose was calculated from thermal, epithermal, fast neutrons, and gamma rays contained in the irradiated neutrons. This is calculated by the equation DB + DN + DH + Dγ, which correspond to 10B(n,α)7Li, 14N(n,p)14C, and 1H(n,n)1H capture reactions and γ-ray, respectively. More specifically, DB is the physical dose of boron derived from the equation of 7.43 × 10− 14 (Gy cm2/µg 10B/g) × boron concentration (µg 10B/g) × thermal neutron fluence (1/cm2). DN is the physical dose of nitrogen derived from the equation of 6.78 × 10− 14 (Gy cm2/weight %) × nitrogen concentration (weight %) × thermal neutron fluence (1/cm2). DH is the elastic scattering between epithermal or fast neutrons and the hydrogen nucleus, and Dγ is the measured value of gamma rays mixed in the neutron beam [20]. Based on the results of the biodistribution experiments for F98 rat glioma models, the physical doses to the brain and the tumor of the F98 rat glioma model were calculated in BNCT using BPA 2.5 h, MID-AC 2.5 h, and MID-AC 24 h. The estimated photon-equivalent dose for BNCT using BPA 2.5 h was calculated by the equation DB × compound biological effectiveness (CBE) + DN × relative biological effectiveness of nitrogen (RBEN) + DH × relative biological effectiveness of hydrogen (RBEH) + Dγ [20]. CBE is a biological effectiveness factor specific for each irradiated tissue and each boron compound at the neutron capture reaction. Based on the survival time of each group and the estimated photon-equivalent doses in BNCT using BPA 2.5 h, the CBE of MID-AC was calculated by the estimated photon-equivalent doses acquired in BNCT using MID-AC.
Statistical Analysis
The Kaplan–Meier curve evaluated the MST from the implantation date of F98 rat glioma cells, and log-rank tests determined the potentially significant differences among the groups. A p-value less than 0.05 were considered statistically significant. Statistical analyses were performed by the JMP® Pro version 15.1.0. software (SAS, Cary, NC, USA).