WO2009126866A2 - Delta-tocotrienol as a radioprotective countermeasure agent - Google Patents

Abstract

The present invention provides compositions comprising delta-tocotrienol, and derivatives thereof, that are effective for protection against radiation-induced injury and radiation-induced mortality. Also provided are methods of using these compositions for protecting the body from radiation-induced injury and radiation-induced mortality.

Description

DELTA-TOCOTRIENOL AS A RADIOPROTECTIVE COUNTERMEASURE AGENT

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made in part with Government support. The Government has certain rights in the invention.

RELATED APPLICATION

[0002] This application claims the benefit under 35 U. S. C. § 119(e) of U.S. Provisional

Application No. 61/043,972 filed on April 10, 2008, which is hereby incorporated by reference herein in its entirety.

FIELD

[0003] The instant invention relates generally to radioprotective agents, compositions comprising such agents, and methods for making and using such agents. In certain aspects, the invention relates to radioprotective compositions comprising delta-tocotrienol and derivatives thereof, and methods of using such compositions for protecting the body from radiation-induced injury.

BACKGROUND

[0004] Exposure to various sources of environmental radiation poses significant health risks. For example, military forces and inhabitants of metropolitan areas are at risk of exposure from a nuclear or radiological attack, and citizens risk exposure from industrial accidents and environmental pollution. In addition, patients are often subjected to radiation in the course of medical care, including X-ray diagnoses and therapies for cell-pro liferative disorders. [0005] Depending on the level of exposure, the effects of radiation can range from nausea and vomiting to immune system compromise to death from radiation-induced tissue damage or infection. Exposure to moderate doses of gamma radiation has been shown to cause chromosomal damage and defects in hematopoiesis and immunosuppression. Successively higher radiation doses compound these effects with gastrointestinal (GI) and neurovascular tissue damage.

[0006] Ionizing radiation can trigger free-radical reactions that lead to the formation of reactive oxygen species (ROS). It is generally believed that production of ROS is a primary mechanism underlying radiation-induced biological damage. The cells of the immune and blood- forming systems are particularly sensitive to changes in oxidant/antioxidant balance due to the high percentage of polyunsaturated fatty acids in their plasma membranes. The oxidant/antioxidant balance is thus an important determinant for both immune and blood- forming functions, not only for maintaining the integrity and function of the plasma membrane, cellular proteins, and nucleic acids, but also for control of signal transduction and gene expression.

[0007] A compound that is safe for human use and capable of preventing the ablation of immune system function and other radiation-induced oxidative tissue damage would provide an effective medical countermeasure against a nuclear or radiological attack or other source of environmental radiation, and provide adjunct therapies to radiation-based medical treatments. However, despite over four decades of effort in this area, no compound has yet been identified and fielded that has the broad-spectrum radioprotective attributes necessary to adequately protect populations against the range of potential radiation exposures, including high dose exposures associated with catastrophic events, both accidental (e.g. , industrial spills) and intentional (e.g. , terrorist attacks).

[0008] Amifostine (WR-2721) is considered the "gold standard" radioprotectant by many investigators, due to its broad spectrum of activity and general efficaciousness. Amifostine is currently FDA-approved and is commercially marketed for use as a normal tissue protectant in cancer patients undergoing intense chemo- and/or radiotherapy. However, amifostine has significant toxicity at therapeutic doses, and can be debilitating when applied at the relatively high doses required for cytoprotection. Amifostine therefore has undesirable dose-limiting side effects that limit its usefulness in clinical settings, and preclude its use for protection of large military or civilian populations which have undergone, or are expected to undergo, significant radiation exposure. [0009] A suitable radioprotectant should have one or more, and preferably all of the following attributes: (i) capability of providing significant protection against lethality from acute and long-term effects of chronic radiation damage; (ii) suitability for oral administration, and rapid absorption and distribution throughout the body; (iii) lack of significant toxico logical effects, including behavioral effects; (iv) ready availability and affordability; and (v) chemical stability that permits easy handling and storage. Unfortunately, no currently available radioprotective agent possesses all of these qualities. Although combinations of radioprotective drugs acting via different mechanisms can improve the degree of protection in small rodents, attempts to use such treatments in large mammals have had limited success, as evidenced by poor levels of radioprotection and considerable toxicity.

[0010] The search for non-toxic radioprotectants has recently led to an examination of natural dietary antioxidants. For example, the dietary antioxidants vitamin E (Weiss and Srinivasan 1992, Roy et al., 1988), vitamin A, and β-carotene (Seifter et al., 1984) have shown varying degrees of radioprotective efficacy. Among these compounds, vitamin E has attracted considerable attention.

[0011] Vitamin E, or alpha-tocopherol, is a member of the "tocol" family of compounds that are present at varying levels in common human foods, with Vitamin E being one of the most abundant in typical diets. The term "tocol" is generally used to collectively refer to all tocopherol and tocotrienol compounds. All members of the tocol family are considered generally recognized as safe (GRAS) or self-affirmed GRAS. For example, in one study measuring oral toxicity in rats, a mixed preparation containing all 8 tocols had a no-observed adverse effect level (NOAEL) of 120-130 mg/kg daily, which corresponds to a human dose of about 9.4 grams/day (Nakamura et al., 2001). Moreover, vitamin E has a long record of safety as a preservative and nutritional supplement in a wide variety of foods, and nutritional supplements containing mixed tocopherols and tocotrienols are currently sold in the US and overseas. [0012] Vitamin E has been shown to confer a degree of protection against radiation- induced lethality in mice. For example, vitamin E (Weiss and Landauer 2000) fed at 3 times the normal mouse requirement for 1 week before, and for 30 days following, an 8.5 Gray (Gy) dose of Cobalt 60 gamma radiation led to 60% 30-day survival, whereas 100% of control animals succumbed by 30 days. At 7.5 Gy the control survival was 10%, whereas 100% of the vitamin E- treated animals survived (Jacobs 1983).

[0013] In another study, subcutaneously administered vitamin E provided a 79% survival rate in mice exposed to a supralethal dose of 10.5 Gy. In contrast, oral administration was found to be ineffective (Kumar et al., 2002).

[0014] In mice exposed to 1 Gy of whole-body Cobalt 60 gamma radiation 2 hr before or

2 hr after oral administration of vitamin E, both bone marrow polychromatic erythrocyte micronucleus formation and chromosome aberrations were significantly suppressed (Sarma and

Kesavan 1993). Suppression of chromosome damage by vitamin E has also been demonstrated in mouse cells (Konopacka 1998) and human lymphocytes (Konopacka and Rzeszowska-Wolny

2001).

[0015] The dose reduction factor (DRF), determined with varying radiation doses and a defined end-point, such as 30-day survival, allows quantitative comparison of radioprotectants.

The DRF for vitamin E in mice (using 30-day survival as the end point) has been variously reported to be 1.06, 1.11, and 1.23 (Srinivasan et al., 1997; Srinivasan and Weiss 1983; Seed et al., 2002).

[0016] Amifostine has a DRF value of 2.7 at a high dosage level (Giambarresi and

Walker, 1984), but the associated toxicity at this dose has led the US Department of Energy to recommend that amifostine not be used as a pretreatment for emergency radiation exposures

(Shea, 2004; Weiss, 1997). At a dose that minimizes side effects, the DRF of Amifostine drops to 1.2, which is within the range reported for vitamin E. Even at this dose, Amifostine continues to have significant side effects, especially in tests conducted with large animal models. In contrast, vitamin E is non-toxic at doses that give a DRF of about 1.2.

[0017] While some tocols other than vitamin E are effective antioxidants, only vitamin E is widely available in pure form and published studies concerning the radioprotective properties of tocols are limited to vitamin E.

SUMMARY

[0018] In one aspect, methods are provided herein for protecting a subject from the effects of radiation exposure, wherein the methods comprise administering to the subject a composition comprising a radioprotective amount of delta-tocotrienol or a derivative thereof. [0019] In another aspect, methods are provided herein for reducing the risk of radiation- induced mortality, wherein the methods comprise administering to a subject that has been exposed to radiation or is at risk of exposure to radiation a radioprotective amount of a composition comprising delta-tocotrienol or a derivative thereof.

[0020] In various methods provided herein, the derivative of delta-tocotrienol is a succinate ester of delta-tocotrienol, a salt of an amino acid ester of delta-tocotrienol, or a glycoside formed from a monosaccharide or disaccharide and delta-tocotrienol. [0021] In various aspects, subjects are protected from the effects of ionizing radiation, including for example, alpha radiation, beta radiation, gamma radiation, X-radiation, ultraviolet radiation, and/or neutrons. In some aspects, subjects are protected from the effects of high doses of ionizing radiation. Scenarios involving radiological hazards include nuclear detonations, covert placement of radioactive substances, and dirty bombs (Waselenko et al. 2004). At doses above about 1 Gy (gamma or X-rays) in humans, hematopoietic function is compromised, leading to decreases in white blood cell counts and increased susceptibility to infection (AFRRI 2003). At doses above 2 Gy, some mortality is likely (AFRRI 2003). The dose at which one half of exposed individuals would die (LD50) without medical support is estimated to be about 3.25 Gy in humans (AFRRI 2003). Doses of 1 to 8 Gy and higher are likely to be experienced by dozens to thousands of people during the scenarios mentioned above (Walelenko et al., 2003). At doses above about 8 Gy, injury to the GI system becomes serious and contributes to mortality (AFRRI 2003). The acute consequences of exposures between about 1 and 8 Gy are termed the "hematopoietic syndrome," while the acute effects after doses of about 8 to 20 Gy are known as the "GI syndrome," with effects after doses of about 20-30 Gy being gastrointestinal and cardiovascular damage with death occurring within 2-5 days (AFRRI 2003). The doses of radiation necessary to cause mortality will be lower in the event of deliberate exposure of civilian populations to pathogenic microorganisms. Combined use of radiological and biological weapons will result in a synergistic effect that will produce much higher rates of mortality than with either type of agent alone (Elliott et al., 2002).

[0022] In various methods provided herein, subjects are protected from injury or damage at the molecular level, the cellular level, the tissue level, the organ level, and/or the system or organism level. [0023] In various methods described herein, a composition comprising a radioprotective amount of delta-tocotrienol or a derivative thereof is administered before a subject is exposed to radiation, after the subject is exposed to radiation, or before and after the subject is exposed to radiation. In further aspects, a composition comprising a radioprotective amount of delta- tocotrienol or a derivative thereof is administered via a subcutaneous, oral, intravenous, or transdermal route of administration, or by insufflations or inhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows a delta-tocotrienol survival study at 8.75 Gy, with drug administered prior to irradiation. Male CD2F1 mice, 12 weeks of age and weighing approximately 27 g were used. The radiation source was Cobalt 60 at an in-house radiation facility. The dose rate was always 0.6 Gy/min. Total radiation dose varied between experiments. The drug was dissolved in Polyethylene glycol -400 (PEG-400) containing 5% Tween 80 as an emulsifying agent, and administered subcutaneously 24 hours prior to irradiation. Animals were monitored for 30 days postirradiation for survival. Survival in treated animals was 100%, as compared to less than 20% survival in controls receiving no tocotrienol. [0025] Figure 2 shows a delta-tocotrienol survival study at 8.75 Gy, with drug administered after irradiation. The experiment was carried out as described, and animals monitored for 30 days post-irradiation for survival. Survival was significantly enhanced in treated animals relative to control animals receiving no delta-tocotrienol. [0026] Figure 3 shows that PEG-400/tween 80 vehicle administered 24 h or 1 h prior to radiation did not support survival at a dose of 9.6 Gy. Thirty day survival was high when the drug was administered 12 or 24 h prior to radiation. Animals that received the drug 48 h prior to radiation did not show enhanced survival. Similarly, although administration of the drug 1 h prior to radiation was more effective than 48 h- treated group, the survival was significantly lower than that for animals that received the drug 12 or 24 h prior to radiation. This indicates that delta-tocotrienol may be working not only as a long-acting membrane-bound antioxidant, but also as a cellular modulator, possibly affecting the signal transduction pathway. Most antioxidants, such as the gold standard radioprotectant, amifostine, are effective only when given 30 min before or up to 4 h after radiation. [0027] Figure 4 shows a time course optimization for post-irradiation delta-tocotrienol administration. Animals were injected with 300 mg/kg of the drug at different time points with reference to radiation to study the beneficial effect of the drug if administered postirradiation. Results revealed that 2 hour appeared slightly superior, although the drug is effective up to 12 h after irradiation. These results suggest that delta-tocotrienol affords protection of immune cells from reactive oxygen species (ROS) dependent damage. One such type of ROS dependent damage relates to protein oxidation. Oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives (Stadtman, 2003). [0028] Figure 5 shows the effect of various doses of delta-tocotrienol, administered 24 h pre-irradiation, on 30 day survival. Animals that received 300 and 75 mg/kg dose exhibited 100% survival. For these experiments, the radiation dose was 9.25 Gy.

[0029] Figure 6 shows the effect of various doses of delta-tocotrienol administered 2 h postirradiation, on 30 day survival. Naϊve group represents animals that didn't receive either the vehicle or the drug. In this study the vehicle group showed higher than normally expected protection. Animals that received 150 or 300 mg/kg dose had higher survival than those animals that received 18.75, 37.5 or 75 mg/kg drug dose.

[0030] Figure 7 shows the results of the dose reduction factor (DRF) study in animals that received 150 mg/kg delta-tocotrienol 2 h postirradiation. Results clearly show that survival of the drug treatment resulted in a statistically significant increase over the vehicle group. Animals were subjected to various doses of radiation and monitored for 30 days postirradiation. A probit curve was developed using mortality data in each group. The DRF of 1.1 indicates significant improvement.

[0031] Figure 8 shows the results of the dose reduction factor study in animals that received 300 mg/kg delta-tocotrienol 24 h pre-irradiation. Results clearly show that survival of the drug treatment group resulted in a statistically significant increase over the vehicle group. Animals were subjected to various doses of radiation and monitored for 30 days postirradiation. A probit curve was developed using mortality data in each group. The DRF of 1.27 indicates significant improvement.

[0032] Figure 9 shows the cell count of white blood cells (A), red blood cells (B), monocytes (C), eosinphils (D), large unstained cells (E), basophils (F), reticulocytes (G), and platelets (H) in animals exposed to radiation and treated with a single 300 mg/kg dose of delta- tocotrienol. Significant results can be seen in the increase in white blood cells, monocytes, eosinphils, basophils, and platelet cells of the drug treated mice in comparison to that of the vehicle group.

DETAILED DESCRIPTION

[0033] Various publications, including issued patents, published patent applications, technical articles and scholarly articles are cited throughout the specification. Each of the cited publications is herein incorporated by reference in its entirety, and for all purposes. [0034] It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0035] Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

[0036] As used in this specification and the appended claims, the singular forms "a,"

"an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like. [0037] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. [0038] As used herein, "measure" or "determine" refers to any qualitative or quantitative determinations.

[0039] "Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, material, or composition, as described herein effective to achieve a particular biological result, such as, but not limited to, biological results disclosed, described, or exemplified herein, in a particular patient. The term

"radioprotective amount" is used herein to refer to an amount effective to treat radiation exposure in a subject.

[0040] As used herein, "treating" includes prevention, amelioration, alleviation, and/or elimination of one or more effects or symptoms of the condition being treated, as well as improvement in the overall well being of a patient, as measured by objective and/or subjective criteria. In some aspects, "treating" refers to reversing, attenuating, minimizing, suppressing, or halting undesirable or deleterious effects of, or effects from the progression of, a condition, including but not limited to, radiation exposure. In various aspects, treating a subject according to methods provided herein includes, but is not limited to, the prevention of radiation-induced injury or mortality in a subject, as determined by any means suitable in the art.

[0041] "Pharmaceutically acceptable" refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxico logical point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.

[0042] "Pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.

[0043] Except when noted, "subject" or "patient" are used interchangeably and refer to mammals such as humans and non-human primates, as well as companion, farm, or experimental animals such as rabbits, dogs, cats, rats, mice, horses, cows, pigs, and the like. Accordingly,

"subject" or "patient" as used herein means any mammalian patient or subject to which the compositions of the invention can be administered.

[0044] "Injury" refers to any physical harm, damage, degeneration, or trauma to any molecule, cell, tissue, or system in the body. [0045] As used herein, the term "vitamin E" refers to the compound d-α-tocopherol, and not to the entire tocol family.

[0046] "Tocol" refers generally to the family of tocopherols and tocotrienols.

[0047] "Tocotrienol" refers to a compound having the general structure:

As provided in the present disclosure, tocotrienols include both the naturally occurring (2R) and racemic forms.

[0048] In "α-tocotrienol," R¹, R², and R³ are each methyl. In "β-tocotrienol," R¹ and R³ are each methyl and R² is hydrogen. In "γ-tocotrienol," R² and R³ are methyl and R¹ is hydrogen. In "δ- tocotrienol," R¹ and R² are each hydrogen and R³ is methyl. Greek letter symbols α, β, γ, and δ are synonymous with alpha, beta, gamma and delta and can be used interchangeably unless otherwise noted (for example, α or alpha, β or beta, γ or gamma, δ or delta). [0049] "Derivatives" of tocotrienols, as referred to herein, include, without limitation, esters of tocotrienols, especially delta-tocotrienol, with organic monobasic or dibasic acids having from 1-30 carbon atoms, linear or branched, functionalized or not functionalized; examples include formate, acetate, proprionate, palmitate, succinate, maleate, citrate, and the like. "Derivatives" also include tocotrienol esters of the naturally-occurring amino acids and their salts, both with organic and inorganic acids, and esters of non-natural amino acids and their salts with both inorganic or organic acids. Examples of this class of derivative could include the hydrochloride of the N,N-dimethylglycine ester of delta-tocotrienol, or the citrate of the lysine ester of tocotrienol. "Derivatives" also include glycosides formed from tocotrienols and natural- and non-natural monosaccharides and disaccharides; examples of this class of derivative would be the alpha- or beta-D-glucosides of delta-tocotrienol. "Derivatives" also include inorganic esters of tocotrienols such as phosphates, sulfates, nitrates, and the like. [0050] The instant invention relates generally to the discovery that tocotrienols, and particularly delta-tocotrienol and derivatives thereof, are effective radioprotective agents with a high degree of efficacy and low toxicity. For example, it has been discovered in accordance with the present invention that delta-tocotrienol has a radioprotective effect when administered to irradiated mice, resulting in significantly enhanced survival and minimized tissue damage in irradiated animals compared to controls.

[0051] Literature studies have primarily focused on the radioprotective effects of the tocopherols, which are more readily available in pure form than tocotrienols. Structurally, tocotrienols differ from tocopherols by the presence of three trans double-bonds in the hydrocarbon tail. Without being limited to a particular theory, it is believed that unsaturations in the isoprenoid side-chain and the resulting shortened functional length of the tocotrienol molecule impart a unique conformation to tocotrienols that results in properties, including but not limited to membrane behavior, that are distinct from those of the tocopherols. Moreover, although tocotrienols have common side chains, the side chain characteristics differ based on the position of the methyl groups on the chroman ring. Thus, delta-tocotrienol has distinct functional properties from other tocotrienols, and is one of the most potent anti-cancer tocotrienol isoforms. [0052] Accordingly, provided herein are compositions and methods relating to the use of tocotrienols, and particularly delta-tocotrienol and derivatives thereof, for treating subjects for radiation exposure. In some embodiments, methods are provided herein for treating the effects of radiation exposure in a subject, including but not limited to radiation-induced tissue damage, by administering a radioprotective amount of delta-tocotrienol or a derivative thereof to the subject. The delta-tocotrienol, or derivative thereof can be administered as part of a composition, for example, as an admixture with a pharmaceutically acceptable carrier. In certain embodiments, compositions comprising delta-tocotrienol or its derivatives may further comprise one or more other members of the tocotrienol or tocopherol family or their derivatives, such as gamma tocotrienol.

[0053] Compositions comprising delta-tocotrienol or derivatives thereof can be prepared, stored, or administered as solid, semisolid, or liquid forms. Solid forms can be prepared according to any means suitable in the art. For example, capsules are prepared by mixing the tocotrienol composition with a suitable diluent and filling the proper amount of the mixture in capsules. Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Non-limiting examples of diluents include various types of starch, cellulose, crystalline cellulose, microcrystalline cellulose, lactose, fructose, sucrose, mannitol or other sugar alcohols, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Non- limiting examples of tablet binders include starches, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

[0054] A lubricant can be used in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. [0055] Tablets can be coated with sugar as a flavor and sealant, or with film- forming protecting agents to modify the dissolution properties of the tablet. The compounds can also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol, flavorants, and/or sweeteners in the formulation

[0056] Compositions described herein can be prepared as liquid formulations or solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. Such liquid forms include solutions, suspensions, syrups, slurries, and emulsions. Liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats or oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid). These preparations can contain, in addition to the tocotrienol, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. Compositions described herein can be in powder form for constitution with a suitable vehicle such as sterile water, saline solution, or alcohol, before use. Compositions described herein can also contain mucosal enhancers. [0057] In various aspects, methods provided herein comprise administering compositions orally, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, vaginally, bucally, and the like, or by inhalation or insufflation. In some embodiments, compositions are administered subcutaneously. In further embodiments, compositions are administered orally.

[0058] Compositions described herein can also be formulated for injection into a subject.

For injection, tocotrienol compositions can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer. Solutions can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Injection formulations can also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.

[0059] Compositions described herein can also be formulated in sustained release vehicles or depot preparations. Such long-acting formulations can be administered subcutaneously or by intramuscular injection. Thus, for example, compositions can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.

[0060] Administration of compositions according to methods provided herein can be by infusion or injection (intravenously, intramuscularly, intracutaneously, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and the like).

[0061] Compositions and methods provided herein can protect a subject from any injury or condition that is caused or exacerbated by non-ionizing or ionizing radiation. In various aspects, methods and compositions provided herein protect subjects from injuries or conditions at the molecular level (e.g., DNA mutagenesis or alteration, or chromosomal damage), the cellular level (e.g., apoptosis, or uncontrolled cell proliferation, including tumor formation and metastasis), the tissue level (e.g., tissue damage, including degeneration, atrophy, fibrosis, and necrosis), the organ level (e.g., organ failure), and/or the system or organism level (e.g., mortality).

[0062] In various embodiments, methods and compositions provided herein provide protection from ionizing radiation. Ionization is the result of the release of orbital electrons from atoms, and radiation that facilitates ionization is termed ionizing radiation. Ionizing radiation includes electromagnetic (X-radiation and gamma-radiation) and particulate (alpha irradiation, beta irradiation, and neutrons) radiation. Ionizing radiation can damage cells and tissues by damaging proteins and DNA, cross-linking biomolecules, and generating free radicals, among other things.

[0063] Exposure to a significant dose of ionizing radiation can also result in systemic effects. For example, high doses of radiation can induce radiation sickness, also referred to as Acute Radiation Syndrome (ARS). ARS is induced as a result of the effect of radiation on various systems of the body, including the hematopoietic system, digestive system, cardiovascular system, reproductive system, and the like. Bone marrow and circulating immune cells, such as leukocytes, are particularly sensitive to the effects of ionizing radiation. Radiation results in immunosuppression leading to opportunistic infection, including bacterial translocation from a damaged gastrointestinal system. In addition, neutropenia and thrombocytopenia are characteristics of exposure to high but acute dose of whole body radiation. [0064] In some embodiments, a radioprotective amount of a composition described herein provides a clinically significant decrease in tissue damage. The nature, extent, time course, and/or other aspects of radiation-induced tissue damage can be measured using various methods and clinical indicators known in the art. For example, in some aspects, the radioprotective effect of a composition described herein is assessed by measuring levels and/or recovery of hematological endpoints, such as lymphocyte, neutrophil, and/or platelet counts. In further aspects, a radioprotective effect can be assessed at the organ or system level by, for example, measuring the occurrence of opportunistic infection and/or bacterial translocation. In yet further aspects, a radioprotective effect can be assessed at the organism level by measuring survival rates, such as the thirty day survival in mice, which is a robust measurement of recovery from radiation.

[0065] The effective amount of a composition to be administered according to a method described herein can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, the dose of radiation received or anticipated, or the amount of time before or the amount of time elapsed since exposure to radiation. The appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques, the skilled and informed judgment of the practitioner, and other factors evident to those skilled in the art. Preferably, a therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity to the subject. [0066] Toxicity and therapeutic efficacy of agents or compounds can be assayed using standard pharmaceutical procedures in a variety of systems and environments, including cell-free environments, cellular environments (e.g., cell culture assays), multicellular environments (e.g., in tissues or other multicellular structures), and/or in vivo (e.g., in experimental animals), e.g., by determining the LD50 (the dose lethal to 50% of the population) and/or the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Agents or compositions which exhibit large therapeutic indices are included. In various aspects, a radioprotective agent used in methods and compositions provided herein have a therapeutic index of at least about 2, such as at least about 5, or at least about 10. [0067] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in the subject. The dosage of such agents or compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0068] In various aspects, compositions used in methods provided herein comprise a radioprotective agent, such as delta-tocotrienol or a derivative thereof, in a range of about 0.01% to about 90% of the dry matter weight of the composition. In some aspects, a radioprotective agent comprises up to about 50%, up to about 40%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, or up to about 10% of the dry matter weight of the composition. [0069] In some aspects, subjects are administered delta-tocotrienol in a daily dose range of about 0.01 mg/kg to about 300 mg/kg of the weight of the subject. The total daily dosage can be divided and administered in portions throughout the day, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more portions throughout the day. Portion-based administration can be at regular intervals, for example, every 0.5, 1, 2, 3, 4, 5, 6, or more hours, or at irregular intervals. The dose administered to the subject can also be measured in terms of total amount of tocotrienol or tocotrienol derivative administered per day. In some embodiments, delta-tocotrienol is administered once daily.

[0070] Compositions described herein can be co-administered with other agents that are known to have radioprotective qualities. Examples of such agents include, without limitation, amifostine, other tocopherols or tocotrienols, potassium iodide, 5-androstenediol, melatonin, aminothiols, selenium (including organoselenium, such as selenomethionine), curcumin, flavonoids, interleukin-1, and other radioprotective agents known in the art. In certain embodiments, compositions described herein can be co-administered with one or more other members of the tocotrienol or tocopherol family or their derivatives, such as gamma-tocotrienol. [0071] In some aspects, a composition comprising delta-tocotrienol or derivative thereof is administered from about 10 minutes to about 96 hours before radiation exposure. In some embodiments, compositions provided herein are administered less than 24 hours before radiation exposure. For example, compositions can be administered 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours, or less before radiation exposure. In some additional aspects, compositions are administered as a single dose, while in other aspects compositions are administered in multiple doses of the same or varying concentrations of delta-tocotrienol, or a derivative thereof. In some embodiments, delta-tocotrienol is administered 12 to 24 hours prior to radiation exposure. [0072] In some aspects, compositions are administered from about 1 minute to about 48 hours after radiation exposure. In some embodiments, compositions are administered less than 24 hours after radiation exposure. For example, compositions can be administered 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, or less hours after exposure to radiation. In some particularly embodiments, compositions are administered less than 2 hours after exposure to radiation. In some aspects, compositions are administered as a single dose, while in other aspects compositions are administered in multiple doses of the same or varying concentrations of delta- tocotrienol, or a derivative thereof.

[0073] Methods and compositions provided herein find wide use in various settings, including anywhere where radiation exposure is likely to be encountered. Such settings include, for example, a solar radiation event, such as those potentially experienced by astronauts and aviation personnel that make high altitude trips; use in conjunction with radiation-based medical diagnostics and therapies, nuclear power plant facilities, food radiation plants, and in the cleanup of radiation dump sites and accidents such as Chernobyl, Ukraine, Tokaimura, Japan, and Three- Mile Island, USA; use by the military in the event of a nuclear radiation event, as well as by civilian civil defense personnel in response to a terrorist radiation event such as a dirty bomb, and civilians exposed to such radiation; and use in reducing the toxic effects of inhaled radionuclides and in reducing toxicity from radiation produced by electronic devices such as cellular telephones.

[0074] The above disclosure generally describes the present invention. The following exemplary aspects of specific aspects for carrying out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXEMPLARY ASPECTS [0075] Example 1 - General Experimental Protocols

[0076] Male CD2F1 mice were used throughout this study. Mice 12-15 weeks of age, weighing approximately 27 g, were purchased from Harlan Labs, IN., held in quarantine for 10 days, tested for Pseudomonas, and maintained in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facility of the Armed Forces Radiobiology Research Institute (AFRRI) before use. Animals were kept in plastic micro-isolator cages (eight per cage) on hardwood-chip contact bedding with free access to food and acidified water, in an air-conditioned room with 12 changes of air per hour. All animal procedures were completed according to the protocol approved by the Institutional Animal Care and Use Committee at AFRRI. Research was conducted according to the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, US National Academy of Sciences.

[0077] Variation in response to radiation has been noted in different strains of mice. For example, CD2F1 and C3H/HeN mice have been demonstrated to be robust and hence have been chosen for use in these studies. The survival response to radiation depends not only on the dose of radiation but to some extent on the health of the specific batch of animals, and age of the animals. These variations are minimized by periodically evaluating a positive control, such as 5- androstenediol (5-AED) as a positive control. The positive controls generally show 90-100% survival under the experimental conditions. [0078] Irradiation. Irradiation of mice was carried out in well-ventilated Plexiglas boxes

(eight per box) with bilateral irradiation in the Cobalt 60 gamma radiation facility of AFRRI at the dose rate of 0.6 Gy/minute. After irradiation, mice were returned to their original cages with free access to food and water. Various doses of radiation were used as indicated, the doses having been selected to test the effect of tocols on a wide range of radiation doses. Doses of 8.75 Gy, 9.25 Gy, and 9.6 Gy were used in preliminary studies. These doses are significantly higher than the range of possible radiation threat scenarios that can be encountered by soldiers, first responders, and civilian radiation victims. Human lethality radiation doses are much less, i.e. the range of 2 to 4 Gy.

[0079] Blood Collection. Blood was drawn from caudal vena cava or by cardiac puncture in mice anesthetized by isofluorane inhalation. A small jar with a lid and removable metal platform was used for isofluorane anesthesia. The chamber was saturated by wetting a small piece of gauze with isofluorane. Individual mice were introduced into the jar and the animal rested on the metal platform without directly coming in contact with isofluorane. Generally the anesthetic effect occurred within 1-3 min after introduction of a given animal into the jar. The animal was then removed from the jar and its nose positioned in a syringe case with isofluorane soaked gauze for anesthetic maintenance. Blood was collected either via percutaneous cardiac puncture or by laparotomy, with blood collected from the caudal vena cava. Animals were euthanized by cervical dislocation immediately after blood collection. [0080] Statistical Methods. Survival response in vivo after drug treatment and irradiation was used to obtain cumulative survival days of irradiated mice, and analyzed by statistical methods associated with Kaplan-Meier product limit survival curves. The data were analyzed using Mantel-Cox log rank statistics for significant difference between the survival curves obtained with various tocols, tocol doses, and vehicle -treated control groups. All mice under test were assigned randomly to various treatment groups.

[0081] Depending on the nature of a given experiment different statistical techniques were required and applied. The generic experimental design was constructed in order to identify an expected probability of 50% or better survival probability in the drug-treatment groups over the expected low survival rates seen in the vehicle-control groups. Student's T tests, Chi-square or Fisher's Exact tests were used for these analyses.

[0082] Example 2 - Delta-Tocotrienol and its Derivatives

[0083] Target purity for these materials is a minimum 95% assay, with the remaining impurities being other tocotrienols and/or plant triglycerides. Neither delta-tocotrienol nor its succinate esters are commercially available at this purity level, however delta-tocotrienol can be extracted from natural rice bran, annatto or palm oils, using standard chemical methods. Various derivatives can be prepared from delta-tocotrienol using methods known in the chemical art. [0084] For the preparation of d-delta-tocotrienol, an initial enrichment to ca. 50-60% assay by open column chromatography is followed by conversion to the acetate esters. This derivitization allows chromatographic separation of tocotrienol acetate from otherwise inseparable sterols and carotenoids. The resulting ca. 80% pure delta-tocotrienol acetate is then saponified and the crude tocotrienol is enriched to about 95% purity by normal or reverse-phase chromatography (applicable to small scales, < ca. 1 g) or (more conveniently on larger scales) by conversion to a crystalline derivative such as the palmitate, stearate, or 4-phenylbenzoate, recrystallization, saponification, and final polishing by chromatography. In addition, supercritical fluid chromatography or simulated moving bed chromatography, either alone or in combination with the techniques above, can also be used in the purification/separation of delta-tocotrienol. [0085] A. Succinate ester of delta-tocotrienol

[0086] Delta-tocotrienol can be converted to the succinate ester by reaction with succinic anhydride in pyridine under 4-dimethylaminopyridine catalysis. Alternatively, it is possible to convert the tocol to the succinate ester using reaction with succinic anhydride catalyzed by potassium acetate in the absence of solvent at temperatures in the range of 50-150 degrees C, and preferably at about 90-100 degrees C. The succinates are characterized and assayed using standard HPLC and NMR techniques. Other derivatives of delta-tocotrienol (i.e. other esters and glycosides) can be prepared using analogous methodologies to well-established published procedures for other tocol derivatives, such as those for alpha tocopherol. [0087] B. Glycosides of delta-tocotrienol

[0088] The compound d-delta-tocotrienyl-beta-D-glucoside may be prepared by reaction of delta-tocotrienol with acetobromoglucose (conveniently used in molar excess compared to the tocotrienol) in the presence of a suitable solvent such as dichloromethane, ether, tetrahydrofuran, and the like, and in the presence of a molar equivalent amount of a strong base such as methanolic potassium or sodium hydroxide. This reaction will provide the tetraacetate of d- delta-tocotrienyl-beta-D-glucoside; this substance may be deacetylated without isolation or purification by addition, after a suitable reaction time, of excess methanolic potassium or sodium hydroxide to provide the title compound in reasonable yield and purity. It is understood that the preparation of the title compound is not limited to the method herein described, but may achieved through other methods and processes of reaction and purification which are well known to those conversant with the art of glycoside synthesis. The title compound is a glassy solid which may be dispersed in water if desired.

[0089] C. Hydrochloride of d-delta-tocotrienyl-N^-dimethylglycinate

[0090] The title compound may be conveniently prepared from delta-tocotrienol by reaction with N,N-dimethylglycine hydrochloride in the presence of a suitable solvent such as anhydrous pyridine in the presence of a reagent known to facilitate ester coupling reactions, such as N,N-dicyclohexylcarbodiimide, diisopropylcarbodiimide, carbonyldiimidazole, and the like. After a suitable reaction time, the by-product dicyclohexylurea is removed and the desired product purified by chromatography or other suitable method, and treated with HCl to provide the title hydrochloride salt as a gummy solid which is dispersible in water. [0091] Example 3 - Determination of 30-Day Survival Rates in Mice Exposed to

Gamma Radiation

[0092] It is accepted in the art that 30 day mortality can be taken as good index of radioprotection. Previous studies have shown that alpha tocopherol, and to a greater extent gamma tocotrienol, protect from radiation lethality when administered subcutaneously (SC) at a dose of 400 mg/kg 24 h before irradiation at 10.5 Gy. To evaluate the radioprotective properties of delta tocotreinol, similar 30 day survival studies were performed with mice injected with this tocol prior to radiation exposure. Although delta-tocotrienol, like other analogs of vitamin E, have general antioxidant properties, it has some unique characteristics, such as inhibition of cholesterol synthesis, similar to statins. Mass spectroscopy studies also suggest that delta- tocotrienol, unlike tocopherols, has a circular structure that allows it to penetrate deeper in to the hydrophilic region of the membrane and thus increase its efficacy in scavenging radicals, such as hydroxyl radicals, generated during radiation exposure. Further, its location in the membrane may facilitate regulation of signal transduction pathways.

[0093] Mice were administered 0.1 ml of the delta-tocotrienol (or vehicle control) dispersion subcutaneously (SC) 24 hours before or 6 hours after irradiation at 8.75 Gy. Sixteen mice were used each for the tocol and the control. Irradiated mice were returned to their original cages and monitored daily for survival and weight loss/gain for 30 days. The results are presented in Figure 1 (24 h pre-irradiation) and Figure 2 (6 h post-irradiation). Figure 1 shows that, remarkably, all mice receiving delta-tocotrienol 24 hours pre-irradiation survived through 30 days. Control mice began to die around day 9, with fewer than 20% of the mice alive at day 30. Figure 2 shows that greater than 80% of mice receiving delta-tocotrienol at 6 hours post- irradiation survived after 30 days. As shown for the pre-irradiation tests, control animals receiving the vehicle began to die after about a week, with significant mortality after two weeks, and fewer than 20% survival at day 30. The percent of animals surviving as a result of tocol pretreatment is considered as a parameter for radioprotective efficacy. [0094] The above-described experiments can be repeated with derivatives of delta- tocotrienol to determine if the derivatives provide even better radioprotective efficacy relative to the parent compound. Previous studies have indicated that orally administered alpha tocopherol succinate is a more effective radioprotectant than its parent alpha tocopherol. We thus hypothesize that analogous derivatives of delta-tocotrienol will likewise exhibit good oral radioprotective activity.

[0095] Example 4 - Determination of Optimum Effective Dose of Delta-Tocotrienol

[0096] In the following study, the optimum dose of delta-tocotrienol and its succinate ester derivatives required for radioprotection was determined. This was accomplished by measuring 30-day survival rate for mice at a series of drug dose levels.

[0097] For these experiments, varying doses of delta-tocotrienol were administered to the mice 24 hours prior to irradiation and 2 hours after irradiation. Mice were irradiated at 9.25 Gy, and monitored for weight loss/gain and survival for 30 days. The following doses were administered to mice in both pre- and post- irradiation groups: 18.75 mg/kg, 37.5 mg/kg, 75 mg/kg, 150 mg/kg, and 300 mg/kg. [0098] The results of the foregoing experiments are shown in Figures 5 and 6. Figure 5 demonstrates the optimized dosing for mice receiving delta-tocotrienol 24 hours prior to radiation exposure. The data show that 300 mg/kg and 75 mg/kg provide the highest protective efficacy, with 100% of mice surviving at day 30. Interestingly, 37.5 mg/kg provided the next highest protective efficacy, with greater than 90% of the mice surviving at day 30. The level of survival was higher, although not significantly, than 150 mg/kg, which demonstrated between 80 and 90% survival between 28 and 30 days after irradiation. 18.75 mg/kg was modestly effective, with approximately 70% of mice receiving this dose surviving at day 30. In these experiments, approximately 40% of control mice were also alive at day 30. This is likely due to batch variation in the mice, which would have an impact on overall survival.

[0099] Figure 6 shows the optimized dosing for mice receiving delta-tocotrienol 2 hours after radiation exposure. The data show that 150 mg/kg and 300 mg/kg of delta-tocotrienol provide the highest rate of survival after 30 days, relative to the other doses evaluated. Fifty percent of mice receiving either of these doses were alive after 30 days. Interestingly, between these two doses, 150 mg/kg showed an apparently higher protective efficacy between the second and third week, with a greater number of mice alive during this period relative the number of mice that received 300 mg/kg. The doses 75 mg/kg and 37.5 mg/kg demonstrated approximately 40% survival at day 30, with the higher dose showing slightly higher and the lower dose showing slightly lower than 40% survival. The 18.75 mg/kg dose demonstrated the least protective efficacy, with approximately 30% of mice alive at day 30. It is noted that in all groups, mice began to die between the first and second week.

[0100] Example 5 - Determination of Optimum Time of Effectiveness for Delta-

Tocotrienol Administration

[0101] The following study defines the window of effectiveness, both pre-and post- irradiation, for delta-tocotrienol. The relationship between time of administration and 30-day survival of mice treated with delta-tocotrienol at the two effective doses was evaluated. [0102] The optimum pre-irradiation time of administration of delta-tocotrienol for

CD2F1 mice was determined. Experimental groups of 16 mice each received SC injection of 75 mg per kg body weight of delta-tocotrienol at time points of 48 hours, 24 hours, 12 hours, and 1 hour prior to radiation exposure. Control mice were administered an equivalent volume of a vehicle only formulation at time points of 24 hours and 1 hour prior to irradiation. Mice were irradiated at a radiation dose of 9.6 Gy (0.6 Gy/min), and monitored for weight loss/gain and survival for 30 days. The results, shown in Figure 3, demonstrate that, at three weeks post- irradiation, mice receiving delta-tocotrienol at 12 and 24 hours pre-irradiation provided the highest level of survival among the time variables investigated. There was no difference in survival rate at 12 and 24 hour administration. In contrast, survival of mice receiving the tocol at 48 hours and 1 hour pre-irradiation more closely paralleled that of the vehicle control mice, in that survival began to drop off between weeks one and two. Interestingly, even one hour pre- irradiation administration of delta-tocotrienol demonstrated some relative protective efficacy, as around 30% of mice in this group were still alive on day 21.

[0103] The optimum post-irradiation time of administration of delta-tocotrienol for

CD2F1 mice was also determined. Experimental groups of 16 mice each received SC injection of 300 mg per kg body weight of delta-tocotrienol at time points of 2 hours, 6 hours, 12 hours, and 24 hours after radiation exposure. Control mice were administered an equivalent volume of a vehicle only formulation at 2 hours after irradiation. Mice were irradiated at a radiation dose of 9.25 Gy (0.6 Gy/min), and monitored for weight loss/gain and survival for 30 days. The results are shown in Figure 4.

[0104] The results demonstrate that administration of 300 mg/kg delta-tocotrienol at 2 hours post-irradiation provided the most protective efficacy, as greater than 70% of mice survived at day 30. Also providing good protective efficacy, was administration of the tocol at six and 12 hours post-irradiation, with greater than 60% of such mice alive at day 30. In contrast, 24 hour post irradiation administration of delta-tocotrienol was not effective, with such mice closely paralleling vehicle control mice, and with fewer than 10% survival after 30 days. The effectiveness of the drug is a function of pharmacokinetics and functional enhancement of specific tissues. Although it has been observed that 24 hour time point before radiation is useful, additional time points described above may provide optimum dose for radioprotection. In some instances additional time points were chosen, and pharmacokinetic data was obtained to support the selection of time points. [0105] Example 6 - Determination of Dose Reduction Factor

[0106] The dose reduction factor (DRF) is the accepted parameter that allows quantitative comparison of anti-radiation agents. A DRF compares survival rates with and without the agent at optimal dose, and it is the ratio of the radiation LD50 with and without the agent. The DRF is specific for animal type, radiation dose and rate, and measured outcome. Since the various tocols are known to differ in blood and tissue deposition, the DRFs for delta- tocotrienol for both hematopoietic and gastrointestinal protection was determined. DRF enables benchmarking of delta-toco trienol radioprotective effectiveness relative to other known agents. [0107] Six radiation doses near 10.5 Gy were selected. These doses were selected in such a way that the lowest radiation dose will provide approximately 100% survival and the highest dose 100% lethality. In between these two extremes, four doses were selected that were expected to provide varying degrees of protection. Similarly, another set of six radiation doses were selected for the vehicle control. Some radiation doses overlapped between vehicle and delta- tocotrienol groups.

[0108] All groups were administered 0.1 ml of the vehicle or 0.1 ml of the delta- tocotrienol formulation SC. Mice were administered 150 mg/kg of delta-tocotrienol at 2 hours post radiation exposure (Figure 7) or 300 mg/kg of delta-tocotrienol at 24 hours pre -radiation exposure (Figure 8). Mice were irradiated at the predetermined doses of radiation at a dose rate of 0.6 Gy/min at the experimentally determined optimum time. The protective efficacy at each radiation dose was determined by the percent survival of mice 30 days post-irradiation. [0109] The results of these experiments are shown in Figures 7 and 8. Probit analysis of the data for vehicle and tocol was carried out by plotting percent survival on the ordinate and radiation doses on the abscissa. From the probit analysis, LD50/30 (lethal dose of radiation that results in the lethality of 50% of the exposed animals in 30 days) radiation dose was calculated for vehicle and tocol. From these data, the DRF was calculated according to the following formula:

[0110] Dose Reduction Factor = LD50/30 Radiation dose for tocol ÷ LD50/30 Radiation dose for vehicle. The DRF for delta-tocotrienol was calculated to be 1.10 for 150 mg/kg of delta- tocotrienol at 2 hours post radiation exposure and 1.27 for 300 mg/kg of delta-tocotrienol at 24 hours pre -radiation exposure. [0111] The dose reduction factor studies described above are straight forward. However, for such studies, factors such as the health and age of animals, may change, in some cases dramatically, the survival profiles. The probit curves may become too steep, indicating a greater mortality even at lower doses of radiation and since the DRF probit curve depends on 2-3 radiation doses that have significant survival. It is also possible the drug (although non-toxic) may have synergistic effect with radiation and in this case the curve will flatten out and hence may not be parallel to the untreated irradiated group. To minimize such problems, the LD50/30 dose was used.

[0112] Example 7 - Effects of Delta-Tocotrienol on Hematological Recovery

[0113] In this study, enhancement of immune recovery was shown in mice treated with a single dose of delta-tocotrienol.

[0114] Figure 9 shows the cell count of white blood cells (A), red blood cells (B), monocytes (C), eosinphils (D), large unstained cells (E), basophils (F), reticulocytes (G), and platelets (H) from 14 week old CD2F1 mice exposed to 7.0 Gy gamma radiation from a cobalt 60 source at a dose rate of 0.60 Gy/min and dosed once subcutaneous Iy with either 0.1 ml 95% PEG-400/5% Tween vehicle (fuchsia) or 300 mg/kg DT3 (blue). Blood was drawn 0, 3, 7, 10, 14, 21, 28 days post irradiation. As shown in Figure 9, significant results can be seen in the increase in white blood cells, monocytes, eosinphils, basophils, and platelet cells of the drug treated mice in comparison to that of the vehicle group.

[0115] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[0116] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. REFERENCES:

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Claims

What is Claimed:

1. A method for treating the effects of radiation exposure in a subject, comprising administering to the subject a composition comprising a radioprotective amount of delta- tocotrienol or a derivative thereof.

2. The method of claim 1 , wherein the radiation is ionizing radiation.

3. The method of claim 2, wherein the ionizing radiation is alpha radiation, beta radiation, gamma radiation, X-radiation, ultraviolet radiation, or neutrons.

4. The method of claim 1 , wherein the composition is administered before the subject is exposed to radiation.

5. The method of claim 1, wherein the composition is administered after the subject is exposed to radiation.

6. The method of claim 1, wherein the composition is administered before and after the subject is exposed to radiation.

7. The method of claim 1, wherein the composition is administered subcutaneously.

8. The method of claim 1 , wherein the composition is administered orally.

9. The method of claim 1, wherein the method significantly enhances subject survivability.

10. The method of claim 1, wherein the derivative is the succinate ester of delta- tocotrienol.

11. The method of claim 1 , wherein the derivative is the salt of an amino acid ester of delta-tocotrienol.

12. The method of claim 1 , wherein the derivative is the hydrochloride of delta- tocotrienol-N,N-dimethylglycinate.

13. The method of claim 1, wherein the derivative is a glycoside formed from a monosaccharide or disaccharide and delta-tocotrienol.

14. The method of claim 1, wherein the derivative is delta-tocotrienyl-beta-D- glucoside.

15. The method of claim 1 , wherein the composition further comprises one or more other members of the tocotrienol or tocopherol family or their derivatives.

16. The method of claim 1, wherein the composition further comprises gamma tocotrienol.

17. The method of claim 1, further comprising administering to a subject one or more agents with known radioprotective qualities.

18. The method of claim 1 , further comprising administering to a subject one or more of amifostine, other tocopherols or tocotrienols, potassium iodide, 5-androstenediol, melatonin, aminothiols, selenium (including organoselenium, such as selenomethionine), curcumin, flavonoids, and interleukin-1.

19. A method for reducing the risk for radiation-induced mortality comprising administering to a subject that has been exposed to radiation or is at risk of exposure to radiation a radioprotective amount of a composition comprising delta-tocotrienol or a derivative thereof.

20. The method of claim 19, wherein the radiation is ionizing radiation.

21. The method of claim 20, wherein the ionizing radiation is alpha radiation, beta radiation, gamma radiation, X-radiation, ultraviolet radiation, or neutrons.

22. The method of claim 19, wherein the composition is administered subcutaneously.

23. The method of claim 19, wherein the composition is administered orally.

24. The method of claim 19, wherein the derivative is the succinate ester of delta- tocotrienol.

25. The method of claim 19, wherein the derivative is the salt of an amino acid ester of delta-tocotrienol.

26. The method of claim 19, wherein the derivative is the hydrochloride of delta- tocotrienol-N,N-dimethylglycinate.

27. The method of claim 19, wherein the derivative is a glycoside formed from a monosaccharide or disaccharide and delta-tocotrienol.

28. The method of claim 19, wherein the derivative is delta-tocotrienyl-beta-D- glucoside.

29. The method of claim 19, wherein the composition further comprises one or more other members of the tocotrienol or tocopherol family or their derivatives.

30. The method of claim 19, wherein the composition further comprises gamma tocotrienol.

31. The method of claim 19, further comprising administering to a subject one or more agents with known radioprotective qualities.

32. The method of claim 19, further comprising administering to a subject one or more of amifostine, other tocopherols or tocotrienols, potassium iodide, 5-androstenediol, melatonin, aminothiols, selenium (including organoselenium, such as selenomethionine), curcumin, flavonoids, and interleukin-1.