Acute exposure to high dose radiation can cause acute radiation syndrome (ARS), a potentially life-threatening illness. Individuals that survive ARS are at risk of developing the delayed effects of acute radiation exposure, with the lungs being particularly susceptible (DEARE-lung). For individuals at risk of radiation exposure, there are no Food and Drug Administration-approved medical countermeasures (MCMs) for prophylactic or post-exposure use that can prevent or mitigate DEARE-lung. BIO 300 is a novel formulation of synthetic genistein that has been extensively studied as a prophylactic MCM for the hematopoietic subsyndrome of ARS (H-ARS). Here, we used a C57L/J mouse model of total-body irradiation (TBI) to investigate whether prophylactic administration of BIO 300 is able to prevent animals from developing DEARE-lung. Oral and parenteral formulations of BIO 300 administered prior to TBI were compared against standard of care, PEGfilgrastim, administered shortly after radiation exposure, and the combination of oral BIO 300 administered prior to TBI and with PEGfilgrastim administered post-exposure. All animals were exposed to 7.75 Gy cobalt-60 gamma-radiation and the primary endpoint was lung histopathology at 180 days post-TBI. Animals treated with BIO 300 had a significant reduction in the incidence of interstitial lung inflammation compared to vehicle groups for both the oral (0% vs. 47%) and parenteral (13% vs. 44%) routes of administration. Similar results were obtained for the incidence and severity of pulmonary fibrosis in animals treated with oral BIO 300 (incidence, 47% vs. 100% and mean severity score, 0.53 vs. 1.3) and parenteral BIO 300 (incidence, 63% vs. 100% and mean severity score, 0.69 vs. 1.7). PEGfilgrastim alone had no significant effect in reducing the incidence of inflammation or fibrosis compared to vehicle. The combination of oral BIO 300 and PEGfilgrastim significantly reduced the incidence of interstitial inflammation (13% vs. 46%) and the severity of pulmonary fibrosis (mean severity score, 0.93 vs. 1.6). Results in the C57L/J mice were compared to those in CD2F1 mice, which are less prone to lung injury following total-body irradiation. Taken together, these studies indicate that BIO 300 is a promising MCM that is able to prophylactically protect against DEARE-lung.
Individuals acutely exposed to high dose radiation are at risk of developing life-threatening complications. Large doses of radiation can cause injury to the whole body and initiate radiation sickness, which is an acute illness also known as acute radiation syndrome (ARS). ARS consists of many health effects that can impact multiple tissues and organs. One of the most radiosensitive organs is the bone marrow which can be damaged by exposure to doses as low as 1 Gy. Acute radiation injury of the bone marrow is known as the hematopoietic subsyndrome of ARS (H-ARS), which can involve anemia, neutropenia and thrombocytopenia, hemorrhaging, infection, and bone marrow failure. Surviving ARS does not preclude individuals from further health complications (1 ). The delayed effects of acute radiation exposure (DEARE) may manifest months after the initial exposure event. DEARE can impact multiple different organs, including the lungs, which are especially radiosensitive, and can present as radiation pneumonitis and pulmonary fibrosis (2 ).
U.S. Food and Drug Administration (FDA)-approved medical countermeasures (MCMs) for high dose radiation exposure are only indicated for H-ARS and have to be administered after radiation exposure. These include the leukocyte growth factors filgrastim (Neupogen), PEGfilgrastim (Neulasta) and sargramostim (Leukine), and the thrombopoietin analog, romiplostim (Nplate) (3 ). There are no FDA-approved MCMs that are effective against DEARE-lung, and there are no approved MCMs that are effective prophylactically and can protect individuals at risk of high dose radiation exposure such as warfighters and first responders. Several DEARE-lung MCMs are currently under investigation, including the angiotensin-converting enzyme (ACE) inhibitor lisinopril (4 ), the transforming growth factor-beta (TGFβ) receptor inhibitor IPW-5371 (5 ), and the superoxide dismutase mimetic AEOL 10150 (6 ). However, all these MCMs are administered post-exposure and have not been evaluated for prophylactic efficacy.
BIO 300 is a MCM under development that has demonstrated prophylactic efficacy against H-ARS and post-exposure efficacy against DEARE-lung in small animal models (7–11 ). The active pharmaceutical ingredient in BIO 300 is synthetic genistein [5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one], which is an isoflavone that functions as a selective estrogen receptor beta (ERβ) agonist (9 ). Multiple formulations of BIO 300 have been developed for different routes of administration such as BIO 300 Injectable Suspension (BIO 300 IS), which is suitable for parenteral (intramuscular, intravenous, and subcutaneous) dosing, and BIO 300 Oral Powder (BIO 300 OP), which was developed for oral dosing. Here, we sought to evaluate the prophylactic efficacy of BIO 300 IS and BIO 300 OP against DEARE-lung in a total-body irradiation (TBI) C57L/J mouse model. C57L/J mice are the ideal small animal model for DEARE-lung studies as they present with a similar pathogenesis of radiation-induced lung injury in a similar timeframe, within the same relative dose range, as humans and nonhuman primates (NHPs) (12–15 ). We also compared the prophylactic efficacy of both BIO 300 formulations to that of PEGfilgrastim administered after TBI, as well as the combination of BIO 300 OP given prior to TBI and PEGfilgrastim administered after TBI. Both BIO 300 formulations, as well as the combination of BIO 300 OP with PEGfilgrastim, demonstrated significant efficacy in preventing radiation-induced lung inflammation and fibrosis, whereas PEGfilgrastim administered alone had no significant impact on lung injury.
MATERIALS AND METHODS
Drug Preparation and Dosing
BIO 300 OP and BIO 300 IS (Humanetics Corporation, Minneapolis, MN), PEGfilgrastim (Neulasta, Amgen Inc, Thousand Oaks, CA) and the corresponding vehicles for each formulation were prepared as previously described (16 ). BIO 300 OP was dosed at 200 mg/kg by oral gavage (po) twice daily for 6 consecutive days prior to TBI with the final dose administered 24 h prior to TBI. BIO 300 OP was administered in a 0.2 ml volume with a 1 ml syringe and an 18-gauge feeding cannula. BIO 300 IS was dosed at 200 mg/kg and administered as a single intramuscular (im) injection in the thigh muscle 24 h prior to TBI. PEGfilgrastim was administered as a single subcutaneous (sc) injection between the shoulder blades at 0.3 mg/kg given 24 h after TBI. All po, im and sc dosing of mice was completed as previously described (16 ).
Mice and Total-Body Irradiation
Male 7- to 9-week-old C57L/J mice (The Jackson Laboratory, Bar Harbor, ME) or male 6-8 week old CD2F1 mice (Envigo, Indianapolis, IN) were housed (N=4/cage) in an air-conditioned facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care-International. All mice were maintained in rooms on a 12-h light/dark cycle, at 21 ± 2°C, with 10 to 15 hourly cycles of fresh air, and a relative humidity of 50% ± 10% (16 ). All animal procedures were performed according to a protocol approved by the Armed Forces Radiobiology Research Institute (AFRRI), Uniformed Services University of the Health Sciences (USUHS) Institutional Animal Care and Use Committee (IACUC). Research was conducted according to the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council, US National Academy of Sciences (17 ).
For TBI, mice were placed in well-ventilated Plexiglas boxes compartmentalized to accommodate eight mice per box and exposed to simultaneous bilateral radiation in the high-level cobalt-60 facility at a dose rate of 0.6 Gy/min to total midline doses of 7.75 Gy (C57L/J) or 9.2 Gy (CD2F1). After the procedure, mice were returned to their cages and monitored for any acute signs of distress. Radiation dosimetry was based on the alanine/electron paramagnetic resonance system as described earlier (16 ).
After TBI, mice were monitored for 180 days, three times daily, with a maximum of a 10 h gap between checks during the critical period, which runs from day 10–20 postirradiation. Outside of the critical period, mice were checked twice daily, and any abnormalities were recorded. Mice found to be moribund (prior to the study endpoint of day 180) were euthanized by CO2 asphyxiation, and cervical dislocation was performed as a secondary method of euthanasia. Lung tissue was not collected from these animals for histopathologic analysis. All animals that survived until study ended (180 days post-TBI) were euthanized by deeply anesthetizing the animal with isoflurane (confirmed with toe pinch) and cervical dislocation. After euthanasia of these animals, lung, sternum, and jejunum tissue was collected for histopathologic analysis.
Lung histopathology was performed to evaluate the effect of BIO 300 formulations and PEGfilgrastim on radiation-induced lung injury. Tissues (lung, sternum, and jejunum) were fixed, embedded in paraffin, and cut with a rotary microtome at 4 microns. All tissue samples were stained with hematoxylin and eosin (H&E), and serial sections of lung tissues were also stained with Masson's Trichome stain to observe collagen deposition in order to evaluate the extent of fibrosis. Tissue slides were evaluated by an independent board-certified veterinary pathologist (ToxPath Solutions, Ponte Vedra, FL). Lesions were scored by severity using a 5-point Likert scale: 1 (minimal), 2 (mild), 3 (moderate), 4 (marked) and 5 (severe). All findings were minimal or mild in severity, which indicates that there were 1–5 foci/lesions (minimal) or 6–10 foci/lesions (mild) for the specified microscopic findings in the entire lung section.
Animal survival at 180 days post-TBI was assessed by Kaplan-Meier curves, which were analyzed by log-rank tests for statistical differences in survival between drug-treated and vehicle-treated groups. Descriptive statistics were used for lung histopathology scores (mean ± SD) and unpaired t-tests were used to compare means. Statistical tests were conducted using GraphPad Prism 9.4 (GraphPad Software, San Diego, CA).
C57L/J mice were exposed to a 7.75 Gy total-body dose in order to ensure animals did not succumb to ARS, and a sufficient number of animals survived until 180 days post-TBI for lung histopathology analysis (18 ). As expected, overall mortality was minimal through 180 days post-TBI; no treatment group lost more than 1 animal and no vehicle group lost more than 3 animals (Fig. 1). There was no statistical difference in survival by log-rank test between any study groups. Furthermore, no histopathological abnormalities were noted within the jejunum and sternum, these tissues were normal in all treatment and vehicle group animals.
Lung sections from the 180-day survivors had two major findings that were related to radiation exposure. These consisted of interstitial inflammation characterized by an increase in monocytic cells within densely packed alveolar structures, and fibrosis which was evidenced by blue-staining collagen fibrils within alveolar walls. Within the BIO 300 treatment groups, a significant mitigation of the chronic effects of radiation exposure in the lungs of C57L/J mice at 180 days post-TBI was observed, when compared to the corresponding vehicle-treated animals. None of the vehicle animals in any group had normal lung histopathology whereas the BIO 300 OP and BIO 300 IS groups had N = 7/15 (47%) and N = 6/16 (38%) animals, respectively, with normal lungs on histopathology examination (Table 1). The BIO 300 OP group was the only group that had mineralization in the lung (N = 2/15; 13%), a microscopic finding defined as small areas of calcium deposition occurring incidentally or as a result of previous tissue necrosis. Both the PEGfilgrastim and BIO 300 OP + PEGfilgrastim groups had only a single animal with normal lungs on histopathology examination. Each vehicle group presented with 40–50% of animals having a minimal degree of interstitial inflammation (Table 1). BIO 300 treatment groups, alone or in combination with PEGfilgrastim, had a significant decrease in the incidence of interstitial inflammation, whereas PEGfilgrastim alone did not significantly reduce lung inflammation (Fig. 2A and B).
Collagen fibrils were often quite thin in mice administered BIO 300 and/or PEGfilgrastim, but often quite thick in mice administered vehicle. Analysis of fibrosis demonstrated that 100% of animals in all vehicle groups had evidence of minimal to mild fibrosis, and the fibrosis tended to consist of a greater number of affected areas, with thicker bands of mature collagen than that observed in mice administered any of the treatment agents (Fig. 2A). In general, mice that received one of the BIO 300 formulations, alone or in combination with PEGfilgrastim, had a decreased incidence and/or severity of fibrosis compared to findings observed in corresponding vehicle groups (Fig. 2C). BIO 300 treated animals had minimal fibrosis with only one or two foci of interstitial fibrosis, usually near the pleural surface, and these would likely have not created a problem with complete oxygenation on inspiration. PEGfilgrastim administered alone appeared to reduce the severity of fibrosis but did not significantly improve the incidence of fibrosis compared to vehicle animals (Fig. 2C). Further, BIO 300 OP administered alone significantly reduced the incidence of fibrosis compared to the combined treatment of BIO 300 OP + PEGfilgrastim.
BIO 300 OP and BIO 300 IS were previously evaluated for the prophylactic efficacy against H-ARS (primary) and against DEARE-lung (secondary), along with PEGfilgrastim administered post-exposure, in a CD2F1 mouse model of TBI (16 ). However, in the CD2F1 model, a 9.2 Gy TBI dose was used, which corresponded to an LD87/180 to LD100/ 180 in vehicle-treated animals. Here we show that all BIO 300 OP and BIO 300 IS-treated animals had minimal signs of immune cell infiltration in the lungs at 180-days post-TBI, and about half of the animals had minimal signs of fibrosis as determined by Masson's trichome staining (Fig. 3). However, the high mortality of the CD2F1 vehicle treated animals precludes an accurate evaluation of BIO 300 OP and BIO 300 IS on DEARE-lung in the CD2F1 model.
We sought to evaluate multiple BIO 300 formulations for prophylactic efficacy against DEARE-lung. Previously, studies were completed using a CD2F1 mouse model (16 ). The limited survival of CD2F1 vehicle-treated animals, and the minimal lung histopathology shown in that study, confounded interpretation of those results and did not allow evaluation of the BIO 300 formulations against DEARE-lung. The previously used CD2F1 mouse model, compared to the current C57L/J model, likely requires a radiation dose greater than 9.2 Gy in order to develop significant radiation-induced lung injury.
C57L/J mice were used for these studies because they have been well-characterized as an excellent model of DEARE-lung that presents with similar pathological features in the lungs as NHPs and humans following radiation exposure (12–15, 18, 19). Importantly, others have shown that a similar non-lethal dose of whole thoracic lung irradiation (WTLI) (7.5 Gy) results in pathophysiological outcomes in the lungs of C57L/J mice (18 ). These studies were extended to other strains of mice as well and found signs of lung histopathologic changes in the absence of significant mortality further emphasizing the radiosensitivity of the lung. Since this was the first evaluation of BIO 300 in this DEARE-lung model, we used only male C57L/J mice. This was done in order to reduce the number of variables that may impact radiation response in this study as there are sex-specific differences in radiation sensitivity between male and female animals (20–23 ). It is acknowledged that this is a major limitation to this study, and that future development efforts will require the inclusion of animals of both sexes. BIO 300 functions as a ERβ agonist, which is expressed in the lungs of both males and females (24 ). So, while there may be sex differences in radiation response, it is not anticipated that there will be sex differences in BIO 300 efficacy, although this will need to be confirmed in future studies. Per FDA communications, TBI was preferentially used over WTLI as a “real-world” scenario that is more applicable for MCM evaluation. More recently, the FDA has been open to the use of partial-body irradiation (PBI) (25 ) and future studies would consider the use of the PBI model as well as testing also in female mice. PEGfilgrastim (Neulasta) was included as an active comparator as well as in combination with BIO 300 OP because the FDA suggests evaluation of MCMs with current standard of care as part of drug development under the FDA Animal Rule (21 CFR 314.600-650).
Consistent with previous studies of C57L/J mice which indicated that this strain of mice develop pneumonitis which can rapidly progress into fibrosis (18, 19, 26 ), 100% of vehicle animals exhibited minimal to mild pulmonary fibrosis, and nearly half of the vehicle animals had evidence of interstitial inflammation at 180 days after TBI. Animals treated with BIO 300 formulation alone had a significant decrease in the incidence of interstitial inflammation and fibrosis, whereas PEGfilgrastim alone did not significantly mitigate radiation-induced lung pathology. The lack of effect on the lungs by PEGfilgrastim alone is not completely unexpected as it is an MCM approved to mitigate H-ARS. Since PEGfilgrastim is currently used as standard of care, we sought to determine its effect on DEARE in this model. Of note, the combination of BIO 300 OP with PEGfilgrastim reduced the severity of pulmonary fibrosis but unlike the monotherapy, the combination did not reduce the incidence of fibrosis as effectively. According to the FDA package insert for Neulasta (PEGfilgrastim), acute respiratory distress syndrome is a serious adverse reaction that may occur following sc administration of PEGfilgrastim. Therefore, it is possible that two different mechanisms (PEGfilgrastim and radiation) are damaging the lung, which may explain the attenuated efficacy of BIO 300 OP when given in combination with PEGfilgrastim. Whether or not PEGfilgrastim impacts the ability of BIO 300 OP to mitigate radiation induced lung injury warrants further investigation.
Taken together, a C57L/J model of DEARE-lung is better than a CD2F1 model, as the C57L/J mice develop signs of pneumonitis and fibrosis at a lower dose of radiation, which allows for more animals to survive for DEARE-related analyses in control as well as in drug-treated groups. This is crucial for MCM development because MCM-treated animals need to be compared to vehicle and standard of care controls. In conclusion, BIO 300 OP and BIO 300 IS were both able to prophylactically prevent the development of DEARE-lung in C57L/J mice exposed to 7.75 Gy total-body dose. The combination of BIO 300 OP and PEGfilgrastim did not improve the efficacy of BIO 300 OP administered prophylactically as a monotherapy. These results continue to support the development of BIO 300 as a prophylactic radiation MCM for DEARE-lung.
The authors thank Allen W. Singer, DVM, DACVP, DABT of Singer & Associates Toxpath Consulting Solutions (Ponte Vedra, FL) for his veterinary pathology expertise in the analysis of the study results. The research reported in this study was supported by the Department of Defense Congressionally Directed Medical Research Programs Grant #W81XWH-17-1-0584 awarded to Humanetics Corporation, subaward to VKS and administered by The Henry M. Jackson Foundation for the Advancement of Military Medicine. The opinions or assertions contained herein are the private views of the authors and are not necessarily those of the Uniformed Services University of the Health Sciences or the Department of Defense.
1 Vijay K. Singh, PhD, Division of Radioprotectants, Department of Pharmacology and Molecular Therapeutics F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814: email: firstname.lastname@example.org; and Artur A. Serebrenik, PhD, Humanetics Corporation, 7760 France Avenue South, Suite 920, Minneapolis, MN 55435; email: email@example.com.