A novel oral formulation of BIO 300 confers prophylactic radioprotection from acute radiation syndrome in mice

Abstract

Purpose

Exposure to high doses of ionizing radiation can result in hematopoietic acute radiation syndrome (H-ARS) and delayed effects of acute radiation exposure (DEARE). There is no radiation medical countermeasure (MCM) approved by the U.S. Food and Drug Administration which can be used prior to radiation exposure to protect exposed individuals. Different formulations containing synthetic genistein (BIO 300) are being developed to counter the harmful effects of radiation exposure.

Materials and methods

We investigated the efficacy of a BIO 300 oral powder (OP) formulation as a prophylactic radiation MCM against a lethal dose of cobalt-60 gamma-radiation in CD2F1 male mice while comparing to other formulations of BIO 300 and Neulasta (PEGylated filgrastim), a standard of care drug for H-ARS.

Results

BIO 300 OP provided significant radioprotection against ionizing radiation in mice when administered twice per day for six days prior to total-body radiation exposure. Its radioprotective efficacy in the murine model was comparable to the efficacy of a single subcutaneous (sc) injection of Neulasta administered after total-body radiation exposure.

Conclusions

Our results demonstrate that BIO 300 OP, which can be administered orally, is a promising prophylactic radiation countermeasure for H-ARS.

Keywords:

• Animal model
• BIO 300
• mice
• prophylactic
• radiation
• radioprotection

Introduction

Exposure to ionizing radiation due to a nuclear accident or incident may cause a spectrum of radiation syndromes known as acute radiation syndrome (ARS) (Gale et al. 2021). Radiation doses of 2 Gy or higher can lead to a multitude of tissue injuries resulting in various sub-syndromes of ARS such as hematopoietic, gastrointestinal, neurovascular, and cutaneous (Singh and Seed 2017). Hematopoietic ARS (H-ARS) in humans occurs following exposure to radiation doses of 2–6 Gy at a high dose rate and is characterized by the development of neutropenia, thrombocytopenia, and anemia. The hematopoietic system is particularly radiosensitive because stem and progenitor cells are actively dividing, increasing their susceptibility to DNA damage by radiation and free radicals generated by oxidative stress (Hall and Giaccia 2012; Heylmann et al. 2021). Surviving acute injury does not eliminate the risk of developing further radiation-induced health complications at a later date. The delayed effects of acute radiation exposure (DEARE) can manifest several months after the initial radiation exposure-induced injury and include a plethora of pathologies affecting multiple organs, particularly the lungs (DiCarlo et al. 2012; Unthank et al. 2015; MacVittie et al. 2019). DEARE-lung is characterized by a chronic inflammatory response to the initial radiological insult that progresses into potentially life-threatening pneumonitis and subsequent pulmonary fibrosis (Garofalo et al. 2014; Unthank et al. 2015).

Currently, there are only four FDA-approved radiation medical countermeasures (MCMs) that are indicated for the treatment of H-ARS. Neupogen (filgrastim/granulocyte colony-stimulating factor (G-CSF)), Neulasta (PEGfilgrastim/PEGylated G-CSF), and Leukine (sargramostim/granulocyte-macrophage colony-stimulating factor (GM-CSF)) are approved to treat H-ARS-associated neutropenia, while Nplate (romiplostim) is approved to treat H-ARS-associated thrombocytopenia (Farese et al. 2013; Farese and MacVittie 2015; Hankey et al. 2015; Singh and Seed 2018; Wong et al. 2020a, 2020b; Zhong et al. 2020; Clayton et al. 2021; Gale and Armitage 2021; U.S. Food and Drug Administration 2021). All four agents require parenteral administration shortly after radiation exposure (24–48 h after radiation exposure), which is not ideal for administration in austere environments. Furthermore, all four agents function as cellular growth factors, and therefore their mechanisms of action only support treatment and not the prevention of H-ARS. Murine studies with G-CSF or GM-CSF showed that prophylactic administration of either cytokine did not improve survival following lethal total-body irradiation (TBI) (Neta et al. 1988; Singh and Seed 2018). Furthermore, Neupogen did not show efficacy in the absence of supportive care in a large animal model (Gluzman-Poltorak et al. 2014). Although this effort has been on going for the last six decades, there is currently no FDA-approved MCM for radioprotection from H-ARS.

Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one) is an isoflavone commonly found in soy and it functions as a non-steroidal, selective estrogen receptor-beta (ERβ) agonist (Landauer 2008; Landauer et al. 2019). The radioprotective properties of genistein have been studied for decades, and it has been shown to confer prophylactic radioprotection from H-ARS in murine models (Landauer 2008; Singh and Seed 2020). However, genistein has failed to be clinically adopted due to its poor bioavailability resulting from its near insolubility in water. The pharmacokinetic (PK) challenges were overcome with the development of a suspension of synthetic genistein nanoparticles named BIO 300. Since its development, BIO 300 has been extensively tested in animal models of H-ARS (Ha et al. 2013; Cheema et al. 2019; Landauer et al. 2019; Girgis et al. 2020; Singh and Seed 2020), DEARE-lung (Jackson et al. 2017; Jones et al. 2017), and solid tumor radiotherapy (Jackson et al. 2019). Additionally, BIO 300 was the subject of a recently completed phase 1b/2a study in patients with non-small cell lung cancer (NSCLC; NCT02567799). In this clinical study, BIO 300 was tested as a supportive care therapeutic for the mitigation of chemoradiotherapy-induced pneumonitis and pulmonary fibrosis.

Separate BIO 300 formulations were developed to facilitate administration of this agent through oral and parenteral routes. BIO 300 injectable suspension (BIO 300 IS) is administered parenterally, while BIO 300 oral suspension (BIO 300 OS) is administered orally (po, per-os). BIO 300 OS has demonstrated excellent oral bioavailability in both mice (unpublished observation) and nonhuman primates (Cheema et al. 2019), and a novel oral solid-dosage formulation is also being developed to enable easy field use. This formulation, BIO 300 oral powder (BIO 300 OP), is a free-flowing powder produced by hot-melt extrusion (HME). Initial H-ARS studies in mice with BIO 300 OP have demonstrated that it has an equivalent efficacy to BIO 300 OS (Singh and Seed 2020).

Here, we continued the development of BIO 300 OP as a prophylactic radiation MCM. First, we assessed the radioprotective efficacy of BIO 300 OP through a dose scheduling study in a TBI murine model. Next, we evaluated the efficacy and durability of the response of BIO 300 OP compared to other formulations of BIO 300 and FDA-approved Neulasta in a 180-day TBI mouse study. The prophylactic oral efficacy of BIO 300 OP is noteworthy and equivalent to the post-exposure parenteral administration of Neulasta.

Materials and methods

Test and control items

BIO 300 injectable suspension (323 mg/mL genistein, 5% povidone K17 (w/w), 0.2% polysorbate 80 (w/w) in 50 mM phosphate buffered saline (61 mM sodium chloride)) was used for intramuscular (im) dosing. BIO 300 oral suspension (325 mg/mL genistein, 5% povidone K25 (w/w), 0.2% polysorbate 80 (w/w), 0.18% methylparaben (w/w), and 0.02% propylparaben (w/w)). BIO 300 OS was dosed by oral gavage (po). Both BIO 300 IS and BIO 300 OS are aqueous liquid suspensions of synthetic genistein nanoparticles with a median particle size of 200 nanometers. BIO 300 OP is an amorphous solid dispersion of genistein (347 mg/g genistein, 65% povidone K12 (w/w)) produced as a free-flowing dry powder with a median particle size of 160 µm. BIO 300 OP is prepared by HME and milled to the final particle size. BIO 300 OP was slowly dispersed into vehicle (0.5% Methocel A4M, 3% PVP K25 (w/w) in water) and kept suspended with continuous stirring until po dosing. Neulasta: was procured from Amgen Inc. (Thousand Oaks, CA) as 6 mg/0.6 mL liquid in a single use prefilled syringe and administered subcutaneous (sc). BIO 300 IS Vehicle: 5% povidone K17 (w/w) and 0.2% polysorbate 80 (w/w) in 50 mM phosphate buffered saline (61 mM sodium chloride) at a pH of 7.4. BIO 300 OS Vehicle: double-distilled water. BIO 300 OP Vehicle: 5 mg/mL methylcellulose A4M, 30 mg/mL povidone K25. Neulasta Vehicle: normal saline (0.9% NaCl).

Drug preparation and administration

BIO 300 IS was resuspended in its vehicle to deliver a 200 mg/kg dose in 0.05 mL. A 0.5 mL insulin syringe with a 28 G needle was used for im drug administration via the thigh muscle. BIO 300 OS was further diluted with its vehicle to deliver a 200 mg/kg dose in 0.2 mL. A 1 mL Luer-lock syringe with a 20 G disposable feeding needle with a silicone-rubber tip was used for the po drug administrations. BIO 300 OP was mixed with its vehicle using pestle and mortar to deliver a 200 mg/kg dose in 0.2 mL. The solution was continuously stirred at room temperature until dosing. Pharmaceutical-grade PEGfilgrastim (Neulasta) was further diluted with its vehicle to deliver a 300 µg/kg dose in 0.1 mL. A 1 mL Luer-lock syringe with a 23 G needle was used for sc drug administrations via the nape.

All oral gavages via the po route were administered by securing the mouse at its scruff to extend the two front legs while immobilizing the head and neck. The gavage needle was then slowly inserted into the left side of the animal’s mouth and directed along the hard palate of the mouth to the back of the throat and guided to the abdominal area to deliver the bolus. The feeding needle was wiped and disinfected between dosing using a gauze sponge that was moistened with 70% ethanol (absolute alcohol diluted to 70%) to reduce the microorganisms and saliva on the feeding needle, and was then wiped with a purified water-soaked gauze sponge to reduce irritation to the mucosa before commencing dosing.

Total-body irradiation of mice

CD2F1 mice were placed in irradiation boxes that consisted of compartmentalized Plexiglas boxes designed to accommodate eight mice. These boxes were then positioned in the irradiation towers, and the towers were hand carried to the irradiation platform where the mice were exposed to a bilateral, midline dose of 9.2 Gy TBI ⁶⁰Co γ-radiation at a dose rate of 0.6 Gy/min. The radiation dosimetry used in this study was discussed in detail earlier (Seed et al. 2014). At the completion of the irradiation procedure, the mice were observed for any adverse reactions and then returned to their home cages and monitored for 30- or 180-days post-irradiation.

Efficacy studies in mice

Male 6–8-week-old CD2F1 mice were purchased from Envigo (Indianapolis, IN). These animals were subsequently housed (four per cage) following arrival in an environmentally controlled facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care-International. Details about animal housing and care have been published earlier (Singh et al. 2014). All animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee (protocol numbers AFR-18-003 and PHA-20-026). 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 (National Research Council of the National Academy of Sciences 2011).

Histopathology of lung tissue

Lung tissue samples were harvested at 180 days post-irradiation and processed for terminal histopathology from all irradiated mice. The lung samples were gently perfused with zinc-buffered formalin, placed in tissue cassettes, and submerged further in zinc-buffered formalin for at least 12 h. All samples were further processed by trimming and sectioning for hematoxylin and eosin-stained slides, which were used for the assessment of morphological changes, and with Masson’s trichrome stained slides, which imparts a blue color to collagen structures, and were used for the assessment of pulmonary fibrosis.

Statistical analysis

The Kaplan–Meier curves were constructed to view overall survival throughout each study. Log-rank tests were performed to compare survival between the drug treated groups and their respective vehicle control group. p values of less than 0.05 were considered statistically significant and have been noted with an asterisk (*). All statistical tests were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA).

Results

BID dosing of BIO 300 OP to identify the optimal dose schedule for prophylactic efficacy

Prior studies with genistein or BIO 300 OP have indicated that multiple days of prophylactic dosing are required to improve survival following myelosuppressive doses of TBI in mice (Landauer 2008; Singh and Seed 2020). However, no dose scheduling study has been performed with BIO 300 OP. Here, we sought to identify the optimal BIO 300 OP dose and treatment schedule in mice for improving survival following lethal TBI exposure. In this study, CD2F1 mice were administered BIO 300 OP (200 mg/kg/dose), BID (administered twice a day, bis in die), po, or its vehicle control consecutively for 6, 5, 4, 3, 2, and 1 day prior to 9.2 Gy TBI, or one day after 9.2 Gy TBI (cobalt-60; 0.6 Gy/min, approximately LD_70/30). As a positive control, BIO 300 IS (200 mg/kg) or its vehicle were administered as a single im injection 24 h prior to 9.2 Gy TBI. All irradiated animals were monitored for 30 days post-TBI.

In this model, vehicle-treated animals started to succumb to ARS between day 7–13 post-TBI, and the 30-day survival mean for the vehicle control groups was 26.6% (median, 31.3%; range, 6.3–50%), confirming that 9.2 Gy TBI is an LD_70/30 (Table 1). All treatment groups resulted in a statistically significant improvement in survival except for dosing 5 d prior to and 1 d after TBI (Figure 1, Table 1). The net percent radioprotection (survival of the BIO 300 OP treated group minus the survival of the respective vehicle control) for the various treatment schedules of this experiment is presented in Figure 1(C). BIO 300 OP dosing for six days prior to TBI showed a net 62.5% improvement in survival, while net radioprotection after dosing for five days was 31%, four days was 50%, three days was 44%, two days was 56%, and one day was 56%. BIO 300 OP dosing 24 h post-irradiation showed 13% net radioprotection (Figure 1(C)), while BIO 300 IS administered 24 h pre-irradiation exhibited 62.5% net radioprotection (drug-treated 93.75%, vehicle 31.25%) (Figure 1(B)). Based on the results of this experiment, the group administered BIO 300 OP for six days prior to TBI demonstrated the highest survival rate as compared to its corresponding vehicle (62.5%, drug-treated 68.75%, vehicle 6.25%) (Figure 1(A)) and provided radioprotection equivalent to BIO 300 IS.

Figure 1. The Kaplan–Meier curves and net radioprotection of 30-day survival of mice administered BIO 300 OP twice daily using various dose schedules. CD2F1 mice (n = 16/group) were administered (A) BIO 300 OP (200 mg/kg) or vehicle po, BID for 6 d prior to TBI or (B) BIO 300 IS (200 mg/kg) or its vehicle administered as a single im injection 24 h prior to TBI. (C) Net radioprotection of BIO 300 OP administered twice daily using various dose schedules or BIO 300 IS administered 24 h prior to TBI. Percent net radioprotection at 30 days was determined by subtracting BIO 300 treatment survival from the respective vehicle group’s survival for each dose schedule and BIO 300 formulation tested. All mice were irradiated with 9.2 Gy cobalt-60 (0.6 Gy/min, approximately LD_70/30 dose). Survival was monitored for 30 d post-irradiation. Statistical significance for 30-day survival was determined by log-rank test (*p<.05).

Table 1. Summary of BIO 300 oral powder 30-day dose scheduling study.

Treatment group	Route of administration	Dosing schedule (30-day survival)
		6 d pre	5 d pre	4 d pre	3 d pre	2 d pre	1 d pre	1 d post
BIO 300 IS	im						93.8%
IS Vehicle	im						31.3%
BIO 300 OP	po, BID	68.8%	62.5%	100%	56.3%	87.5%	93.8%	25%
OP Vehicle	po, BID	6.25%	31.3%	50%	12.5%	31.3%	37.5%	12.5%

BIO 300 OP (oral powder, 200 mg/kg, twice a day for different days as mentioned prior to irradiation) and BIO 300 IS (injectable suspension, 200 mg/kg, 24 h prior to irradiation) were evaluated in CD2F1 mice using 9.2 Gy total-body cobalt-60 gamma-radiation exposure.

Efficacy of BIO 300 OP on mouse survival in mice

The current standard of care for H-ARS is Neupogen, Neulasta, Leukine, or Nplate. These drugs are administered parenterally and are only indicated for administration after radiation exposure. Since there are no FDA-approved prophylactic treatments for H-ARS, the radioprotective efficacy of BIO 300 IS, OP, and OS was compared to the radiomitigative efficacy of Neulasta in CD2F1 mice exposed to lethal TBI. To better resolve differences in survival following TBI between the different therapeutics, the following studies were carried out for 180-days post-TBI using CD2F1 mice. This endpoint was chosen based on previous DEARE-lung studies that reported the development of radiation pneumonitis in mice within the first 3–6 months post-irradiation, and demonstrated significant collagen deposition in the lungs at 180 days post-irradiation (Jackson et al. 2012). Furthermore, by monitoring animals for 180 days rather than the standard 30 days for H-ARS studies, the durability of response for BIO 300 or Neulasta can be evaluated, as well as the efficacy of the therapeutics for protecting or mitigating against DEARE-lung. Mice were treated with BIO 300 OP (200 mg/kg), BIO 300 OS (200 mg/kg) or their corresponding vehicle controls BID, po, for six consecutive days prior to TBI, or with BIO 300 IS (200 mg/kg) or its vehicle control as a single im injection 24 h prior to TBI. Neulasta (300 mg/kg) or its vehicle control were administered as a single sc injection 24 h after TBI. All animals in this study received 9.2 Gy (0.6 Gy/min) cobalt-60 gamma-irradiation.

Interim survival analysis was performed on day 30 post-TBI to assess the efficacy of the BIO 300 formulations (Figure 2(A)) and Neulasta (Figure 2(B)) against H-ARS. The 30-day survival for all 4 vehicle groups was 18.8%, indicating that 9.2 Gy was an LD_80/30 in this study. Survival for BIO 300 IS, BIO 300 OP, BIO 300 OS, and Neulasta was 100%, 87.5%, 62.5%, and 81.3%, respectively (net 30-day radioprotection for BIO 300 IS, BIO 300 OP and BIO OS was 81.3%, 68.8% and 43.8%, respectively, and net 30-day radiomitigation for Neulasta was 62.5%). There was no statistical difference between 30-day survival of Neulasta and any of the BIO 300 groups.

Figure 2. The Kaplan–Meier curves of 30-day survival of CD2F1 mice administered BIO 300 IS, OP, OS, or Neulasta. (A) Mice (n = 16/group) were administered BIO 300 IS (200 mg/kg) or its vehicle by im 24 h prior to TBI. BIO 300 OP (200 mg/kg) and BIO 300 OS (200 mg/kg) or their respective vehicle controls were administered BID, po to mice for 6 d prior to TBI. (B) Mice (n = 16/group) were administered Neulasta (300 µg/kg) sc or its vehicle 24 h post-TBI. All animals were irradiated with 9.2 Gy cobalt-60 (0.6 Gy/min, approximately LD_70/30). Survival was monitored for 30 d post-TBI. *Statistical significance at day 30 between the drug treated group and respective vehicle control as determined by log-rank test (*p<.05, **p<.01, ***p<.001).

To assess the radioprotective efficacy in terms of enhanced survival due to mitigation of DEARE, all above-mentioned irradiated mice were monitored until day 180 post-TBI (Figure 3). The mean ± SD 180-day survival for all four vehicle groups was 7.8%±6.0 (median, 9.4%; range, 0–12.5%) indicating that 9.2 Gy was approximately an LD_90/180. After the initial 30 days, BIO 300 IS-treated animals continued to succumb to disease and 180-day survival dropped to 81.3% for the drug-treated group and 0% for the vehicle-treated group (net radioprotection was unchanged at 81.3%) (Figure 3(A)). For the BIO 300 OP treatment group, survival was 81.3% while survival for the vehicle group was 12.5% (net radioprotection was unchanged from day 30 at 68.8%) (Figure 3(A)). As for BIO 300 OS, survival at 180 days was unchanged at 62.5%, while survival for the vehicle-treated group dropped to 6.3% (net radioprotection improved to 56.3%) (Figure 3(A)). Percent survival for Neulasta-treated animals dropped to 56.3%, while survival for the vehicle-treated animals dropped to 12.5% (net radiomitigation dropped from day 30 to 43.8%) (Figure 3(B)). There was no statistical difference between 180-day survival of Neulasta and any of the BIO 300 groups.

Figure 3. The Kaplan–Meier curves of 180-day survival of CD2F1 mice administered BIO 300 IS, OP, OS, or Neulasta. Mice that survived 30 d post-TBI in the above study were monitored for an additional 150 d until 180 d post-TBI. (A) Mice (n = 16/group) were administered the indicated BIO 300 formulation or vehicle as described above. (B) Mice (n = 16/group) were administered Neulasta or vehicle as described above. Survival was analyzed at 180 d post-TBI. *Statistical significance at day 180 between the drug treated group and respective vehicle control as determined by log-rank test (*p<.05, **p<.01, ***p<.001).

Furthermore, the durability of response can be determined by comparing animal survival from 30 to 180 d post-TBI. BIO 300 OP exhibited improved survival durability compared to Neulasta and BIO 300 IS, with survival dropping by only 6.3% from day 30 to day 180, while Neulasta and BIO 300 IS survival dropping 25% and 18.8%, respectively, in the same time frame. No animals died after day 30 in the BIO 300 OS group, although it is important to note that this group had the worst 30 d survival among the treatment groups. These results indicate that prophylactic administration of BIO 300, particularly BIO 300 IS and BIO 300 OP, is equivalent, if not modestly improved, when compared to Neulasta administered post-exposure.

Histopathological evaluation

Histopathology analysis of CD2F1 lung tissue collected at 180 days post-irradiation (Table 2) revealed that all mice had minimal or mild mononuclear inflammatory cell infiltrates either in the interstitial wall of alveoli or presenting as increased macrophages within alveolar spaces. Neutrophils were rarely present in the alveolar spaces. A few mice in the BIO 300 IS treatment group (n = 1) and the Neulasta group (n = 2) had evidence of minimal type II pneumocyte hyperplasia, indicative of an ongoing repair of alveolar cell damage. One mouse from the BIO 300 IS-treated group had excessive neutrophil infiltrate within alveolar spaces, and this was coded ‘alveolar mixed cell infiltrate’. In general, BIO 300 or Neulasta-treated animals exhibited minimal mononuclear cell infiltrate, while the few vehicle-treated animals that survived until day 180 had mild mononuclear cell infiltrate.

Table 2. Histopathological effects of total-body irradiation on lungs of CD2F1 male mice at day 180 after total-body irradiation.

Lung (microscopic findings)	Treatment group (number of animals)
	BIO300 OS (n = 10)													Vehicle OS (n = 1)
Mononuclear cell infiltrate	1	1	1	1	1	1	1	1	1	1				2
Fibrosis, interstitial, alveolar wall	1	0	1	1	1	0	1	1	1	1				2
	BIO300 OP (n = 13)													Vehicle OP (n = 2)
Mononuclear cell infiltrate	1	1	1	1	1	1	1	1	1	1	1	1	1	2	2
Fibrosis, interstitial, alveolar wall	0	0	1	1	1	1	0	0	1	0	0	1	1	2	2
	BIO300 IS (n = 13)													Vehicle IS (n = 0)
Mononuclear cell infiltrate	1	2	1	2	1	1	1	1	1	1	1	1	1
Fibrosis, interstitial, alveolar wall	0	0	1	1	0	1	1	0	0	0	1	0	0
Lymphoid hyperplasia	5	0	0	0	0	0	0	0	0	0	0	0	0
Type II pneumocyte hyperplasia	0	1	0	0	0	0	0	0	0	0	0	0	0
Alveolar mixed cell infiltrate	0	1	0	0	0	0	0	0	0	0	0	0	0
Thymic lymphosarcoma													P*
	PEG-G-CSF (n = 9)													Vehicle PEG-G-CSF (n = 2)
Mononuclear cell infiltrate	1	1	1	1	1	1	1	1	1					2	1
Fibrosis, interstitial, alveolar wall	0	0	0	0	1	0	0	0	0					1	1
Lymphoid hyperplasia	0	0	0	0	0	0	0	0	5
Type II pneumocyte hyperplasia	0	0	0	0	1	1	0	0	0

Values in the table are histopathological scores from an individual lung harvested from CD2F1 male mice exposed to 9.2 Gy that underwent scheduled euthanasia on completion of the study (SD180 post-irradiation). Surviving mice at 180 days post lethal radiation totaled 50; these mice were terminated with lungs collected, fixed in preservative, trimmed, and sectioned for histopathology. Two slides were cut from each block; one slide was stained with hematoxylin and eosin (HE) and the other slide was stained with Masson’s trichrome stain that imparted a blue color to collagen structures. Lesions coded were semi-quantitatively scored as 1 (minimal), 2 (mild), 3 (moderate), 4 (marked), or 5 (severe) with the exception of the thymic lymphosarcoma noted in animal #13 of BIO300 IS group, which was coded as P* (present). Sections were evaluated by a board-certified veterinary pathologist. All mice had minimal or mild mononuclear inflammatory cell infiltrates either in the interstitial wall of alveoli or presenting as increased macrophages within alveolar spaces. Rarely, a few neutrophils were also present. Rather than code these morphologies separately, the single term ‘mononuclear cell infiltrate’ was used. This finding is expected in mice exposed to lethal radiation. A few mice also had evidence of type II pneumocyte hyperplasia, indicative of an ongoing repair of alveolar cell damage. One mouse had excessive numbers of neutrophils within alveolar spaces, and this was coded ‘alveolar mixed cell infiltrate’. The trichrome stained slides were examined for evidence of mature collage formation within alveolar walls; this was found in many mice but not all at this 180-day period. Finally, two mice had marked lymphoid hyperplasia in addition to the mouse with thymic lymphosarcoma. While such findings may be seen in older mice, it is possible that the radiation insult was related to these findings.

Masson’s trichrome staining of lung tissue slides was used to visualize collagen deposition within alveolar walls as an indicator of pulmonary fibrosis. BIO 300-treated animals had evidence of minimal fibrosis at day 180 post-TBI (n = 20/36), and n = 1/9 Neulasta-treated animals had evidence of minimal fibrosis. Out of the five vehicle-treated animals that survived until day 180 post-TBI, n = 3/5 had mild fibrosis, and n = 2/5 had minimal fibrosis. Similar to mononuclear infiltrate, vehicle-treated animals had slightly worse alveolar fibrosis compared to the BIO 300- or Neulasta-treated animals. However, the degree of fibrosis detected in all animals was marginal and tended to be focal to multifocal at best.

Additionally, a single mouse in both the BIO 300 IS- and Neulasta-treated groups had marked lymphoid hyperplasia, while a single mouse in the BIO 300 IS group presented with thymic lymphosarcoma. While such findings may be common in older mice, it is possible that these histopathological findings were related to DEARE.

Discussion

Though the development of nontoxic, safe, and effective radiation MCMs has been the focus of investigators during the last several decades, no radioprotector for ARS has been approved by the US FDA which can be used prior to radiation exposure to prevent either ARS or DEARE. This is a critical need for the protection of our armed forces and first responders who can potentially be exposed to radiological/nuclear threats and for those who are involved in responding to radiological/nuclear events (Gale and Armitage 2018; DiCarlo et al. 2021; Gale et al. 2021). To date, the US FDA has approved only four agents (Neupogen, Neulasta, Leukine and Nplate) as radiomitigators for H-ARS, which are indicated for use only after radiation exposure (Stone et al. 2004; Farese et al. 2013; Farese and MacVittie 2015; Hankey et al. 2015; Singh and Seed 2018; Wong et al. 2020a, 2020b; Zhong et al. 2020; Clayton et al. 2021; Gale and Armitage 2021; U.S. Food and Drug Administration 2021). Genistein, as well as other promising radioprotectors, are under investigation to fill this gap (DiCarlo et al. 2011; Singh and Seed 2017; Singh et al. 2017a, 2017b).

BIO 300 is a promising radioprotector demonstrating cell cycle effects, anti-inflammatory activity, as well as estrogen-like pharmacological action with a 2000-fold higher binding preference for ERβ over ERα (Landauer et al. 2019). Either alone or in combination with other drugs, genistein has shown beneficial effects in protecting various organs from harmful radiation exposure (Deviatkina et al. 1989; Calveley et al. 2010; Kim et al. 2012; Jackson et al. 2017; Hanedan Uslu et al. 2018; Jackson et al. 2019; Singh and Seed 2020). Since genistein has poor water solubility, it was developed into a nanosuspension and as a solid dosage formulation, known as BIO 300, using a wet-nanomilling or an HME process, respectively. These technologies have made it possible to administer therapeutic doses of genistein through multiple routes (po, sc, and im) (Ha et al. 2013; Jackson et al. 2017; Landauer et al. 2019; Singh and Seed 2020). One of the nanosuspension products, BIO 300 OS, has been evaluated for safety and efficacy in a phase 1b/2a clinical study in NSCLC patients (NCT02567799) and is currently being evaluated in a phase 2 study for the mitigation of long-term pulmonary complications due to coronavirus disease 2019 (COVID-19; NCT04482595). The new solid dosage product, BIO 300 OP, is being investigated for the prevention of H-ARS as well as DEARE (Singh and Seed 2020). Additionally, BIO 300 OP has been assessed for safety, PK and pharmacodynamics in a phase 1 study of healthy volunteers (NCT04650555). BIO 300 has also demonstrated clinical utility in mitigating DEARE-lung in mice. For this study, BIO 300 was administered orally daily for 6 weeks, starting 24 h post-whole-thorax lung irradiation (WTLI) (Jackson et al. 2017).

In the studies presented here, we sought to determine the optimal dose schedule for prophylactic BIO 300 OP radioprotection in mice. We demonstrate that BIO 300 OP can confer radioprotection in a mouse model when administered for at least 1 d BID prior to TBI, although 6 d BID prophylactic treatment provided optimal efficacy. Interestingly, 5 d of BID dosing did not significantly improve survival, while both 6 and 4 d of treatment conferred substantial radioprotection. Given the large range in survival within the different vehicle groups among the different treatment schedules, the minimal improvement in survival for the 5 d treatment group needs to be validated with repeat studies to gain confidence. Considering 6 d BID dosing provided the best radioprotection in the dose scheduling study and that these results reinforced previous findings that 6 d of BID oral dosing of BIO 300 confers significant radioprotection, this was the dose schedule chosen to proceed with in subsequent efficacy studies.

Finally, our results demonstrate that prophylactic dosing of BIO 300 IS, OP, and OS provide equivalent radioprotection to Neulasta administered after lethal radiation exposure. This is a critical finding, as the clinical adoption of BIO 300 will likely require demonstration of clinical benefit beyond current standard of care. Furthermore, since this study was carried out past the H-ARS phase, the durability of response was evaluated for the three BIO 300 formulations and Neulasta. Ultimately, it was found that BIO 300 OP and BIO 300 OS displayed the best durability in response. When considering survival through the H-ARS phase and the durability of response through the DEARE phase, BIO 300 OP performed the best out of the four drugs evaluated. These findings are corroborated by the histopathological analysis of lung tissue from 180 d survivors, which showed that BIO 300 OP-treated animals had minimal evidence of immune infiltrate or fibrosis, while vehicle-treated animals exhibited mild signs of lung injury. Similar results were observed in the BIO 300 IS-, OS-, and Neulasta-treated groups compared to their respective vehicle controls. Nevertheless, while the histopathological results trend appropriately, they need to be interpreted with caution, as CD2F1 mice are not a clinically relevant model of DEARE-lung. Future studies will be dedicated to testing BIO 300 OP in the C57L/J mouse model, which presents with similar DEARE-lung clinical manifestations as humans or nonhuman primates (Jackson et al. 2017).

While BIO 300 formulations improved 180-day survival, and histopathological results moderately reinforced these results, there are several limitations to the DEARE-lung studies. Namely, the CD2F1 strain, while an excellent model for ARS studies, is not ideal for DEARE-lung studies and may require higher radiation doses than those used in this study (9.2 Gy) to observe lung injury. One CD2F1 study reported modest signs of pulmonary inflammation 6 months post-TBI doses ≥11.5 Gy (Sharma et al. 2020). However, this study did not have vehicle-treated animals survive to 6-months post TBI, a similar problem observed in our studies, indicating that future work with the CD2F1 strain will likely require a significant increase in animal numbers in order to have an adequate number of survivors in control groups. Based on mouse model development work conducted at Duke University, the best mouse strains for DEARE-lung studies are C57L/J and CBA/J mice because they develop radiation-induced lung injury similarly to humans (Jackson et al. 2012). In order to definitively investigate the efficacy of BIO 300 on DEARE-lung, future work will need to be conducted in these mouse strains.

In the current phase 1 study of BIO 300 OP in healthy human volunteers (NCT04650555), the drug is being dosed as a free flowing powder in sachets. This formulation is ideal for early development efforts and is amendable for field-use by military personnel. However, BIO 300 OP can also serve as the basis for a potential final product (e.g. tablet, capsule, chewable, resuspendable powder) in order to further improve its utility and adoption in austere environments.

In summary, these studies provided the first attempt at identifying the optimal oral dose schedule for BIO 300 and are the first to compare the prophylactic efficacy of BIO 300 and Neulasta. Overall, BIO 300 OP is efficacious when dosed for 6 d po, BID prior to TBI. However, there is opportunity to shorten the therapeutic window, as these initial studies demonstrated efficacy with as little as a single BID dose prior to irradiation. Additionally, prophylactic administration of BIO 300 OP has equivalent, if not slightly improved, efficacy compared to Neulasta given post-exposure. The results of our present study support the advanced development of BIO 300 OP as an effective prophylactic radiation MCM for H-ARS.

Author contributions

Study design: V.K.S., M.D.K., A.A.S. Performance of the study: V.K.S., S.Y.W., O.O.F. S.N., A.C., A.A.S., M.D.K. Drafting of the manuscript: V.K.S., O.O.F., S.Y.W., A.A.S., M.D.K. Revision of manuscript content: V.K.S., A.A.S., M.D.K. All authors have read and approved the final submitted manuscript.

Disclosure statement

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, USA.

Dr. Artur Serebrenik and Dr. Michael D. Kaytor are employees of Humanetics Corporation, Edina, MN, USA. The other authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Data availability statement

All data generated or analyzed during this study are included in this article.

Additional information

Funding

This study was supported by funding from Congressionally Directed Medical Research Programs Grant # W81XWH-17-1-0584 awarded to MDK (subawared to VKS by MDK/Humanetics Corporation) and Joint Program Committee-7/Radiation Health Effects Research Program (RHERP) Grant # 1I80 VP00262-01 awarded to VKS and administered by The Henry M. Jackson Foundation for the Advancement of Military Medicine.

Notes on contributors

Vijay K. Singh

Vijay K. Singh, PhD, is a Professor at the School of Medicine/USUHS and a well-recognized radiation biologist involved in the development of promising radiation countermeasures following US FDA Animal Rule. The primary focus of his research is to investigate countermeasures for efficacy, and identify and validate biomarkers for radiation injury and countermeasure efficacy. His research is well-funded by various US government agencies of the Department of Defense and Department of Health and Human Services.

Oluseyi O. Fatanmi

Oluseyi O. Fatanmi, MLS, is a Research Biologist at the Scientific Research Department, AFRRI/USUHS and conducts studies with various radiation countermeasures. He primarily performs and oversees the relevant biochemical, hematologic, microbiological, and immunologic studies to advance radiation medical countermeasures. His focus is to study the effects of radiation injury using various animal models and methodologies to investigate countermeasure efficacy.

Stephen Y. Wise

Stephen Y. Wise, B.Sc., is a Research Associate at the Scientific Research Department, AFRRI/USUHS. His primary focus is to develop promising radiation countermeasures using various animal models to combat radiation injury. His knowledge is focused on the identification of biomarkers, improvement of methodologies, and validation of various biomarkers for the development of radiation countermeasures.

Alana Carpenter

Alana Carpenter, B.Sc., is a Research Assistant at the Scientific Research Department, AFRRI/USUHS. Her primary focus is to investigate countermeasures for efficacy and the identification and validation of various biomarkers for radiation injury and countermeasure efficacy. She is responsible for research data management, quality control and assurance, and interpretation of research data.

Sara Nakamura-Peek

Sara Nakamura-Peek, B.A., is a Research Assistant at the Scientific Research Department, AFRRI/USUHS. Her primary focus of her research is to develop radiation countermeasures. She works with various animal models to identify biomarkers, utilize data to determine efficacy, and interpret data for the study of the effects of radiation injury.

Artur A. Serebrenik

Artur A. Serebrenik, PhD, is a Senior Research and Development Scientist at Humanetics Corporation, Edina, MN, USA.

Michael D. Kaytor

Michael D. Kaytor, PhD, is the Vice President of Research and Development at Humanetics Corporation, Edina, MN, USA.