BIO 300: a promising radiation countermeasure under advanced development for acute radiation syndrome and the delayed effects of acute radiation exposure

ABSTRACT

Introduction

There are no radioprotectors currently approved by the United States Food and Drug Administration (US FDA) for either the hematopoietic acute radiation syndrome (H-ARS) or for the acute radiation gastrointestinal syndrome (GI-ARS). There are currently, however, three US FDA-approved medicinals that serve to mitigate acute irradiation-associated hematopoietic injury.

Area covered

We present the current status of a promising radiation countermeasure, BIO 300 (a genistein-based agent), that has been extensively investigated in murine models of H-ARS and models of the delayed effects of acute radiation exposure (DEARE) and is currently being evaluated in large animal models. It is also being developed for the prevention of radiation-induced toxicities associated with solid tumor radiotherapy and is the subject of two active Investigational New Drug (IND) applications. We have included a listing and brief review of significant investigations of this promising medical countermeasure.

Expert opinion

BIO 300 is a leading radioprotector under advanced development for H-ARS and DEARE, as well as for select oncologic indication(s). Efficacy following oral administration (po), lack of clinical side effects, storage at ambient temperature, and intended dual use makes BIO 300 an ideal candidate for military and civilian use as well as for storage in the Strategic National Stockpile.

KEYWORDS:

• Acute radiation syndrome
• BIO 300
• biomarkers
• delayed effects of acute radiation exposure
• estrogen receptor
• radiation countermeasures

1. Introduction

Radiation accidents, large and small alike, serve as warning signs of the potential health and environmental hazards associated with catastrophic nuclear/radiological events [1–6]. The risk of life-threatening exposures to high doses of radiation following terrorist-driven nuclear/radiological weapon attacks have become a rising concern in recent years [7,8]. These threats are amplified by the lack of available medical countermeasures for protecting the public against radiation exposure-related illness and death [9,10]. In addition, under such radiation-exposure scenarios, the capacity to assess precise levels of exposure to victims would not only be limited, but there would also be likely substantial delays in delivering medical care to those in need. Therefore, there is little question about the need for new types of lifesaving medical countermeasures that are safe, effective, and that have extended time windows of effectiveness relative to radiation exposure [11]. Although efforts to develop radiation countermeasures for acute radiation syndrome (ARS) were initiated more than six decades ago, only an extremely limited number of safe and effective medical countermeasures have been fully approved by the United States Food and Drug Administration (US FDA) for unwanted overexposures to radiation [12–14]. Moreover, these are drugs largely repurposed from their original oncology indications. This situation has prompted increased research efforts to identify a new generation of radiation countermeasures.

Radiation countermeasures are classified as radioprotectors, radiomitigators, and radiation therapeutics based on the time of administration in relation to radiation exposure. Radioprotectors are agents that are administered prior to radiation exposure to protect individuals who may get exposed to radiation at a later time [15]. To date, no radioprotector specifically designed for either hematopoietic ARS (H-ARS) or gastrointestinal ARS (GI-ARS) has been approved by the US FDA [12]. If such putative radioprotectors were to be fully developed and approved by the US FDA, their labeled use would be to protect soldiers, first responders, or civilians in anticipation of radiation exposure. Such agents would also be helpful for cancer patients to protect against collateral tissue/organ system damage when undergoing radiotherapy for various malignancies. By contrast, radiomitigators are agents used shortly after radiation exposure and before the appearance of the overt symptoms of radiation injury, and serve to minimize injury, largely by stimulating tissue recovery [15]. Lastly, radiotherapeutics are defined by the timing of the medicinal’s initial use; namely, once symptoms of radiation exposure fully manifest [15].

ARS develops in humans following total-body or partial-body radiation exposures at acute radiation doses generally estimated at 1 Gray (Gy) or higher and delivered at relatively high dose rates (~0.05 Gy/h or higher). Clinical manifestations of ARS include several well-documented sub-syndromes, namely H-ARS (1–6 Gy), GI-ARS (>6 Gy), and the neurovascular sub-syndrome (>10 Gy) [16]. Once elicited by extremely high exposure levels, the neurovascular sub-syndrome is extremely difficult to clinically manage and is essentially incurable, with rapid and inevitably fatal outcomes (~within 24–48 h), mainly due to systemic vascular accidents and multi-organ failure [17]. Victims exposed to radiation doses resulting in H-ARS and GI-ARS are, by contrast, expected to respond to medical countermeasures. Consequently, these two sub-syndromes of ARS have been the focus of the development of medical countermeasures. In addition, victims manifesting delayed and late effects of radiation exposure are likely to benefit from early, post-exposure interventions using select types of medical countermeasures [12].

All considering, there is a need for a judicious approach to not only develop, but to also ‘stockpile’ and possibly deploy essential medical countermeasures, particularly radioprotectors for ARS, for future radiological/nuclear contingencies involving both military personnel and civilians alike [18]. The development of such medical countermeasures is clearly vital for national security. It is important to note that radioprotectors would be used by healthy individuals in anticipation of radiation exposure at a later time point. This means that the use of such agents prophylactically would impose more of an inherent safety risk when compared to the radiomitigative agents administered after exposure. For such prophylactically administered agents, safety is of paramount importance and must be well-established. Therefore, radioprotectors need additional regulatory stringency for FDA approval.

The soybean (Glycine max) is a major source of plant-derived phytoestrogenic compounds. Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4 H-chromen-4-one) is a soy-derived isoflavone with a multitude of health-promoting effects. Two benzene rings form the basis of its flavone nucleus (Figure 1). Due to structural similarity, genistein competes with 17β-estradiol (Figure 2) in estrogen receptor binding assays [19].

Figure 1. Molecular structure of genistein, C₁₅H₁₀O₅, molecular weight: 270.24 Da.

Figure 2. Molecular structure of 17β-estradiol, molecular weight: 272.4 Da.

Genistein’s efficacy as a radioprotectant was discovered by researchers at the Armed Forces Radiobiology Research Institute (AFRRI), where it was studied in rodent models of ARS [20]. Genistein is a challenging molecule to manufacture into a drug as it is virtually insoluble in most excipients used for such purpose and has very poor oral bioavailability. To overcome these limitations, Humanetics Corporation (Edina, MN, USA) has developed a patented formulation that contains synthetic genistein nanoparticles (Table 1). This genistein nanosuspension is named BIO 300. The genistein in BIO 300 has been wet-nanomilled to a fine particle size, which provides significant enhancement to its oral bioavailability and efficacy as a radioprotector [21].

Table 1. List of genistein-related product details.

Drug Substances and Formulations	Manufacturer	Description
Genistein	DSM Nutritional Products	A naturally occurring soy isoflavone that is commonly delivered in a standardized isoflavone mixture derived by extraction.
Bonistein®	DSM Nutritional Products	Pharmaceutical grade synthetic genistein present in all BIO 300 drug product formulations and manufactured by DSM Nutritional Products
geniVida®	DSM Nutritional Products	Nutraceutical grade synthetic genistein manufactured by DSM Nutritional Products and available commercially.
BIO 300 capsules	Humanetics Corporation	Bonistein®capsule without excipients (particle size median diameter 8 microns)
BIO 300 injectable formulation	Pharmaceutics International, Inc.	Bonistein® nanoparticles in a sterile aqueous suspension (particle size d(50) ≤ 0.2 microns). Has been used in both parenteral and oral nonclinical studies. The route of administration for each study mentioned in this manuscript is explicitly stated.
BIO 300 oral formulation	Pharmaceutics International, Inc.	Bonistein® nanoparticles in an aqueous suspension with preservatives (particle size d(50) ≤ 0.2 microns).
BIO 300 oral formulation (powder)	Lonza Group AG	Bonistein® solid formulation

BIO 300 is being developed following the US FDA Animal Rule [22] (Box 1). The Animal Rule was issued by the US FDA in 2002 to expedite the development of new drugs and biologics as medical countermeasures against chemical, biological, radiological, and nuclear (CBRN) threats. This rule applies only to new countermeasures for which conclusive human efficacy investigations under phase 2 and phase 3 clinical trials cannot be performed due to ethical reasons [22]. According to this rule, the FDA can approve new drugs that have been shown to be safe in humans based on well-controlled animal efficacy studies. However, data from animal efficacy studies is not as convincing as human data, as it is uncommon that an animal model perfectly mirrors any human disease. For this reason, it is imperative to understand how and why a medical countermeasure works in order to ensure that such an agent will be effective in humans for the desired indication. Biomarkers for radiation injury and countermeasure efficacy are particularly important [23,24]. Such biomarkers are helpful for drug dose conversion from animals to humans [25].

Here, we review the attributes of BIO 300 and suggest that it is a strong candidate as a medical countermeasure for unwanted radiation exposure. We intend to summarize the drug’s pharmacology, safety, efficacy, biomarkers, and outcome of clinical trials.

2. Mechanism of action: pharmacology

The molecular structure of genistein bears resemblance to estrogen and hence it is known as a phytoestrogen [26]. Phytoestrogens have been shown to bind with three estrogen receptors: estrogen receptor-α (ERα), estrogen receptor-β (ERβ), and the G-protein coupled estrogen receptor-1 (GPER1). ERα and ERβ are very similar and act as ligand-dependent transcription factors, while GPER1 is a less characterized G-coupled protein receptor and mediates signal transduction in response to an estrogenic signal [27]. ERα and ERβ are antagonistic; ERα is responsible for cellular growth and differentiation, while ERβ partly functions as a repressor of cell growth [28]. ERβ is found throughout the body of males and females, even in many tissues that do not express ERα. Such distribution of ERβ suggests it has other roles in addition to negative regulation of ERα signaling [29]. ERα contributes to tumor development as an oncogene-driven moiety, while ERβ activation inhibits cell growth and proliferation via upregulation of its target genes in normal tissue and cancer cells [30–33]. Anti-cellular growth characteristics of ERβ label it as a tumor suppressor. Furthermore, it is commonly mutated in advanced human cancers [27]. ERβ is known to activate DNA damage response pathways, reduce the cell cycle, and suppress inflammatory pathways. GPER1 is ubiquitously expressed throughout the body of males and females including the hematopoietic and pulmonary systems [34]. Its biological role in estrogenic reproductive health is more similar to ERβ than ERα as GPER1 knockout mice have normal reproduction and fertility phenotypes [35]. In addition, GPER1 acts as a tumor suppressor, with high levels of expression commonly linked to better treatment outcomes in multiple tumor types [36,37]. Similar to ERβ, radiation induces GPER1 expression, and is able to inhibit nuclear factor-κB (NF-κB) expression and NF-κB target genes [37–41]. This suggests that genistein may be exerting some of its function through GPER1, though to a lesser extent than ERβ.

Genistein has estrogenic activity and binds to all three estrogen receptors, but it has selective affinity to ERβ [42]. A structural comparison of ERα- and ERβ-bound genistein reveals that key amino acids in similar locations of the ligand binding domain differentially interact with genistein and contribute to its selectivity for ERβ [19]. Studies have demonstrated that genistein binds to ERβ with a higher affinity than it does to either ERα or GPER1: the EC₅₀ (concentration giving half of the maximal response) of genistein for ERα, GPER1, and ERβ is 145 nM, 133 nM, and 8.4 nM, respectively [43,44]. Studies conducted with the BIO 300 injectable formulation in Chinese hamster ovary (CHO) cells expressing the human ERα or human ERβ full-length isoforms demonstrated that nanoparticulate genistein activates ERβ at EC₅₀ of 0.9 nM and ERα at EC₅₀ of 2,000 nM [45]. This indicates that BIO 300 exhibits a 2,000-fold higher selectivity for ERβ than ERα. Furthermore, the estrogenic specificity of genistein is affirmed by its relative binding compared to daidzein, another isoflavone which differs from genistein by the lack of a hydroxyl group at the C5 position of genistein, but has no estrogenic activity [46]. Only one isoform of ERβ has a ligand binding domain, namely ERβ1. Thus, this is the therapeutic target of agonists such as genistein.

Genistein controls hematopoietic progenitor cell depletion after irradiation by increasing expression of cell cycle checkpoints and DNA repair enzymes. There are several reports that genistein upregulates the cell cycle checkpoints p21, p27, and GADD45a [47–51]. NF-κB is induced by irradiation and is shown to promote proliferation through the activation of c-myc, which is a negative regulator of p21, p27, and GADD45a [52,53]. Genistein has also been shown to inhibit expression of NF-κB and c-myc [54–56]. Genistein may be promoting cell cycle arrest by activating ERβ and thereafter inactivating NF-κB or directly inducing expression of cell cycle checkpoints.

The path for drug development and approval by the FDA is full of hurdles. Only a small percentage of drugs that enter clinical trials are eventually approved by the FDA. Having a clear understanding of the mechanism of action of a drug is very helpful before it enters clinical trials. Mechanistic studies take time and delay the drug development process; nevertheless, mechanistic knowledge is valuable in the long run because it increases the chances of drug approval, it saves time and money, and may also perhaps contribute to saving of lives. The well understood mechanism of action of a drug facilitates better dosing through monitoring of the drug’s effects on the target pathway in the patient. The BIO 300 mechanism of action is being elucidated using various models.

3. Nonclinical studies for efficacy

BIO 300 is being developed as a radioprotector to be administered prior to radiation exposure in order to prevent H-ARS [57–59]. Currently, additional formulations of BIO 300 intended for intramuscular (im) and oral (po) administrations are being developed for use as radioprotectors for H-ARS as well as radiomitigators for DEARE (Table 1 details all products and formulations tested). A large number of studies using animal models have been completed in order to establish the efficacy of these formulations [60]. These studies were performed with different doses of BIO 300 administered parenterally or orally prior to irradiation to evaluate the drug’s efficacy against H-ARS. For its use in countering DEARE, this agent was used after radiation exposure as a radiomitigator. While the parenteral formulation and its corresponding oral formulations are effective, these formulations are suboptimal for field-use and stockpiling. Recently, Humanetics has developed a new formulation for oral dosing and called it BIO 300 oral powder, which is easy to use, stable at ambient temperature, and amenable for field-use in severe weather conditions. It is important to note that all formulations of BIO 300 have the same active pharmaceutical ingredient (API), genistein.

3.1. Radioprotective efficacy of BIO 300 for H-ARS

Various formulations of BIO 300 and their API, genistein, have been studied for efficacy in murine models for ARS. There is also a study reporting its efficacy in a canine model. A brief description of important studies is provided below.

3.1.1. Efficacy studies in murine models

Early studies carried out using murine models established genistein’s radioprotective efficacy for H-ARS [61]. A single subcutaneous (sc) administration of genistein 24 h prior to irradiation provided significant radioprotection to the hematopoietic progenitor cell compartment [62]. When administered sc 24–12 h before total-body irradiation (TBI) (9.25 Gy ⁶⁰Co γ-radiation), a single dose of BIO 300 significantly improved 30 d survival in mice [63]. The BIO 300 nanosuspension also attenuated the radiation-induced enhancement of proinflammatory cytokines; interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 (COX-2) in mouse bone marrow and spleen. This may contribute to hematopoietic stem and progenitor cell protection. A single im injection of BIO 300 was also effective when administered 48 h to 12 h prior to 9.25 Gy TBI. The optimal efficacy of BIO 300 was observed when administered 24 h prior to TBI. In a murine model of H-ARS, genistein protected bone marrow progenitor cells from radiation-induced injury, leading to the prevention of hematopoietic stem cell pool exhaustion, and thereby augmenting subsequent recovery of blood neutrophil and platelet levels [61,62,64].

There are several studies demonstrating radioprotective efficacy of the BIO 300 im and po formulations, although some earlier studies have used only the API genistein [45,61–63,65,66] (Table 2). The dose reduction factor (DRF) of genistein/BIO 300 at a dose of 150 mg/kg administered im 24 h prior to irradiation is 1.16 [45]. Unlike im, po administration required twice a day dosing to demonstrate significant levels of protection.

Table 2. Summarized nonclinical ARS efficacy studies.

Species	Formulation	Route	Study	Doses	Duration	Comments	Reference
Mouse	Genistein in PEG 400	sc	Efficacy	400, 200, 100, 50, 25, 12.5, 6.25, 3.12 mg/kg	Single dose 1 h and 24 h before 9.5 Gy TBI	Doses of at least 25 mg/kg showed significant improvements in survival when administered 24 h before TBI	[61]
Mouse	Bonistein® in PEG 400	sc	Efficacy	200 mg/kg	Single dose 24, 8, 4, 2, 1 and 0.5 h before 8.75 Gy TBI	24 h prior to TBI treatment group had improvement in 30-day survival relative to vehicle	Landauer MR et al. - Unpublished
Mouse	Genistein in PEG 400	sc	Efficacy, hematol.	200 mg/kg	Single dose 24 h prior to 8.75 Gy TBI	Significant improvement in CBC and accelerated hematologic recovery in irradiated mice	[62]
Mouse	Bonistein® in PEG 300/castor oil, PEG 300/cottonseed oil or PEG 300/sesame oil	sc	Efficacy with different excipients	200 mg/kg	24 h prior to 8.75 Gy	All Bonistein® groups showed significant increases in survival	Landauer MR et al. - Unpublished
Mouse	Bonistein® in PEG 400 or BIO 300 injectable suspension (IS)	sc	Efficacy	200 mg/kg (Bonistein®) 150 mg/kg (IS)	Single dose 24 h prior to 8.75 Gy, 9.0 Gy or 9.25 Gy TBI	Both formulations had similar efficacy	Landauer MR et al. - Unpublished
Mouse	BIO 300 IS	sc	Efficacy	200 mg/kg	Single dose 24 h prior to 9.0 Gy TBI	Significant improvement in survival	Landauer MR et al. - Unpublished
Mouse	Genistein in PEG 400	sc	Efficacy	200 mg/kg	Single dose 24 h before 7.75 Gy TBI	Significant improvement in survival	[66]
Mouse	Genistein in PEG 400	po	Efficacy	100 and 400 mg/kg	QD 4 d prior to 9.5 Gy TBI	Improved survival only at 400 mg/kg	[59]
Mouse	Bonistein® in PEG 400	po	Efficacy	400 mg/kg	Once daily for 4, 7, 14, and 21 days prior to 8.75 Gy TBI	Daily dosing for 7, 14 or 21 d improved survival	Landauer MR et al. - Unpublished
Mouse	Bonistein® in PEG 400	po	Efficacy	200 mg/kg twice daily (BID) or 400 mg/kg QD	2, 4 or 6 days prior to 8.75 Gy TBI	Optimal treatment regime was 200 mg/kg BID for 6 days prior to TBI	[58]
Mouse	Bonistein® in PEG 400	po	Efficacy and DRF	200 mg/kg BID	BID for 6 days prior to 8.75–10.25 Gy TBI	Estimated DRF 1.11 with 95% confidence intervals of [1.09–1.14]	Landauer MR et al. - Unpublished
Mouse	Bonistein® in PEG 400	po	Efficacy	133.3 mg/kg thrice daily (TID) or 200 mg/kg BID	TID for 1,2,4,6 days prior to 9.0 Gy TBI and 6 d for BID prior to TBI	No improvement with TID over BID dosing	Landauer MR et al. - Unpublished
Mouse	Bonistein® in PEG 400	po	Efficacy	400 mg/kg	QD 1,2,4,6 d prior to 8.75 Gy TBI	Survival similar to sc dosing	Landauer MR et al. - Unpublished
Mouse	Bonistein® in PEG 400 or BIO 300 Oral Suspension (OS)	po	Efficacy	200 mg/kg BID	6 days prior to 9.25 Gy TBI	Both formulations had similar survival	Landauer MR et al. - Unpublished
Mouse	BIO 300 OS	po	Efficacy	200 mg/kg	BID 6 days prior to 8.75 Gy to TBI	6 d prior to irradiation, po efficacy equivalent to im dosing	Landauer MR et al. - Unpublished
Mouse	BIO 300 Oral Powder	po	Efficacy	200 mg/kg	BID 6 and 3 days prior to 9.2 Gy TBI	Significant improvement in survival when administered for 6 days prior or 3 days prior	Unpublished – manuscript under preparation
Mouse	Bonistein® in PEG 400 or BIO 300 IS	im	Efficacy	200 mg/kg Bonistein® 150 mg/kg IS	Single dose 24 h prior to 9.25 Gy TBI	Similar survival results with both formulations	Landauer MR et al. - Unpublished
Mouse	BIO 300 IS	im	Efficacy	150 mg/kg	Single dose 24 h prior to 9.5, 9.75, 10 and 10.25 Gy TBI	LD50 is between 10 and 10.25 Gy	[45]
Mouse	BIO 300 IS	im	DRF	150 mg/kg	Single dose 24 h prior to 9.5, 9.75, 10, 10.25, 10.5, 10.75, 11 Gy TBI	DRF of 1.16	[45]
Mouse	BIO 300 IS	im	Efficacy	200 mg/kg	QD 24 h prior to 9.2 Gy TBI	Significant improvement in survival	Unpublished – manuscript under preparation
Mouse	BIO 300 IS	im	Efficacy	150 mg/kg	QD 1, 2, 3, 4 or 5 days prior to 9.25 Gy TBI	Single dose provides radioprotection for up to 48 h prior to TBI	[45]
Mouse	BIO 300 IS	im	Efficacy	150 mg/kg	QD 24 h prior to 9.0 Gy TBI	BIO 300 IS efficacy is dependent on ER-binding	[45]
Mouse	BIO 300 IS	im	Efficacy	150, 75, 37.5, 18.75, 9.375 mg/kg	Single dose 24 h prior to 9.25 Gy TBI	Significant improvement in survival down to 37.5 mg/kg	[63]
Canine	Bonistein® in PEG 400	sc	Efficacy	370 mg/kg	Single dose prior to 2.6 Gy TBI	Significant improvement in survival, amelioration of leukopenia, neutropenia, and thrombocytopenia	[68]

Summary of published and unpublished efficacy studies with genistein, Bonistein® and various formulations of BIO 300 using murine and canine models. DRF, dose reduction factor; im; intramuscular; sc, subcutaneous; po, oral; TBI, total-body irradiation.

Recent mouse studies with the BIO 300 injectable formulation confirmed earlier studies of radioprotection. Furthermore, these studies confirmed that genistein’s efficacy is mediated through the estrogen receptor. Pre-treatment of mice with ERα/ERβ antagonist, ICI 182,780, significantly abrogated the radioprotective efficacy of BIO 300 [45]. The residual protection in the BIO 300-treated group could be due to the antioxidant properties of genistein or genistein-mediated activation of GPER1. Similar to the canine study described below, a single im treatment of BIO 300 to mice 24 h prior to irradiation led to an increase in bone marrow cellularity at 1, 7, and 14 days post-irradiation [45].

Oral administration of radiation countermeasures is preferred, particularly by the US Department of Defense for their military personnel. As a consequence, a solid-dose oral formulation of BIO 300 was developed (BIO 300 oral powder). This new formulation was tested for its survival enhancing effects (30 d survival) in acutely irradiated mice and was compared to the BIO 300 injectable formulation. Twice daily po administration of BIO 300 oral powder formulation was initiated 6 days prior to TBI and was shown to have significantly improved 30 d survival in mice (unpublished observation). The administration of BIO 300 oral power resulted in 63% survival compared to 6% in vehicle-treated animals, whereas the BIO 300 injectable formulation (used as a positive control) resulted in 75% survival compared to 13% in its vehicle group. This study was repeated for confirmation and similar survival results were observed. Based on the above study, it was concluded that 6-day consecutive prophylactic treatment is still the optimal treatment window for orally administered formulations of BIO 300.

In summary, the BIO 300 oral powder formulation had comparable efficacy to the extensively investigated BIO 300 injectable formulation. Based on the efficacy data discussed above and favorable pharmacokinetic (PK) data (not presented), the BIO 300 injectable formulation as well as the oral powder formulation are moving forward to testing in large animal models using nonhuman primates (NHPs) for investigation of its efficacy as a radioprotector for H-ARS with the objective to gain FDA approval following the Animal Rule.

Captopril is an angiotensin converting enzyme (ACE) inhibitor that has also shown efficacy as a radioprotectant in murine models of H-ARS [67]. Co-administration of captopril in drinking water, from 1 h through 30 days post-irradiation, to mice receiving a single dose of genistein im increased survival in total-body irradiated mice compared to animals receiving either captopril or genistein alone [65]. The combination of genistein and captopril treatment enhanced bone marrow progenitor recovery as assessed by colony forming units for erythroid and myeloid lineages. Genistein alone and genistein plus captopril protected hematopoietic progenitors from irradiation-induced micronuclei formation, while captopril alone had no effect. Captopril alone and genistein plus captopril, but not genistein alone, suppressed radiation-induced erythropoietin production. This study suggests that the radioprotective mechanisms of captopril and genistein on hematopoietic tissues are most likely distinct.

3.1.2. Efficacy study in a canine model

Genistein’s efficacy as a radioprotectant has also been demonstrated in a canine model. This study used pharmaceutical grade Bonistein®, the BIO 300 drug substance, dissolved in polyethylene glycol-400 (PEG 400) and administered sc 24 h prior to TBI. Bonistein® treatment improved 30-day survival and mitigated radiation-induced grade 3 leukopenia and thrombocytopenia in canines. Furthermore, Bonistein® completely prevented grade 3 neutropenia [68].

3.2. Radiomitigative efficacy – DEARE

BIO 300 has undergone further evaluation as a radiomitigator for DEARE [21]. Studies were performed to assess the ability of BIO 300 to mitigate radiation-induced pneumonitis/fibrosis in a murine model of whole-thorax lung irradiation (WTLI) [69–72]. Mice were orally treated with BIO 300 (400 mg/kg), initiated 24 h following a single dose of WTLI and continued once daily for 4–6 weeks, and had significantly improved 180 d survival compared to those that were untreated [72]. In addition to improved survival, BIO 300 also mitigated radiation-induced lung injury and improved overall lung function [72,73]. Moreover, other studies have shown that treatments with genistein increased murine survival following thoracic irradiation [73]. Genistein, administered after irradiation, also reduced micronuclei in Lin⁻ marrow cells and primary lung fibroblasts, suggesting a direct reduction of radiation-induced DNA damage [65,66,73,74].

3.3. Prevention of toxicities associated with solid tumor radiotherapy

Radiation induced erectile dysfunction (RiED) is one of the most common sequelae associated with radiotherapy of prostate cancer. The Sprague-Dawley rat model for RiED appears quite useful and appropriate to model clinically analogous to radiotherapy elicited (comparable to human males) decreases in apomorphine-induced erectile responses. BIO 300 was tested in this model to determine whether it improves the therapeutic index in prostate cancer treatment by preventing radiotherapy-induced erectile dysfunction without affecting tumor radiosensitivity [75]. BIO 300 was given daily by po administration starting 3 days prior to radiotherapy or 2 h after radiotherapy and continued for the duration of the study. BIO 300 given before radiotherapy was able to improve the erection frequency of male rats at nine and fourteen weeks after irradiation compared to animals who were treated by radiotherapy alone or radiotherapy with non-nanomilled genistein (geniVida®). The erection frequency in recipients of BIO 300 following irradiation (starting at 2 h post exposure) was comparable to controls suggesting, therefore, that administration post-exposure does not mitigate RiED. Furthermore, BIO 300 administration in prostate cancer xenograft models did not affect radiation-induced tumor growth delay in hormone sensitive or insensitive prostate tumor bearing mice; thus, confirming the suggestion that BIO 300 does not confer radioprotection to prostate tumor cells in this model [75]. Further and most notably, the drug itself had anti-tumor activity and when combined with radiotherapy, BIO 300 appeared to sensitize the tumor to radiation-induced cell killing.

Additional studies using human A549 adenocarcinoma non-small cell lung cancer (NSCLC) xenografts implanted in athymic CD1 nu/nu mice have demonstrated that BIO 300 can protect normal lung tissue from injury following irradiation of the implanted tumorous xenografts. In this model, tumors were placed subcutaneously so that a single dose of radiotherapy would irradiate both the tumor and the normal lung. BIO 300 did not protect the tumor from radiation injury but sensitized the tumor to radiotherapy. Histopathology revealed that BIO 300 treated animals had less normal lung tissue damage compared to those treated with radiotherapy alone. Animals receiving BIO 300 also gained weight which countered the deleterious effect of radiation exposure [76].

4. Biomarkers

As mentioned above, to expedite the development of medical countermeasures for life-threatening conditions where human clinical trials for efficacy are neither ethical nor feasible, the US FDA has developed the Animal Rule [22]. The Animal Rule applies to the development and approval of medical countermeasures, drugs and biologics, to mitigate or prevent life-threatening conditions triggered by exposure to lethal or permanently disabling agents. To develop radiation countermeasures following the Animal Rule, important landmarks need to be established that include the identification of essential biomarkers for countermeasure efficacy and radiation injury. Such a biomarker should be measurable and indicate a specific pathological, biological, or therapeutic process of concern. Such biomarkers can include peripheral blood cell counts, patterns of cell receptor expression, proteins, cytokines, chemokines, growth factors, chromosomal aberrations, genetic sequences, messenger RNA (mRNA), microRNA (miRNA), long non-coding RNAs (lncRNAs), citrulline, lipidomes, metabolites, microbiota, tooth enamel- and fingernail-based biomarkers, radiographic/imaging-based measurements, and electrocardiographic parameters [23]. For the Animal Rule, biomarkers can be used to identify the human dose of the countermeasure that should closely correlate with efficacious doses from well controlled animal studies. Biomarkers should be linked to the mechanism by which the drug prevents or decreases the radiation-induced injury, and they should correlate with the desired clinical outcome such as reduced mortality or morbidity. There are ongoing and completed studies using different animal models to identify biomarkers of efficacy and dose conversion for BIO 300. A few of these approaches are discussed below.

4.1. Cytokines/chemokines/growth factors

Several cytokines, chemokines, and growth factors have been identified as candidate biomarkers of radiation injury and countermeasure efficacy over the last few years using total-body and partial-body radiation exposure in murine and NHP models [23,77–81]. These biomarkers have been investigated for BIO 300 efficacy and their utility in dose conversion. Administration of sc formulated genistein has been shown to induce various cytokines using quantitative PCR for the quantification of mRNA and cytokine assays for expressed protein in serum samples and tissues of irradiated and unirradiated mice [82]. Genistein stimulated granulocyte colony-stimulating factor (G-CSF) production in irradiated as well as unirradiated mice [82]. In a recent NHP study, po as well as im formulations of BIO 300 stimulated production of cytokines including interleukin-1 receptor antagonist (IL-1ra) (unpublished observation). IL-1ra is a competitive antagonist of IL-1 receptor and prevents IL-1 binding and signaling that lead to the inhibition of IL-1 mediated inflammatory response. IL-1ra expression is important for mitigating the detrimental effects of IL-1 as observed in cancer patients undergoing radiotherapy [83]. In the above NHP study, IL-8 levels appeared to be suppressed in serum during the 1–8 h time window after BIO 300 formulation was administered either po or im (unpublished observation). IL-8 is known to promote vascular endothelial permeability and attract myeloid cells that promotes reactive oxygen species production to induce apoptosis of damaged cells [84]. These potential cytokine biomarkers warrant continued investigation in future studies.

4.2. Metabolites and lipidomes

Metabolomics is the discipline of qualitative and quantitative assessments of small molecules within biological samples; this relatively new technology has emerged as a promising analytical tool for the rapid analysis of the individual’s level of radiation exposure, as well as determining the irradiated individual’s metabolic phenotype [24]. Biomarkers in easily accessible biofluids and biospecimens (e.g. urine, blood, saliva, sebum, and feces) have offered a potential (the basis for determining a radiation exposure signature) key determinant in assessing the necessity for medical intervention. Without question, such biomarkers have been and will continue to be helpful for countermeasure development. For example, metabolomic studies have been undertaken in an attempt to identify potential biomarkers that link radiation-induced lung injury and the efficacy of BIO 300 for mitigating tissue damage. A targeted metabolomic study of lung tissue from mice exposed to 12.5 Gy WTLI has been reported [72]. In this study, mice were treated daily with 400 mg/kg BIO 300 for 2 weeks or 6 weeks starting 24 h post-irradiation [85]. A panel of lung metabolites (such as amino acids, sphingomyelins, glycerophosphatidylcholine, and their combinations) was identified to be responsive to irradiation and, in turn, served to distinguish an efficacious treatment schedule of BIO 300 from a non-efficacious treatment schedule for the 180 d survival end point in male C57L/J mice.

In an another example of the technology’s utility, serum samples from NHPs treated with single doses of BIO 300 were analyzed for global metabolomic/lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS). Comparisons were made to determine how im and po administered BIO 300 changed metabolomic profiles [86]. Transient alterations in phenylalanine, tyrosine, glycerophosphocholine, and glycerophosphoserine were observed which reverted back to near pretreatment levels seven days following BIO 300 administration. There was significant overlap in the changes of the metabolite profiles induced by each route of administration. The po route of administration elicited fewer metabolic changes compared to the im route; this is perhaps due to differences in the drug’s absorption and distribution following po versus im routes of administration. The results of this study clearly revealed that the administration of BIO 300 caused significant metabolic swings and that these metabolic perturbations might have provided an overall benefit in limiting the extent and duration of radiation injury. This initial assessment in NHPs also highlights the use of metabolite and lipidome analyses to determine the underlying physiological mechanisms involved in the radioprotective efficacy of BIO 300.

4.3. Gamma-H2AX

Genistein treatment is known to activate p53, ATM, CHK2, and BRCA1 which are involved in DNA break repair [49,50,87–89]. BIO 300 injectable formulation treatment of human lung epithelial cells (BEAS-2B) prior to irradiation resulted in a significant reduction in γH2AX foci formation which is indicative of fewer DNA breaks (Dr. Michael D. Kaytor, Humanetics Corporation – personal communication). A combination of cell cycle arrest and elevated DNA repair capacity prevents damaging lesions from persisting, which likely promotes cell survival.

5. Pharmacokinetics (PK), safety and toxicology

Usually, free (unconjugated/non-glucuronidated/aglycone) genistein is linked with therapeutic efficacy and is used as the primary measure for PK analysis [90]. Genistein is primarily glucuronidated upon administration. The drug can also be bound by plasma proteins.

5.1. Preclinical studies

The API in BIO 300 oral formulation, BIO 300 injectable formulation, and BIO 300 oral powder is synthetically prepared genistein (Bonistein®) [91–94]. Its manufacturer has completed several safety and toxicology studies (including acute, sub chronic, and chronic safety studies) in mice, rats, and canines which supports the safety of the BIO 300 formulations [91–94]. Based on these studies, it has been concluded that genistein has no observable teratogenic effects in vivo, even at extremely high doses up to 1,000 mg/kg/day by the po route of administration in rats, using embryo-fetal toxicity endpoints, or up to 500 mg/kg/day by dietary admix in a prenatal developmental study in rats. However and by contrast, in vitro, genistein exhibited teratogenic potential at high concentrations using the whole embryo culture assay.

BIO 300 has a robust multi-species safety and toxicology database that has enabled this agent to proceed to human clinical trials. Safety and PK data have been collected with various routes of administration including po, im, and intravenous (iv) across multiple species such as mice, canines, NHPs, and humans. Absorption, distribution, metabolism, and excretion studies have been accomplished using BIO 300 formulations in animal models. In mice, BIO 300 was tested at a dose of 300 mg/kg, while 400 mg/kg of the native, active component, genistein, was tested as well. Several doses (30, 150 and 500 mg/kg) have been used in canine studies. In a recently published PK study, the doses used in NHPs was 100 mg/kg for po administration and 50 mg/kg for im administration [86]. Mice and canine studies using BIO 300 formulations have demonstrated C_max and area under the curve (AUC) profiles sufficient to support therapeutic efficacy.

In an initial PK study in NHPs, a dose of 100 mg/kg BIO 300 oral formulation [86] resulted in lower total drug exposure (lower C_max and AUC) compared to an allometrically scaled mouse BIO 300 dose. In the NHP study, PK parameters for BIO 300 were determined by serum levels of total genistein and genistein aglycone (active form) following administration through im and po routes. The serum level of genistein aglycone was dependent on the route of administration of BIO 300; higher levels of serum genistein aglycone were observed in animals receiving BIO 300 (50 mg/kg) im as compared to those animals receiving the drug by the po route (100 mg/kg) [86]. A time window of 2–8 h post-BIO 300 administration had highest serum concentrations of the drug. In the im administered group, T_max, C_max, AUC_0–48, and AUC_0–∞ for total genistein were higher than genistein aglycone. Similarly, in the po administered group, C_max, AUC_0–48, and AUC_0–∞ for total genistein were also higher than genistein aglycone. Both routes of administration are able to deliver the putative therapeutic dose of the active drug to the blood stream (i.e. serum); however, these results could explain why a single dose of BIO 300 delivered by im is sufficient for radioprotection, while multiple doses of po administration are required [45].

5.2. Clinical studies

There are several reports of safety and PK studies with different doses of Bonistein® in human volunteers where glucuronidated and non-glucuronidated forms have been measured in blood. In these studies, Bonistein® has been found to be safe and well tolerated with dose dependent linear PK [95–97]. No clinically significant changes were observed in physical parameters, blood chemistry, vital signs, or electrocardiograms (EKG/ECG) in drug-treated individuals. In brief, Bonistein® has been found to be safe, well tolerated, and demonstrated no unexpected/serious adverse effects when administered orally as a single dose as high as 300 mg or a dose of 120 mg/day for 14 days. Additional studies have also been conducted with geniVida® (a pure and nutritional grade genistein aglycone manufactured by DSM) and was reported to be safe [98]. There are additional studies for safety and PK using other forms of genistein in humans and these products were found to be safe [99–101].

Humanetics Corporation has conducted two clinical trials using different formulations of BIO 300. The first (NCT00504335) was a phase 1 single dose escalation safety and PK study with BIO 300 capsules (non-nanomilled Bonistein®) without any excipients. This study reported no serious adverse effects with a single dose as high as 2,000 mg (Dr. Michael D. Kaytor, Humanetics Corporation – personal communication). The second study (NCT02567799) is ongoing. This is a phase 1b/2a study in NSCLC patients receiving chemoradiotherapy. This is a study of safety and PK using three ascending doses: 500 mg/day, 1,000 mg/day, and 1,500 mg/day of BIO 300 oral formulation administered daily for 6–8 weeks during concurrent radiotherapy [21].

5.2.1. Phase 1 study in healthy human volunteers

This study was conducted to evaluate the safety, tolerability, and PK of BIO 300 capsules (non-nanomilled Bonistein®). In this study, the PK of genistein and its metabolites, based on the total serum concentration, in both genders appeared non-linear (less than proportional increase in both C_max and AUC with increasing dose) over a wide range of single drug doses (500 to 2,000 mg). The non-linearity was observed with increasing dose (Dr. Michael D. Kaytor, Humanetics Corporation – personal communication).

5.2.2. Phase 1b/2a study in NSCLC patients

It is important to note that genistein has been shown to decrease adverse effects of radiotherapy and chemotherapy in clinical trials with cancer patients [102,103]. Nonclinical studies have demonstrated that BIO 300 protects normal tissue from adverse effects of irradiation while acting additively with radiation exposure to kill tumor cells while increasing tumor growth delay compared to the control [21].

There is an on-going study with BIO 300 (NCT02567799) evaluating the oral formulation in cancer patients undergoing chemotherapy (carboplatin and paclitaxel) and radiotherapy (radiation exposure of 1.8–2 Gy fractions for a total of 60 − 70 Gy) for NSCLC. In this ascending repeat dose study, patients received BIO 300 oral formulation daily (500 mg, 1,000 mg, or 1,500 mg dose) for up to eight weeks. This study is being conducted at several centers: Henry Ford Health System – Detroit, MI, Medical College of Wisconsin – Milwaukee, WI, and University of Maryland School of Medicine – Baltimore, MD [21,104].

Pharmacokinetics, clinical, and laboratory parameters are being investigated to assess PK, PD, and the safety profile of BIO 300. After the first dose of BIO 300 prior to chemotherapy, the PK profile of BIO 300 was determined to establish drug exposure. There was no evidence of BIO 300, paclitaxel, or carboplatin drug accumulation or any evidence of drug-drug interactions.

Other endpoints in this study include measurement of normal tissue protection, tumor response, and quality of life. All patient dosing for this study has been completed and no dose limiting toxicities have been reported. The most commonly reported adverse events that have a possible association with BIO 300 were mild. Patients are being followed for thirteen months following the conclusion of chemoradiotherapy to examine delayed effects of radiotherapy [21].

6. Conclusion

The radioprotective efficacy of genistein was discovered by scientists at AFRRI with an objective to develop medical radiation countermeasures for military use. The commercial rights to develop genistein as a radiation countermeasure were exclusively licensed to Humanetics Corporation in 2005. Humanetics is pursuing the API, genistein, and several suitable pharmaceutical formulations (collectively called BIO 300) for use as a pre-radiation exposure medical countermeasure and as a post-radiation mitigator of exposure-related injury. Furthermore, Humanetics has a program to develop this agent for use in patients with solid tumors undergoing radiotherapy to protect their normal tissues. In brief, these intended medical products are being developed to increase survival in patients acutely exposed to myelosuppressive doses of radiation (H-ARS) to increase survival in individuals acutely exposed to pulmonary-toxic doses of radiation (DEARE-lung), and to prevent radiation toxicities associated with solid tumor radiotherapy (NCT02567799).

Various formulations of BIO 300 administered im or po have been extensively investigated in murine models and have demonstrated consistent and significant radioprotective efficacy for H-ARS and DEARE. Based on its encouraging efficacy in murine models as well as in canines, this agent is moving forward for further evaluation in the NHP model. In parallel, BIO 300 is being evaluated in clinical studies for PK, safety, toxicity, and efficacy in patients receiving chemoradiotherapy for NSCLC. To date, the results of clinical studies are encouraging, with no serious side effects being observed [21].

7. Expert opinion

As stated earlier, no radioprotector for H-ARS has been approved by the FDA, although three injury-mitigative countermeasures have received regulatory approval for H-ARS; these three radiomitigators are all growth factors, namely Neupogen®, Neulasta®, and Leukine®.

As a testament to the clinical effectiveness of these radiomitigative recombinants to help restore lost hematopoietic function and to limit a fatal outcome following acute radiation exposure, the prospects of long-term survival of such treated patients is clearly improved, but as such, long-term survival carries additional health risks and accordingly, the need of additional monitoring. Specifically, the manifestation of late-arising diseases (e.g. pulmonary DEARE) becomes a reality that needs to be dealt with clinically. Much like the situation mentioned above, there are currently no FDA approved radioprotective or radiomitigative medicinals for DEARE, pulmonary DEARE or otherwise. In this regard, we suggest that BIO 300, once fully approved by the FDA, might well prove to be clinically useful in either protecting against or mitigating the development of DEARE.

Radioprotectors are expected to be administered to healthy individuals in anticipation of radiation exposure at a later time point. Radiomitigators, which are administered after radiation exposure, likewise, may be used by individuals that are otherwise healthy and may not have been exposed. Due to this reason, FDA approval of radioprotectors and radiomitigators must demonstrate, without question or concern, exceedingly well-established safety profiles in humans. This contrasts to the therapeutics for radiation injuries that are administered after radiation exposure symptoms manifest and that must solely demonstrate a favorable therapeutic benefit relative to the potential health risk. Therefore, the bottom line is that FDA approval of any given radioprotector and radiomitigator requires additional regulatory rigor. This may be the very reason that no radioprotector for H-ARS or radiomitigator for DEARE-lung has yet been approved by the US FDA. Nevertheless and much to the credit of a number of rather small, pharmaceutical-based research initiatives, critical work in these biomedical areas continues, providing some hope that current gaps and short-falls in the array of medicinals available for use in the event of major radiological/nuclear exposure contingencies.

Humanetics Corporation is developing BIO 300 with a Target Product Profile (TPP) as a pre-exposure prophylaxis to improve survival by preventing or mitigating radiation exposure-induced myelosuppression to protect military and civilian personnel, including first responders during any radiological/nuclear scenario. At this moment, BIO 300 is at an advanced stage in the development pipeline compared to any other orally effective radiation countermeasure for H-ARS. Humanetics Corporation has an open IND to develop BIO 300 as a prophylactic radioprotectant for ARS. The API of BIO 300, genistein (IUPAC 5,7-dihydroxy-3-(4-hudroxyphenyl)-chromen-4-one), has been granted Orphan Drug Designation (#07-18 June 2411, 2007) for the prevention of H-ARS [105]. In addition, Humanetics Corporation also has another open IND for oncological indications and has an active phase 1b/2a study in patients with NSCLC.

Unlike im, po administration of BIO 300 requires twice a day dosing to result in significant protection of irradiated animals. Although once a day po administration does provide a degree of radioprotection, the level of radioprotection is increased with twice daily dosing. Further, for still seemingly higher levels of protection in humans, BIO 300 can be taken orally for weeks without significant safety/toxicity concerns. The goal behind the development of BIO 300 oral formulation is to produce a countermeasure that can be easily deployed for field use under harsh environmental conditions. This agent is moving toward FDA approval following the Animal Rule. Based on its promising radioprotective efficacy in a large number of murine and canine studies, it has moved forward to NHP models for PK/PD and efficacy evaluation.

The identification and validation of robust biomarker(s) which can be used for its dose conversion from animal models to human for FDA approval under the Animal Rule is a significant next step [23,80]. At this moment, there are no biomarkers for this agent that have been identified and validated to be either upregulated or down-regulated and/or linked to its efficacy or to its’ mechanism(s) of action. The latter is an essential element of ongoing research for this potentially useful radiation countermeasure. Though there are several biomarkers validated/qualified by the US FDA for other indications, there are no FDA qualified biomarkers for any radiation-induced injury or for the response to countermeasures used to mitigate radiation-induced injury. There are numerous reasons for this [80,106], making approval of new radiation countermeasures somewhat difficult. On a positive note, the mechanism of action of this countermeasure and its cellular receptor have been well-documented.

There is also interest from the National Aeronautics and Space Administration (NASA) in BIO 300 for its use to protect astronauts from space radiation exposure during space flights. In the past, BIO 300 has received research support from NASA as well as from the National Space Biomedical Research Institute (NSBRI) for evaluation of its radioprotective effects for radiation exposure encountered in deep space.

Article Highlights

• BIO 300 is a promising medical countermeasure being developed under the US FDA Animal Rule as a radioprotector for H-ARS and as a radiomitigator for DEARE. The drug is the subject of two open and active INDs.

• No radioprotector, to be used prior to radiation exposure in a threat environment, for either H-ARS or for GI-ARS has been approved by the FDA.

• Drug safety data collected in rodents, canines, NHPs, and humans in the clinic is encouraging, as indicated by drug’s tolerance and safety profiles.

• BIO 300 has been found to be efficacious against H-ARS when administered through oral, intramuscular, and subcutaneous routes of administration.

• Based on promising efficacy results using murine models of H-ARS and DEARE, this drug is currently under investigation in NHP models.

• The BIO 300 oral formulation is being developed for an oncologic indication and is currently in a phase 1b/2a study in non-small cell lung cancer patients.

• A rugged and stable solid dosage form of BIO 300 has been developed and is being investigated for use by military personnel.

• The drug’s efficacy through the oral route, lack of side effects, storage at ambient temperature, and intended dual use make it an ideal candidate for both military and civilian use.

Box 1. Drug summary

Drug name	BIO 300
Phase	Phase 1 (safety), Phase 1b/2a (oncology indication)
Indication	Acute radiation syndrome and the delayed effects of acute radiation exposure, Protection against normal tissue toxicities resulting from cancer radiotherapy
Pharmacology/mechanism of action	Ligand for estrogen receptor-β, Controls hematopoietic progenitor cell depletion and lung damage after irradiation by increasing expression of cell cycle checkpoint proteins, DNA repair enzymes, and reducing expression of pro-inflammatory proteins
Routes of administration	Oral, intramuscular, intravenous, subcutaneous
Chemical structure
Pivotal trials	Phase 1 (ClinicalTrials.gov Identifier: NCT00504335) Phase 1b/2a (ClinicalTrials.gov Identifier: NCT02567799) in non-small cell lung cancer (NSCLC) patients receiving chemoradiotherapy

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose

Acknowledgments

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. Mention of specific therapeutic agents does not constitute endorsement by the U.S. Department of Defense, and trade names are used only for the purpose of clarification. We are thankful to Ms. Alana Carpenter and Ms. Sara Nakamura-Peek for editing the manuscript.

Additional information

Funding

The authors gratefully acknowledge the research support from the Joint Program Committee 7, Department of Defense (project # DM178016) to VK Singh.