Development of gamma-tocotrienol as a radiation medical countermeasure for the acute radiation syndrome: current status and future perspectives

ABSTRACT

Introduction

The possibility of exposure to high doses of total- or partial-body ionizing radiation at a high dose rate due to radiological/nuclear accidents or terrorist attacks is increasing. Despite research and development during the last six decades, there is a shortage of nontoxic, safe, and effective radiation medical countermeasures (MCMs) for radiological and nuclear emergencies. To date, the US Food and Drug Administration (US FDA) has approved only four agents for the mitigation of hematopoietic acute radiation syndrome (H-ARS).

Area covered

We present the current status of a promising radiation countermeasure, gamma-tocotrienol (GT3; a component of vitamin E) as a radiation MCM that has been investigated in murine and nonhuman primate models of H-ARS. There is significant work with this agent using various omic platforms during the last few years to identify its efficacy biomarkers.

Expert opinion

GT3 is a newer type of radioprotector having significant injury-countering potential and is currently under advanced development for H-ARS. As a pre-exposure drug, it requires only single doses, lacks significant toxicity, and has minimal, ambient temperature storage requirements; thus, GT3 appears to be an ideal MCM for military and first responders as well as for storage in the Strategic National Stockpile.

KEYWORDS:

• Countermeasure
• gamma-tocotrienol
• mice
• proteomics
• metabolomics
• nonhuman primates
• polypharmacy
• radiation injury

1. Introduction

Undesired exposures to radiological or nuclear materials that arise from accidents or intentional releases by terrorist events seem inevitable [1]. Such radiological or nuclear exposures to individuals or to specific populations would undoubtedly lead to significant health consequences. Exposures of humans to radiation doses of >1 Gy can lead to serious health issues, often expressed as distinct pathologies that commonly involve various organ systems of the body; these pathologies are categorized generally as organ-specific ‘sub-syndromes’ and are collectively known as acute radiation syndrome (ARS) [1–4]. These sub-syndromes most certainly include, but are not limited to, three major, clinically relevant and vital organ systems: hematopoietic (H-ARS), gastrointestinal (GI-ARS), and neurovascular (NV-ARS) systems [5,6]. All ARS sub-syndromes (the above-listed three in addition to pulmonary, cutaneous, etc.) are driven by the same molecular changes occurring at the time of the initial radiation exposure and shortly thereafter. These molecular events are of a direct (ionizations of cellular molecules) and indirect (ionizing cellular water and the production of free radicals) nature, that can damage cellular as well as genomic constituents. A sizable number of promising agents have the potential to counter ARS, not only in terms of preventing or mitigating radiation injuries but also to function therapeutically when administered long after symptoms of radiation exposure appear [5,7–9].

Radiation medical countermeasures (MCMs) are categorized generally into three broad groups based on their time of administration in relation to radiation exposure: radioprotectors (administered prior to exposure), radiomitigators (administered shortly after exposure and before symptoms appear), and radiation therapeutics (administered after overt symptoms of radiation exposure) [10]. Only four MCMs have been approved by the United States Food and Drug Administration (US FDA) for ARS and all these agents are recombinant growth factors/cytokines that are for use after exposure (radiomitigators): Neupogen, Neulasta, Leukine, and Nplate [11–20]. No MCM has been approved by the FDA that can be used prophylactically prior to non-clinical-associated radiation exposures in order to protect individuals or populations from unwanted/unexpected radiation injuries [21,22]. Furthermore, there are no fully approved and effective MCMs available for the treatment (either as prophylaxis or mitigatior) of GI-ARS, NV-ARS, pulmonary injury (delayed effects of acute radiation exposure (DEARE)), or cutaneous injury [5,23,24]. Several candidate radioprotectors and radiomitigators have been identified and are currently being developed following the Animal Rule for FDA approval [25].

A large number of natural products have been studied, with several specific patents garnered for the prophylaxis and treatment of various human diseases, and are generally recognized as safe (GRAS) [26]. Such agents are well accepted as having injury-countering potential and minimal toxicity. Members of the vitamin E family are well recognized for their anti-inflammatory, anti-oxidant, and neuroprotective properties [27]. These agents protect cells from oxidative damage caused by radiation-induced free radicals [28,29]. Vitamin E has appeared as an essential, fat-soluble nutrient that functions as an antioxidant in the human body. It is important since the body cannot manufacture its own vitamin E, so foods and supplements must offer it. At present, vitamin E represents a generic term for all tocopherols and their derivatives with naturally occurring and biologically active stereoisomeric compounds of α-tocopherol [30–32].

Vitamin E represents two subgroups, tocopherols and tocotrienols, that are antioxidants. These agents regulate peroxidation and also restrict free-radical production within the body [33,34]. This family of compounds has eight different isoforms that belong to two categories: four saturated analogues (α, β, γ and δ) called tocopherols and four unsaturated analogues referred to as tocotrienols. These two subgroup of agents share common structural features of a chromanol ring and a 15-carbon tail at the C-2 position and are known as tocols. Tocotrienols differ structurally from tocopherols by the presence of three trans-double bonds in the hydrocarbon tail.

2. Gamma-tocotrienol (GT3) as a radiation MCM

GT3, delta-tocotrienol, tocopherol succinate, and their derivatives have been shown to effectively protect mice against high, acute and potentially fatal doses of ionizing radiation [27]. GT3 has been most extensively evaluated for its radioprotective efficacy in both rodents (mice) and nonhuman primates (NHPs) [35,36]. After comparing various attributes of different tocotrienols and tocopherols, GT3 was selected for advanced development as a radiation MCM [27,35,36]. The initial studies showed GT3 to have significant levels of radioprotectiveness for both H-ARS and GI-ARS when tested in a murine model of acute radiation injury. These initial findings prompted investigators at the Armed Forces Radiobiology Research Institute and elsewhere to initiate advanced, large animal studies (e.g. NHPs) [36–42]. As such and with further development, this candidate MCM may prove to be highly useful and have safe prophylaxis for use in warding off potentially lethal effects of acute radiation exposure and associated injuries. Current research and development on this agent (GT3), along with the positive findings of the agent’s safety and efficacy profiles, seem to be setting the stage for possible approval by the US FDA for human use. This article provides the current status of GT3 as a radioprotective MCM.

3. Important attributes of GT3

The salient features of GT3 as a MCM for radiation exposure and associated injuries are as follows: i) protection against potential lethal radiation injury; ii) protection against specific organ injuries (hematopoietic and gastrointestinal); iii) pharmacokinetic and pharmacodynamic characteristics; and iv) toxicological and safety profiles (Table 1).

Table 1. Major radioprotective attributes of GT3.

Tests and metrics	Major findings of GT3 prophylaxis	References
Survival protection	Estimated ‘Dose reduction Factor’ (DRF) of ~1.29	[43]
Protective hematopoietic effects	Minimizes H-ARS component of ARS	[43]
Blood	Limits life-threatening pancytopenias	[43]
	Mitigates acute neutropenias	[43]
Bone marrow	Promotes recovery of radiation injured vital hematopoietic compartments in bone marrow	[35,43]
	Promotes survival and recovery of hematopoietic stem and progenitor cells	[35,38,49,50]
	Mobilizes marrow stem cells & progenitors into circulation	[35,52]
Protective GI system effects	Minimizes GI component of ARS	[37,44,56]
	Bolsters integrity of intestinal mucosa	[44,56]
	Promotes intestinal crypt cell survival	[44,56]
	Enhances recovery of mucosal surface area	[44,56]
	Limits bacterial translocation across intestinal wall	[35,44]
Protective vascular effects	Promotes integrity of endothelium	[35]
	Serves to quench oxidative stress	[35,44]
Metabolomic changes	Dampens radiation-induced metabolic fluctuations	[41,64]
	Principal metabolites affected: carnitine, propionylcarnitine, xanthine, creatine	[41,64]
Polypharmacy approach	Additive radioprotective efficacy of GT3 by combination with amifostine, pentoxifylline and Simvastatin	[35,58,61,62]

3.1. Protection afforded by GT3 against potentially lethal radiation injury

As demonstrated in small animal models of ARS, the life-sparing efficacy of GT3 prophylaxis following acute, high-dose irradiation was clear and unambiguous. In one study for example, CD2F1 male mice were prophylaxed with optimal doses of GT3 (via subcutaneous (sc), 200 mg/kg injections) 24 h (the optimal time for prophylaxis) prior to supralethal doses of whole-body irradiation (11 Gy) and subsequently monitored for survival over 30 days. The survivability of the GT3-treated mice was remarkable, as evidenced by complete survival (100%) of the treated mice relative to the absence of such survival (0%) in untreated control animals [43]. The effectiveness of GT3 prophylaxis was demonstrated still further by a) the relatively high estimated dose reduction factor (DRF) for GT3 of ~1.29 and b) noted protection by the agent of acute, radiation-induced pancytopenia (via the sequential monitoring of peripheral blood cell counts in individual irradiated vs. unirradiated animals) [43].

Additional small animal studies (CD2F1 male mice) further demonstrated, in a convincing manner, that the positive, radioprotective efficacy of GT3 prophylaxis was not only limited to hematopoietic tissues, but also extended to the gastrointestinal system as well [44]. Such studies have confirmed GT3ʹs capacity to improve the prospects of the irradiated animals to survival (Figure 1) [45]. Furthermore, these studies revealed alternative mechanisms of radioprotection; namely, the capacity of GT3 to accumulate and to reduce oxidative stress within epithelia and endothelia of various radiosensitive tissues. This radioprotective effect has been linked to GT3ʹs capacity to inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [44,46–48]. However, it needs to be noted that despite the clear and unambiguous survival benefit afforded by GT3 prophylaxis of supralethally irradiated mice, a comparable benefit has yet to be confirmed in larger animal models (e.g. NHPs) [36].

Figure 1. Efficacy of single GT3 doses (200 mg/kg) prophylactically administered on mouse survival following exposure to 9.2 Gy or 11.0 Gy cobalt-60 gamma-radiation (0.6 Gy/min). CD2F1 mice were administered GT3 24 h prior to irradiation (n = 16 per treatment group for 9.2 Gy). Mice were observed for survival for 30 days post-irradiation. Statistical analysis was performed by log rank test between the 9.2 Gy GT3 and Vehicle groups (p < .001) and the 11.0 Gy GT3 and Vehicle groups (p < .001).

3.2 Protection afforded by GT3 against specific acute radiation injuries of the hematopoietic and GI systems

GT3 has been shown to protect both mice and NHP hematopoietic and GI tissues from ionizing radiation-induced injury [35].

3.2.1 Hematopoietic system and small animal studies in mice

As reported above, a single, prophylactic dose (200 mg/kg sc) of GT3 administered a day prior to acute, potentially lethal irradiation not only increased the prospects of survival but also significantly reduced the extent of life-threatening pancytopenia. As evidenced by the subsequent report, the radioprotection afforded by GT3 prophylaxis appeared to extend to vital hematopoietic tissues, particularly the more primitive hematopoietic compartments of bone marrow [35]. This protection by GT3 prophylaxis was inferred by the noted improved rates of recovery of the radiation depleted progenitorial marrow compartments (e.g. cKit⁺ lin⁻ hematopoietic stem cells) and not necessarily by GT3ʹs blocking the initial depletion of those compartments [35,49,50]. Follow-up histopathology of bone marrow samples seemed to support the concept that GT3 treatments promoted the regeneration of myeloid tissues following acute irradiation. Mobilization of progenitor cells in peripheral blood by GT3 indicates that GT3 can be used as an alternative to G-CSF to mobilize hematopoietic progenitor cells [49,51–53]. These mobilized cells provide a significant survival benefit to irradiated mice when administered after total-body radiation exposure [52,54,55]. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were infused with antibodies to G-CSF prior to blood collection. Administration of a G-CSF antibody to GT3-injected mice significantly abrogated the efficacy (relative to GT3-survival benefit afforded to recipient animals) of blood or peripheral blood mononuclear cells (PBMC) obtained from GT3 administered donors. Furthermore, GT3-mobilized PBMCs also inhibited the translocation of intestinal bacteria to the various organs and increased colony forming units-spleen (CFU-S) in irradiated mice. In brief, GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. Such an approach using mobilized progenitors from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation [52].

3.2.2 Hematopoietic system and large animal studies in NHPs

GT3 prophylaxis (37.5 or 75 mg/kg) of acutely irradiated NHPs (5.8 to 6.5 Gy) resulted in significant alleviation of severe, life-threatening neutropenia, and thrombocytopenia [36]. A single dose of GT3 without any supportive care was equivalent, in terms of improving hematopoietic recovery, to multiple doses of Neupogen and two doses of Neulasta with full supportive care (including blood products) in the NHP model [15,19,35]. A recent study conducted with 4 and 5.8 Gy total-body gamma irradiation using the NHP model showed significant changes in the frequencies of B and T-cell subsets, including the self-renewable capacity of hematopoietic stem cells (HSCs). Importantly, GT3 accelerated the recovery in CD34⁺ cells, increased HSC function as shown by improved recovery of CFU-granulocyte macrophages (CFU-GM) and burst-forming units-erythroid (BFU-E), and aided the recovery of circulating neutrophils and platelets [38].

3.2.3 GI system and small animal studies

GT3 prophylaxis (single sc dose 24 h prior to exposure) of acutely irradiated (11 Gy) mice was shown to significantly reduce intestinal radiation injury and to accelerate its recovery. This was demonstrated by several distinct assays, namely direct histopathological assessments of mucosal area, quantitative crypt cell assays, and indirectly by assays of plasma citrulline levels reflecting changes in GI-associated epithelial cell mass and by measures of the extent of GI-associated bacterial translocation. These GT3-mediated improvements of GI structure and function coincided with the marked reduction in early, GI-associated deaths and the pronounced extension of survival times of the GT3 prophylaxed animals [44].

3.2.4 GI system and large animal (NHPs) studies

GT3-prophylaxed NHPs that were subsequently acutely irradiated with a supralethal dose of radiation (12 Gy) appeared to respond with a ‘quicker recovery of the small intestine’ than did untreated, irradiated control animals, as evidenced by measures of histopathological parameters and by assayed plasma citrulline levels [37,56]. Similar observations were made with total-body irradiation with gamma-ray as well as linear accelerator (LINAC)-derived X-ray for partial-body irradiation in NHPs.

3.3. GT3ʹs pharmacokinetic and pharmacodynamic characteristics

NHPs were used to develop GT3ʹs pharmacokinetic profiles [36]. With increased doses of GT3 (from low doses of ~9.4 mg/kg, to midrange doses of ~18.8, to high doses of ~37.5 mg/kg), the basic pharmacokinetic parameters, e.g., area under the curve (AUC) and GT3 half-life increased [36]. This study elegantly presented maximum blood plasma concentrations (C_max), the time at which C_max is obtained (T_max), half-life (T_1/2), AUC, clearance rate (Cl), and mean retention time (MRT) for the various doses of GT3. These pharmacokinetic parameters were helpful to determine the dose of GT3 for the efficacy study.

3.4. GT3ʹs toxicological and safety profiles

Considering the widespread use and availability in the marketplace of the tocopherol group, including GT3, as nutritional supplements, in cosmetics, and in oral care/hygienic products, they are recognized as safe for various applications in humans, including medicinal purposes [35]. The literature on this subject is extensive, going well beyond the scope of this article, but suffice it to say that GT3/tocopherol is exceedingly well tolerated when administered topically or orally, even at the upper reaches of dose administrations. However, this general statement concerning GT3/tocopherol’s lack of toxic potential is far less certain when these agents are administered by injection. Despite the lack of reports of observing toxic effects associated with GT3/tocopherol administration in small experimental animals, there has been at least one report of adverse effects at the site of GT3 injections of large experimental animals (NHPs) when doses were at 75 mg/kg or greater [36]. Regardless, additional toxicological assessments of injected GT3 are warranted and would encompass both large animal models as well as human volunteers (Phase 1 clinical trials).

3.5. GT3ʹs mode and mechanisms of radioprotection

GT3 confers protection against radiation-induced lethality but the molecular targets that underlie such protection are not well identified. Several laboratories are investigating its mode of action for radioprotection.

3.5.1 Protection of the hematopoietic system

GT3 has been shown to induce high levels of G-CSF, and the use of G-CSF antibody completely abrogates GT3ʹs radioprotective efficacy in the murine model, demonstrating the cooperative role GT3 plays in fostering radioprotective/radiomitigative actions by G-CSF [57]. This study suggests that G-CSF itself may serve as a useful biomarker for GT3ʹs efficacy [35]. Furthermore, it has been demonstrated (as previously discussed in section 3.2.1) that GT3 induces G-CSF that mobilizes hematopoietic progenitors, which in turn can be collected (within whole blood or mononuclear cell fractions) and administered for reparative purposes in order to salvage acutely irradiated, critically injured animals [35,52].

3.5.2 Protection of the GI and vascular systems

GT3 is known to be an inhibitor of HMG-CoA reductase, and it reduces vascular peroxynitrite generation through its inhibition. Consequently, the radioprotective efficacy of GT3 is thought to be, at least in part, due to its ability to concentrate in endothelial cells and inhibit HMG-CoA reductase. Such inhibitors of HMG-CoA reductase have significant vasculoprotective, anti-inflammatory, as well as anti-fibrotic efficacy [35,44,47]. These activities are mediated by endothelial nitric oxide synthase. Like other HMG-CoA reductase inhibitors, GT3 also upregulates thrombomodulin, an anticoagulant with radioprotective efficacy. The radioprotective efficacy of GT3 has been shown to be dependent on its ability to upregulate endothelial cell thrombomodulin. It has been speculated that additive or synergistic radioprotection can be obtained by the use of GT3 or other HMG-CoA reductase inhibitors in combination with thrombomodulin and/or activated protein C [58].

3.5.3 CEBPD mediated protection conferred by GT3

The role of CCAAT enhancer binding protein delta (CEBPD) has been investigated for its role in radioprotective efficacy of GT3 [59]. Using CEBPD^−/− and CEBPD^+/+ (C57BL/6 background) mice, a study has been conducted to identify a novel role for CEBPD in GT3-mediated protection against radiation-induced injury. In brief, CEBPD was identified to participate in radioprotection of GT3 via modulating radiation-induced oxidative and nitrosative stress. The exact mechanism of CEBPD upregulation by GT3 and irradiation remains unclear [59].

3.5.4 Effect on cytogenetic damage

Prophylaxis with GT3 has been shown to reduce double-strand break formation and decrease chromosomal aberrations in human umbilical vein endothelial cells as well as mouse bone marrow cells after radiation exposure [60]. Furthermore, GT3 enhanced the expression of RAD 50 (a DNA-repair gene) and attenuated radiation-induced RAD50 suppression.

3.5.5 Utility of using a ‘poly-pharmaceutical approach’ in radioprotection

A few agents with alternative modes of action have been investigated for synergistic, radioprotective effects when combined with GT3. Some of these medicinals include but are certainly not limited to pentoxifylline, simvastatin (cholesterol-lowering drug and inhibitor of HMG-CoA reductase), and amifostine [35,58,61,62]. Initial results using small animal models of such poly-pharmaceutical studies are indeed encouraging.

GT3 in combination with pentoxifylline significantly improved survival compared to GT3 alone and also improved bone marrow CFUs, spleen colony counts, and platelet recovery. GT3 and combined treatment were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Combined treatment increased post-irradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of endothelial nitric oxide synthase (eNOS) [48]. It has also been shown that mevalonate had no effect on the radioprotection afforded by GT3-pentoxifylline and calmodulin abrogated the synergistic radioprotection by the GT3-pentoxifylline combination [62]. Mevalonate reverses the inhibitory effect of GT3 on 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and calmodulin reverses the inhibition of phosphodiesterase by pentoxifylline. Above observations suggest that the mechanism of radioprotection by GT3-pentoxifylline may involve phosphodiesterase inhibition.

A study has been conducted to investigate the potentiation of survival protection afforded by relatively low-dose amifostine prophylaxis against total-body γ-irradiation in combination with GT3 [61]. This survival study demonstrated that combined treatments of GT3 and amifostine resulted in significantly higher survival compared to single treatments. Such combination treatment in mice also confirmed prior reports suggesting GT3 induces G-CSF. This polypharmacy approach of the combination of amifostine and GT3 acting through different mechanisms shows promise [61].

The combined treatment of GT3 and simvastatin has been shown to enhance irradiated mouse survival, accelerate hematopoietic recovery, and to augment restoration of bone marrow cellularity when compared to the animals treated with either of the two drugs given singly [58].

4. Recent biomarker studies using various omic platforms

Different omic testing platforms have been recently utilized in the evaluation of GT3ʹs general protective/therapeutic attributes, with promising results to be helpful for the identification and validation of biomarkers of the agent’s efficacy that are essential for FDA approval of MCMs following the Animal Rule [25].

4.1 MicroRNA (miRNA) modulation by GT3

As stated above, the administration of GT3 (37.5 and 75 mg/kg) a day prior to irradiation enhances hematopoietic recovery in irradiated NHPs [36]. Serum levels of microRNA-30a(miR-30a), miR-126, and miR-375 correlated with the radioprotective efficacy of GT3 (miRNA in the GT3-treated irradiated NHP resembled the unirradiated animals) while levels of miR-133a, miR-215, miR-150, and miR-133b demonstrated an increased response (stronger upregulation of miR-133a/b and stronger downregulation of miR-215 and miR-150) (Table 2) [40]. In the murine model, GT3 was shown to reduce radiation-induced levels of miRNA-126 as well [63].

Table 2. Impact of GT3 treatment on radiation-induced miRNA expression in NHPs.

miRNA	p value: pre-rad vs GT3 treated and irradiated	p value: pre-irradiation vs irradiated	p value: irradiated vs GT3-treated and irradiated	Comments
miR-30a-5p		0.0009	0.0283	Expression in GT3-treated and irradiated NHPs overlapped with that in pre-irradiation samples
miR-126-5p		0.0026	0.004
miR-375		0.0001	0.0002
miR-133a-3p	0.0001	0.0001	0.0375	Expression demonstrated more extreme radiation-induced changes in GT3-treated NHPs in comparison with vehicle-treated NHPs
miR-215-5p	0.0001	0.0138	0.002
miR-150-5p	0.0001	0.0021	0.0491
miR-133b	0.0001	0.0001	0.0368

4.2 Metabolomic/Lipidomic changes in NHPs

Global metabolomic changes in serum samples of unirradiated NHPs administered GT3 (37.5 mg/kg) demonstrated altered metabolites at 24 h post-GT3 administration. The changes were linked to transient increases in the bioavailability of antioxidants, cholic acid, lactic acid, anti-inflammatory metabolites 3 deoxy-vitamin D3 and docosahexaenoic acid [64]. Analysis of serum samples of irradiated NHPs administered GT3 also demonstrated that several metabolites were altered after radiation exposure, including compounds involved in fatty acid β-oxidation, purine catabolism, and amino acid metabolism. In brief, GT3 administration appeared to diminish radiation-induced fluctuations in metabolites, suggesting a probable beneficial effect in animals exposed to gamma-radiation (Table 3) [41].

Table 3. Altered levels of important proteomes and metabolites by GT3 in irradiated or unirradiated mice and NHPs.

Proteomic changes in irradiated mice
Affected proteomes	Comments
Beta-2-glycoprotein 1, alpha-1-acid glycoprotein 1, alpha-2-macroglobulin, complement C3, mannose-binding protein C, and major urinary protein 6 Upregulation of the Wnt signaling pathway and actin-cytoskeleton linked proteins in mediating the radiation injury response in spleen	Significant expression changes after irradiation were reversed with GT3 treatment GT3 efficacy is mediated via alleviation of radiation-induced alterations in biochemical pathways with wide implications on overall hematopoietic injury.
Metabolomic changes in irradiated NHPs
Carnitine, propionylcarnitine, xanthine, creatine	GT3 administration reduced radiation-induced high fluctuations in these metabolite levels
Metabolomic changes in unirradiated NHPs
Stearic acid, Pro leu, cholic acid, lactic acid, hexadecyl acetyl glycerol, PI(20:4), alpha-N-phenylacetyl-L-glutamine, tryptophan, propionyl-L-carnitine, 9-octadecenal, 2-hexadecenal, arachidonic acid, docosahexaenoic acid, 10-oxo-nonadecanoic acid, PS(16:0/21:0), phenylalanine, L-histidine, PC(20:5/20:5), butyryl carnitine, 3-deoxyvitamin D3	Top 20 dysregulated metabolites

4.3 Proteomic changes by GT3 in mice

There are several murine model-based studies conducted for proteomics using different analytic methods [45,65,66]. Analyses of serum samples from irradiated mice demonstrated changes in expression of 19 proteins in response to exposure, and that GT3 prophylaxis appeared to reverse these changes. Pathway analysis (i.e. Ingenuity analyses) revealed a network of associated proteins involved in immune cell trafficking, cellular movement, and inflammatory response [45]. Significant changes in α-1-acid glycoprotein-1, α-2-macroglobulin, β-2-glycoprotein 1, complement C3, major urinary protein 6, and mannose-binding protein C were observed after irradiation and reversed by GT3 prophylaxis. This study reported the untargeted approach, the network, and specific serum proteins that could be translated as biomarkers for MCMs. GT3 administration has also demonstrated to attenuate radiation-induced proteomic changes in the spleen of CD2F1 mice (Table 3) [66]. Specifically, this study demonstrated that 24 h pretreatment with GT3 to CD2F1 mice attenuated γ-radiation-induced hematopoietic injury in the spleen by modulating various cell signaling proteins.

4.4 Transcriptomic study in NHPs administered GT3

There is a recent report using GT3 in the NHP model to investigate the effect of a supralethal dose of 12 Gy total- and partial-body radiation exposures on alterations in the lung transcriptome [39]. High-level cobalt-60 γ-radiation source and LINAC were used for total- and partial-body irradiation, respectively, of rhesus NHPs. There were considerable radiation-induced alterations in the lung transcriptome for total- as well as partial-body irradiation. Several common signaling pathways including PI3K/AKT, GADD45, and p53 were upregulated in both exposure types. This study provides insights into the molecular pathways that might be applicable for biomarker discovery. There was limited influence of GT3 on radiation-induced transcriptome changes in total- or partial-body exposed animals at such a supralethal dose [39].

5. Conclusion

The pursuit of an optimal radioprotective agent to be used prior to radiation exposure under various radiological/nuclear scenarios has continued for more than 60 years. To date, we do not have any US FDA-approved radioprotector that can be used for protecting individuals at high risk of lethal radiation exposures resulting from nuclear/radiological accidents and deliberate terrorist activity. Due to the scientific community’s sustained interest and the improved funding situation in the US, post 9/11, for terrorism-associated radiation MCM research, substantial progress has been made in continuing to identify, validate, develop, and to eventually gain FDA-approval for MCMs that can protect the population at large from ARS as a result of exposure to ionizing radiation.

There are several promising agents that have demonstrated potential to be used in the future, but these agents need to be systematically evaluated and developed for FDA approval following the Animal Rule. The radioprotective efficacy of GT3 was discovered by investigators with an objective to develop this radiation MCM for military and civilian use. Its formulation administered sc to mice and NHPs has been extensively investigated and has demonstrated consistent and significant radioprotective efficacy in the countering of H-ARS. Based on its encouraging efficacy results for H-ARS in small and large animal models, this agent is moving forward for further evaluation in the large animal model. Various laboratories are investigating different aspects of its radioprotective efficacies, as well as pursuing work with molecular targets [67–69], pharmacology [70], ways to make it orally effective [71], and developing modern strategies for drug delivery [72]. Although a great deal is known about the potential mechanism by which GT3 acts as a radiation MCM, there are ongoing investigations to further determine the mechanisms by which a single administration of GT3 provokes such a powerful radioprotective response. In recent past, there are several reports using various omic platforms identifying its biomarkers which is a critical step for approval of such MCMs following the US FDA Animal Rule (Figure 2) [25,35,73–77]. We strongly believe this candidate MCM, with its several encouraging attributes, merits further development to get FDA approval for generalized use during radiation exposure emergencies.

Figure 2. Important attributes of GT3 as a radiation medical countermeasure. This agent has been found to be effective when administered prior to radiation exposure. Various omic platforms are being used to identify its biomarkers.

6. Expert opinion

Though tocotrienols were characterized six decades ago, their major biological attributes were only discovered during the last 20 years and tocotrienol evaluation has gained attention in the recent past. Most of the preclinical studies with GT3 for its radioprotective efficacy in various animal models have been conducted using this agent for prophylaxis against ARS. GT3 has proven to be the most efficacious and potent radioprotector of all the tocols examined to date [35].

As there is currently a void within the US National Pharmaceutical Stockpile of safe and effective MCMs that could be used prophylactically to ward off serious, life-threatening radiation injuries that stem from unwanted exposures from nuclear/radiological events, it would seem that aggressive research and development (R&D) of GT3 is both timely and appropriate. The identification, development, and stockpiling of suitable radioprotective agents would fall most certainly into the high priority R&D category relative to national security interests. In this regard, the various species of tocotrienols, GT3 in particular, appears to be sufficiently promising as a safe and effective radioprotector to warrant further consideration, especially in terms of advanced preclinical animal studies, as well as initial clinical safety and toxicological assessments in humans. It should be pointed out that although there is at least one potential radioprotector currently in clinical use (e.g. amifostine, an aminothiol used for the protection of normal tissues during radiotherapeutic procedures), this agent tends to be quite toxic when applied at effective dosing levels and hence, is generally considered unsuitable for non-clinical applications. In this regard, radioprotectors such as GT3 and tocols are sufficiently nontoxic and well tolerated, which is required to be seriously considered by advanced clinical evaluations.

The development and fielding of prophylactic agents such as GT3 would be particularly useful for selected government agencies tasked with the responsibility of securing the health and safety of their citizens. For example, the US Defense Department needs to protect forward deployed personnel during anticipated or ongoing radiological/nuclear events. Details of how and when agents such as GT3 would be deployed remain to be fully developed; e.g. the time-window for effective administration of the protective agent. Clearly, advanced R&D will be needed on developing standard working protocols for GT3ʹs fielding and application.

The multiple ways by which GT3 exerts its radioprotective effects provide a clear advantage over chemical agents that function largely by free radical quenching. As noted, GT3 affords not just a single radioprotective mechanism, but rather multiple, well-defined mechanisms at its disposal, allowing for the protection of several, at risk organ systems/tissues of the body following acute irradiation (Table 1).

The administration of GT3 induces high levels of G-CSF that, in turn, have a pronounced radiation injury mitigating effect. As demonstrated in a murine model of radiation injury, this induction by GT3 and its apparent radioprotective effect is completely abrogated with the use of G-CSF antibody, suggesting that GT3ʹs radioprotectiveness (in terms of survival protection) is an indirect one, mediated largely by GT3ʹs induction of G-CSF. This experimental observation clearly suggests G-CSF’s involvement in GT3ʹs capacity to protect acutely irradiated animals, and that this induction of the hematopoietic growth factor may serve as a biomarker for GT3ʹs efficacy. However, unlike the latter observation in the murine model, the level of G-CSF induced by GT3 in the NHP model is less, but still significant and can be medically leveraged. For example, GT3 has been used in combination with several other potential radioprotective agents at low doses using the polypharmacy approach with a positive outcome. Its combination with small doses of essentially nontoxic amifostine is particularly remarkable for augmenting the radioprotective state in properly prophylaxed animals prior to acute, potentially lethal irradiation [35].

Still yet another distinct radioprotective feature of GT3 relates to its capacity to ameliorate radiation-induced GI injury by improving the survival of crypts, improving the mucosal surface area, and by reducing the HMG-CoA reductase independent vascular oxidative stress after radiation exposure. It is important to note that the ability of GT3 to decrease radiation-induced oxidative stress was reversed by the use of mevalonate. This observation has important implications for GT3, specifically for its development for injury where vascular damage is expected to play an important role (intestinal and lung injury). GT3 decreases vascular peroxynitrite production through HMG-CoA reductase inhibition. Such inhibitors mediate their efficacy by endothelial nitric oxide synthase, with tetrahydrobiopterin (BH4) as an important cofactor.

Under any mass casualty scenario, hospital-based care including blood transfusions will be limited. Thus, there is a need for an optimal radiation countermeasure which should be effective with minimal required number of doses, without intensive medical care (blood transfusion, etc.) available in a hospital, and that can be stored at ambient temperature. The demonstrated efficacy of a single dose of GT3 in NHPs without supportive care is encouraging, particularly because the radiation doses used in that study were equivalent to the lethal doses for humans. Since GT3 is being pursued as a prophylactic agent, its field application might be most appropriate for first responders and military personnel responding to nuclear/radiological contingencies. Additional positive attributes of GT3 include stability at room temperature and not requiring storage at very low temperatures [27,35,71]. Furthermore, GT3 is regarded by the US FDA as GRAS when used as a food additive (i.e. used orally) at lower doses compared with its parenteral use as a MCM.

Article highlight

• No radioprotector to be used for prophylaxis for either H-ARS or for GI-ARS has been approved by the US FDA.

• GT3 is a promising radiation MCM being developed under the US FDA Animal Rule as a radioprotector for H-ARS.

• The drug safety profile based on studies conducted in murine and NHP models appear encouraging.

• GT3 has been found to be efficacious in countering H-ARS in murine and NHP models when administered through subcutaneous route.

• Biomarkers are being identified using various omic platforms in both murine and NHP models.

• The drug’s efficacy in murine and NHP models, lack of side effects, and storage at ambient temperature make GT3 an ideal candidate for use by both military and first responders.

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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.

Author contributions

VKS and TMS performed literature searches, drafted the manuscript, revised, and finalized for publication.

Acknowledgments

The opinions or assertions contained herein are the private views of the authors and are not necessarily those of the Armed Forces Radiobiology Research Institute, or the Department of Defense. The 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 apologize to those having contributed substantially to the topics discussed herein that we were unable to cite because of space constraints. We are thankful to Ms. Alana Carpenter for editing the manuscript.

Additional information

Funding

This work was supported by funding from the Armed Forces Radiobiology Research Institute (RBB29173) awarded to VK Singh.