Methods and compositions for radiation protection using RRx-001

Abstract

Provided herein are therapeutic methods, kits, and pharmaceutical compositions for protecting a subject from radiation using a therapeutic agent selected from the group consisting of RRx-001 and pharmaceutically acceptable salts thereof. An exemplary method of treatment includes administering RRx-001 to the subject prior to exposure of the subject to the radiation to protect the subject from radiation, such as ionizing radiation containing alpha-rays, beta-rays, gamma-rays, neutron radiation, or a combination thereof.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980016865.8 (application date: January 8, 2019, invention name: Methods and compositions for radiation protection using RRx-001).

[0002] priority

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 614,592, filed January 8, 2018, and U.S. Provisional Application Serial No. 62 / 737,093, filed September 26, 2018, the contents of each of which are incorporated by reference in their entirety.

[0004] Federal Funding Statement

[0005] This invention was made with government support under Grant No. RAB2436118 awarded by the Armed Forces Radiobiology Research Institute (AFRRI). The U.S. Government has certain rights in this invention. Technical Field

[0006] Methods of treatment, kits, and pharmaceutical compositions for protecting a subject from radiation using a therapeutic agent selected from 2-bromo-1-(3,3-dinitroazetidin-1-yl)ethan-1-one (RRx-001) and pharmaceutically acceptable salts thereof are described, wherein an exemplary method of treatment comprises administering RRx-001 to a subject prior to exposing the subject to radiation to protect the subject from radiation, such as ionizing radiation containing alpha rays, beta rays, gamma rays, neutron radiation, or a combination thereof. Background Art

[0007] Ionizing radiation causes damage to normal tissues, ranging from genetic mutations to cell death. The deleterious effects of ionizing radiation on normal tissues are a major concern for military and emergency responders to nuclear accidents and terrorist incidents due to the risk of acute and delayed radiation injury. Furthermore, radiation protection is a critical issue in cancer treatment. Despite significant technological advances in radiation delivery in recent years, normal tissue toxicity remains a major dose-limiting factor in therapeutic radiology.

[0008] Developing safer and more effective radiation protection technology is important for protecting civilians and military personnel from unexpected radiation exposure. This radiation may be caused by nuclear power sources, nuclear emergencies, medical instruments that emit high levels of radiation, exposure to sunlight and other sources that have not yet been filtered through each layer of the earth's atmosphere. It is well known that radiation exposure may cause cancer (such as leukemia). High doses of radiation may also be fatal to human and animal subjects. For these reasons, there is a need for safer and more effective radiation protection technology.

[0009] RRx-001 (also known as ABDNAZ), whose chemical name is 2-bromo-1-(3,3-dinitroazetidin-1-yl)ethan-1-one, is a small cyclic nitro compound that has previously been found to induce numerous enzymatic and epigenetic changes in tumor cells. RRx-001 has been used clinically as a chemo- and radiosensitizer in combination with chemotherapy and / or radiation and is described, for example, in International Patent Application Publication No. WO 2007 / 022225, which describes various compounds and their use in treating medical disorders such as cancer. Exemplary scientific publications describing the benefits observed in human clinical trials evaluating the efficacy of RRx-001 in treating patients with cancer include Carter et al. in Respir. Med. Case Rep. (2016) Vol. 18, pp. 62-65; Kim et al. in Transl. Oncol. (2016) Vol. 9(2), pp. 108-113; and Reid et al. in Case Rep. Oncol. (2014) Vol. 7(1), pp. 79-85. Summary of the Invention

[0010] The present disclosure provides methods of treating, kits, and pharmaceutical compositions for protecting a subject from radiation using a therapeutic agent selected from RRx-001 and pharmaceutically acceptable salts thereof, wherein an exemplary method of treating comprises administering RRx-001 to a subject prior to exposure to radiation to protect the subject from radiation, such as ionizing radiation comprising alpha rays, beta rays, gamma rays, neutron radiation, or a combination thereof. The methods of treating are particularly useful for protecting civilians and military personnel from unintended radiation exposure, such as protecting first responders to nuclear emergencies, cosmic radiation associated with expanded space habitats or travel, or other hazards involving hazardous radiation levels. In addition, the methods of treating can be used in combination with radiation therapy for cancer to protect normal tissue. The therapeutic agent is ideally administered to the subject at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before the subject is exposed to radiation that could cause harm to the subject, and ideally provides protection against the harmful effects of radiation for at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. The therapeutic agent is ideally administered to the subject by a procedure that minimizes the pain experienced by the patient due to receiving RRx-001, such as by slowly administering RRx-001 or by administering RRx-001 after mixing with blood to reduce the pain experienced by the patient. The invention, having been generally described, is explained in more detail in the following aspects and embodiments and detailed description.

[0011] Thus, one aspect of the present disclosure provides a method for treating a subject in need of radiation protection. The method comprises administering to a subject in need thereof an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof by a route selected from parenteral administration, oral administration, and topical administration, thereby protecting the subject from radiation for at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. The therapeutic agent is ideally RRx-001, which is ideally administered to the subject by intravenous injection, intraperitoneal injection, subcutaneous injection, oral administration, or topical administration. Ideally, at least one dose of the therapeutic agent is administered to the subject prior to exposure to radiation.

[0012] Another aspect of the present disclosure provides a method for reducing the damage of radiation exposure to a subject. The method comprises administering to a subject in need thereof an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof by a route selected from parenteral administration, oral administration, and topical administration, thereby reducing the damage of radiation exposure to the subject for at least 6 hours. The therapeutic agent is ideally RRx-001, and is ideally administered to the subject by intravenous injection, intraperitoneal injection, subcutaneous injection, oral administration, or topical administration. Ideally, at least one dose of the therapeutic agent is administered to the subject prior to exposure to radiation.

[0013] Another aspect of the present disclosure provides a method for protecting a material (such as an isolated cell, tissue, or organ) from the damaging effects of radiation. The method comprises exposing the biomaterial to an effective amount of a therapeutic agent selected from RRx-001 and pharmaceutically acceptable salts thereof, thereby protecting the biomaterial from the damaging effects of radiation for a period of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. The therapeutic agent is ideally RRx-001. Ideally, the biomaterial is exposed to at least one dose of the therapeutic agent prior to exposure to the radiation.

[0014] The therapeutic agents described herein can be formulated into pharmaceutical compositions. As further described herein, one or more of the foregoing can be included in a kit with instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A Shown is the effect of RRx-001 treatment (10 mg / kg) on survival advantage after radiation (9.35 Gy (LD70 / 30) at 0.6 Gy / min) compared to irradiated vehicle controls. Data are presented as 30-day survival Kaplan Meyer curves. N=24 / group; **p<0.005. Figure 1B A scatter plot of survival time by treatment group is provided. The mean ± standard error for the vehicle and RRx-001 groups was 20.2 ± 1.6 and 27.2 ± 1.1, respectively. ***p < 0.0006.

[0016] Figure 2 Shown is the effect of RRx-001 treatment on bone marrow recovery after a sublethal dose of TBI (7 Gy at 0.6 Gy / min) compared to irradiated vehicle controls. Sham = no irradiation control. N = 6 / group / day; **p < 0.005; Error bars are mean ± SEM.

[0017] Figure 3Shown is the effect of RRx-001 treatment on the number of colony forming units generated from mouse bone marrow following a sublethal dose of TBI (7 Gy at 0.6 Gy / min) compared to irradiated vehicle controls. Three mice / group / day were combined into one sample and plated in triplicate.

[0018] Figure 4 The radioprotective effect of RRx-001 treatment on blood cells following a sublethal dose of TBI (7 Gy at 0.6 Gy / min) is shown compared to irradiated vehicle controls. White blood cell counts ( Figure 4 A), absolute neutrophil count ( Figure 4 B) Lymphocyte count ( Figure 4 C), monocyte count ( Figure 4 D) Reticulocyte count ( Figure 4 E) and hematocrit % (the percentage of red blood cells by volume relative to the volume of whole blood) ( Figure 4 F) Data. N = 4-6 mice / group / day; *p < 0.05; error bars are means with 95% CI.

[0019] Figure 5 Representative sternal bone marrow micrographs are shown, showing increased bone marrow recovery in the RRx-001 + radiation group relative to the irradiated vehicle control at days 7 and 14. All glass slides were stained with hematoxylin and eosin (H&E). Dark gray staining (center bar) is bone marrow (center gray horizontal bar), white is adipocytes, and light gray staining (top and bottom horizontal bars) is muscle.

[0020] Figure 6 shows the antioxidant pathway ( Figure 6A ) or metabolic stress response ( Figure 6B ) Exemplary potential mechanisms of radiation protection by RRx-001.

[0021] Figure 7 Shown is the in vitro model experimental design used to characterize the radioprotective effects of RRx-001.

[0022] Figure 8 Western blot of genes harboring antioxidant response elements (AREs) following irradiation of human mesenchymal stem cells treated with RRx-001 or vehicle control is shown ( Figure 8 A) and quantification of protein expression ( Figure 8 B- Figure 8 E). Heme oxygenase-1 (HO-1) ( Figure 8 B), quinine oxidoreductase-1 (NQO-1) ( Figure 8 C), superoxide dismutase-1 (SOD-1) ( Figure 8D) and superoxide dismutase 2 (SOD-2) ( Figure 8 Data from E). β-actin expression was used as a control.

[0023] Figure 9 Shown are the effects of RRx-001 on superoxide dismutase activity in irradiated human mesenchymal stem cells compared to vehicle control.

[0024] Figure 10 A and Figure 10 B shows a Western blot showing the expression of U937 mononuclear cell fraction ( Figure 10 A) and U937 macrophage fraction ( Figure 10 B) Protein expression of HO-1, NQO-1, and SOD-1 in the culture medium: Lane 1: vehicle / no irradiation; Lane 2: vehicle / 10 Gy irradiation; Lane 3: RRx-001 (3 μM) / no irradiation; Lane 4: RRx-001 (3 μM) / 5 Gy irradiation. Figure 10 C- Figure 10 H shows quantification of protein expression of each gene (HO-1, NQO-1, and SOD-1) expressed as fold change relative to vehicle / non-irradiated control U937 monocyte fraction and U937 macrophage fraction.

[0025] Figure 11 A and Figure 11 B shows a Western blot showing the THP-1 mononuclear cell fraction ( Figure 11 A) and THP-1 macrophage fraction ( Figure 11 B) Protein expression of HO-1, NQO-1, and SOD-1 in the culture medium: Lane 1: vehicle / no irradiation; Lane 2: vehicle / 5 Gy irradiation; Lane 3: RRx-001 (3 μM) / no irradiation; Lane 4: RRx-001 (3 μM) / 5 Gy irradiation. Figure 11 C- Figure 11 H shows quantification of protein expression of each gene (HO-1, NQO-1, and SOD-1) expressed as fold change relative to vehicle / non-irradiated control THP-1 monocyte fraction and THP-1 macrophage fraction.

[0026] Figure 12 A shows dot blot expression of various cytokines and inflammatory regulators in irradiated THP-1 monocytes or THP-1 macrophages (5 Gy) treated with RRx-001 (3 μM) or vehicle control in THP-1 monocyte fraction and THP-1 macrophage fraction. Figure 12 B- Figure 12E shows quantification of cytokine expression of selected genes in THP-1 monocyte fraction and THP-1 macrophage fraction, expressed as fold change relative to vehicle control.

[0027] FIG. 13 shows exemplary data on the effect of RRx-001 on reducing mucositis in a hamster model. Figure 13A Mean daily mucositis scores for the twice-weekly dosing groups are presented. Figure 13B The mean daily mucositis scores for the weekly dosing groups are presented. The mean group mucositis scores were calculated for each day of evaluation.

[0028] FIG. 14 shows exemplary data on the effect of RRx-001 on reducing mucositis in a hamster model. Figure 14A Data are presented for the twice-weekly dosing group for the percentage of days with a mucositis score ≥ 3 over the entire study period. Figure 14B Provide once a week administration group during the whole study period mucositis score ≥ 3 days percentage data.In order to check the level of clinically significant mucositis (as defined by the occurrence of open ulcers (score ≥ 3)), the total number of days that animals show elevated scores is added together and expressed as the percentage of the total number of days scored during the whole study period (day 6-day 28).The statistical significance compared with the vehicle control was evaluated using the chi-square test.***p < 0.001.

[0029] Figure 15 Exemplary data comparing daily mucositis scores are shown (dosing group days -4, -1, 1, 4, 7, 11, 14, 18, 21, and 25). Figure 15 A presents data for the twice-weekly dosing group. Figure 15 B provides the data of weekly administration group.The Mann-Whitney rank sum test is used to determine the significance of the group differences observed in daily mucositis scores.This nonparametric statistic is applicable to the visual mucositis rating scale.The p value calculated each time is shown.Compared with the vehicle group, light grey shading represents that mucositis score reduces (disease improves), and dark grey represents that mucositis score raises (disease worsens).Bold represents the significant difference of mucositis score.

[0030] Figure 16 Exemplary data showing the percentage of animals with ulcers with a mucositis score of ≥3 by a given day are shown. To examine the level of clinically significant mucositis, as defined by the presence of open ulcers (score ≥3), the percentage of animals from each treatment group that exhibited open ulcers was determined on each day of the study. Light shading indicates a decrease in mucositis scores (improved disease) and dark shading indicates an increase in mucositis scores (worsened disease) compared to the vehicle group. DETAILED DESCRIPTION

[0031] definition

[0032] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide general definitions of many terms used in the present invention for technical personnel: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al., (ed.), Springer Verlag (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them below. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit this disclosure.

[0033] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0034] As used herein, the terms "patient" and "subject" refer to an organism to be treated by the methods of the present disclosure. Such organisms are preferably mammals (e.g., marine animals, monkeys, horses, cattle, pigs, canines, felines, etc.), and more preferably humans.

[0035] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present disclosure) sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

[0036] As used herein, the term "treating" includes any action that results in improvement or alleviation of the symptoms of the condition, disease, disorder, etc., such as reducing, lowering, modulating, alleviating, or eliminating.

[0037] As used herein, the terms "alleviate" and "alleviating" refer to reducing the severity of the disorder, such as reducing the severity by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0038] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use, either in vivo or ex vivo.

[0039] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions (such as, for example, oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA

[1975] .

[0040] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present disclosure that, when administered to a subject, is capable of providing a compound of the present disclosure or its active metabolite or residue. As known to those skilled in the art, a "salt" of a compound of the present disclosure can be derived from an inorganic or organic acid and an inorganic or organic base. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids (e.g., oxalic acid), while not pharmaceutically acceptable in themselves, can be used to prepare salts that can be used as intermediates in obtaining the compounds of the present disclosure and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metal (eg, sodium) hydroxides, alkaline earth metal (eg, magnesium) hydroxides, ammonia, and compounds of the formula NW4+, wherein W is C1-4 alkyl, and the like.

[0041] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, etc. Other examples of salts include anions of the compounds of the present disclosure complexed with suitable cations such as Na+, NH4+, and NW4+ (wherein W is a C1-4 alkyl group), etc.

[0042] For therapeutic use, salts of the compounds of the present disclosure are considered pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also be useful, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0043] As used herein, the term "about" when referring to a measurable value (eg, weight, time, and dosage) is intended to encompass variations such as ±10%, ±5%, ±1%, or ±0.1% of the specified value.

[0044] The chemical name of compound RRx-001 (also known as ABDNAZ) is 2-bromo-1-(3,3-dinitroazetidin-1-yl)ethan-1-one, which has the following chemical structure:

[0045]

[0046] Throughout this specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that there are additional compositions of the disclosure consisting essentially of, or consisting of, the recited components, and that there are processes and methods according to the disclosure consisting essentially of, or consisting of, the recited process steps.

[0047] In general, compositions specifying percentages are by weight unless otherwise indicated. Additionally, if a variable is not accompanied by a definition, the preceding definition of the variable controls.

[0048] Overview

[0049] Prophylactic radiation protection compounds that can protect normal tissues from the effects of ionizing radiation are unmet needs for military and first responders, space exploration, and cancer treatment. RRx-001 is a small cyclic nitro compound 1-bromoacetyl-3,3-dinitroazetidine that forms RRx-001-hemoglobin adducts in red blood cells (RBCs). RRx-001 is a systemic non-toxic anticancer agent that has been used as a chemo- and radiosensitizer for various tumor types in multiple clinical trials. In contrast to its ability to achieve tumor radiosensitization, RRx-001 has been shown to protect normal cells from radiation (Scicinski et al., Redox Biology 2015; 6: 1). As described in the examples provided herein, administration of RRx-001 before exposure to lethal radiation significantly increased the survival rate of mice. In addition, in sublethally irradiated mice, it was found that prophylactic administration of RRx-001 can significantly enhance cell recovery in the bone marrow, as demonstrated by accelerated bone marrow reconstruction and improved bone marrow cell structure. Furthermore, RRx-001 treatment was found to increase the expression of antioxidant response element proteins such as heme oxygenase 1 (HO-1) in macrophages, monocytes, and mesenchymal stem cells. The induction of antioxidant response element genes may be driven by the transcription factor Nrf2, which has previously been shown to have increased nuclear presence in tumor cells exposed to RRx-001 (Ning et al., Oncotarget 2015; 6(25): 21547). Without wishing to be bound by theory, RRx-001 may provide protection to cells from oxidative damage via oxidative preconditioning, whereby transient changes in redox balance induce a preconditioned state of expression of compensatory genes for the cytoprotective antioxidant response (see Figure 6A In addition, RRx-001 reduces cell surface expression of the transmembrane protein CD47 (cluster of differentiation 47), which may provide local radioprotection of soft tissues and bone marrow, as CD47 expression after exposure to ionizing radiation is known to limit the ability of cells and tissues to survive and recover from damage caused by ionizing radiation (Miller et al. (2015) J. Biol. Chem. 290: 24858-24874) (see Figure 6B ).

[0050] The present disclosure provides therapeutic methods, kits, and pharmaceutical compositions for protecting a subject from radiation using a therapeutic agent selected from RRx-001 and pharmaceutically acceptable salts thereof.

[0051] In an exemplary method of treatment, RRx-001 is administered to a subject before the subject is exposed to radiation to protect the subject from radiation, such as ionizing radiation containing alpha rays, beta rays, gamma rays, neutron radiation, or a combination thereof. The method of treatment is particularly suitable for protecting civilians and military personnel from unexpected radiation exposure, such as protecting first responders to nuclear emergencies, cosmic radiation associated with expanded space habitats or travel, or other dangers involving harmful radiation levels. In addition, the method of treatment can be used in combination with radiation therapy for cancer to protect normal tissue. Ideally, the therapeutic agent is administered to the subject at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before the subject is exposed to radiation that may cause harm to the subject, and ideally provides protection against the harmful effects of radiation for a duration of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer.

[0052] Methods for protecting against radiation and reducing the effects of radiation

[0053] Provided herein are methods for protecting a subject from radiation using a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof. Various features of the methods are described in the following sections. The sections are arranged for convenience, and information in one section is not limited to that section but may be applied to other sections.

[0054] One aspect of the present disclosure provides a method for treating a subject in need of radiation protection. In some embodiments, the method includes administering an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof to a subject in need thereof by a route selected from parenteral administration, oral administration, and topical administration, thereby protecting the subject from radiation for a duration of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. The method is particularly useful for protecting civilians and military personnel from unexpected radiation exposure, such as protecting first responders to nuclear emergencies, cosmic radiation associated with expanded space habitats or travel, or other dangers involving harmful radiation levels. For the purpose of protecting the subject from radiation, the method can also be used in combination with the radiation therapy for cancer of the subject.

[0055] Another aspect of the present disclosure provides a method for reducing radiation exposure damage to a subject. In some embodiments, the method comprises administering an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof to a subject in need thereof by a route selected from parenteral administration, oral administration, and topical administration, thereby reducing radiation exposure damage to the subject for a duration of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. The treatment method is particularly suitable for protecting civilians and military personnel from unexpected radiation exposure, such as protecting first responders to nuclear emergencies, cosmic radiation associated with expanded space habitats or travel, or other risks involving harmful radiation levels. The treatment method can also be used to reduce radiation exposure damage associated with radiation therapy for cancer in a subject.

[0056] In some embodiments, administration of RRx-001 reduces or inhibits radiation exposure damage to one or more cells, systems, organs, or normal tissues in a subject. In some embodiments, administration of RRx-001 reduces or inhibits radiation exposure damage to one or more of the bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin, and brain. In some embodiments, administration of RRx-001 reduces or inhibits one or more radiation-induced conditions, such as, but not limited to, oral mucositis, dermatitis, rash, ulcers, alopecia, gastrointestinal discomfort, or proctitis.

[0057] Another aspect of the present disclosure provides a method for protecting a biological material (such as an isolated cell, tissue, or organ) from the damaging effects of radiation. In some embodiments, the method comprises exposing the biological material to an effective amount of a therapeutic agent selected from RRx-001 and pharmaceutically acceptable salts thereof, thereby protecting the biological material from the damaging effects of radiation for a duration of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer.

[0058] Another aspect of the present disclosure provides a method for treating a subject in need of radiation protection using an agent that alkylates hemoglobin beta cysteine 93. In some embodiments, the agent that alkylates hemoglobin beta cysteine 93 is selected from RRx-001 and a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a therapeutic agent that alkylates hemoglobin beta cysteine 93, thereby protecting the subject from radiation for a duration of at least 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. In certain embodiments, the therapeutic agent is administered by a route selected from parenteral administration, oral administration, and topical administration. In certain embodiments, the method protects the subject from radiation for a duration of at least 6 hours. The treatment method is particularly suitable for protecting civilians and military personnel from unexpected radiation exposure, such as protecting first responders to nuclear emergencies, cosmic radiation associated with expanded space habitats or travel, or other hazards involving harmful radiation levels. The treatment method may also be used in combination with radiation therapy for cancer in a subject for the purpose of protecting the subject from radiation.

[0059] Another aspect of the present disclosure provides a method for reducing radiation exposure damage to a subject using an agent that alkylates hemoglobin beta cysteine 93. In some embodiments, the agent that alkylates hemoglobin beta cysteine 93 is selected from RRx-001 and a pharmaceutically acceptable salt thereof. The method comprises administering to a subject in need thereof an effective amount of a therapeutic agent that alkylates hemoglobin beta cysteine 93, thereby reducing radiation exposure damage to the subject for a period of at least 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer. In certain embodiments, the therapeutic agent is administered by a route selected from parenteral administration and topical administration. In certain embodiments, the method reduces radiation exposure damage to the subject for a period of at least 6 hours. The treatment method is particularly suitable for protecting civilians and military personnel from unintended radiation exposure, such as protecting first responders to nuclear emergencies, cosmic radiation associated with expanded space habitats or travel, or other hazards involving harmful radiation levels. The treatment methods may also be used to reduce radiation exposure damage associated with radiation therapy for cancer in a subject.

[0060] In certain other embodiments, the therapeutic agent that alkylates hemoglobin beta cysteine 93 comprises a maleimide. In certain embodiments, the therapeutic agent comprises an N-alkylmaleimide. In certain embodiments, the therapeutic agent comprises an N-ethylmaleimide. In certain other embodiments, the therapeutic agent comprises a compound selected from α-haloacetate, α-haloacetamide, and α-halomethylketone. In certain embodiments, the therapeutic agent is an α-haloacetate. In certain embodiments, the therapeutic agent comprises α-bromoacetate or α-iodoacetate. In certain other embodiments, the therapeutic agent comprises α-haloacetamide. In certain embodiments, the therapeutic agent comprises α-bromoacetamide or α-iodoacetamide. In certain other embodiments, the therapeutic agent comprises α-halomethylketone. In certain embodiments, the therapeutic agent comprises α-bromobenzophenone or α-iodobenzophenone. In certain embodiments, the therapeutic agent comprises bromomethylketone. In certain embodiments, the therapeutic agent comprises an α-iodo-dinitroazetidine or an α-chloro-dinitroazetidine.

[0061] In certain embodiments, provided herein are methods that achieve at least 12 hours of radiation protection. In certain embodiments, the method achieves a radiation protection duration of at least 48 hours. In yet other embodiments, the method achieves a radiation protection duration of from about 6 hours to about 12 hours, from about 6 hours to about 24 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 48 hours. In certain embodiments, provided herein are methods that achieve a radiation protection duration of at least 1 week. In certain embodiments, provided herein are methods that achieve a radiation protection duration of at least 1 month.

[0062] In certain embodiments, exemplary anticipated benefits of treatment methods may include, but are not limited to, (i) limiting symptoms of acute radiation exposure, (ii) reducing long-term complications from radiation exposure, and / or (iii) preventing the development of cancers known to be caused by radiation exposure (e.g., leukemia and thyroid cancer).

[0063] Types and sources of radiation

[0064] The methods provided herein can be characterized by the type of radiation. For example, in certain embodiments, the radiation is ionizing radiation. In certain embodiments, the radiation comprises alpha rays, beta rays, gamma rays, neutron radiation, or a combination thereof. In certain other embodiments, the radiation comprises x-rays.

[0065] The method can also be characterized based on the source of the ionizing radiation. For example, in some embodiments, the radiation is ionizing radiation from sunlight.

[0066] In certain other embodiments, the radiation is ionizing radiation from a radionuclide.In certain embodiments, the radiation is ionizing radiation from an explosive device.

[0067] In certain other embodiments, the radiation is from a medical device that emits therapeutic radiation, such as for the treatment of cancer. Exemplary ionizing radiation therapy modalities may include, for example, external beam radiation therapy; intensity modulated radiation therapy (IMRT); image guided radiation therapy (IGRT); X-ray irradiation (e.g., photon beam therapy); electron beam (e.g., beta irradiation); local and whole-skin electron beam therapy; megavoltage photon therapy (about 4 to 10 MeV); proton irradiation; high linear energy transfer (LET) particles; stereotactic radiosurgery; gamma knife; linear accelerator-mediated frameless stereotactic radiosurgery; robotic-controlled x-ray irradiation delivery system; organ-specific or cancer cell-specific uptake radioisotope radiotherapy; radioisotopes bound to monoclonal antibodies for tumor-targeted radiation therapy (or radioimmunotherapy, RIT); brachytherapy (interstitial or intracavitary) high dose rate radiation source implantation; permanent radioactive seed implantation for organ-specific dose delivery.

[0068] Methods for administering therapeutic agents

[0069] The therapeutic method can be characterized according to the time of administration of the therapeutic agent. For example, in certain embodiments, at least one dose of the therapeutic agent is administered to the subject before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 48 hours before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 24 hours before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 12 hours before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 6 hours before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 3 hours before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 2 hours before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 1 hour before exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 30 minutes prior to exposure to radiation. In certain embodiments, at least one dose of the therapeutic agent is administered to the subject within 15 minutes prior to exposure to radiation.

[0070] In certain other embodiments, the first dose of the therapeutic agent is administered to the subject during exposure to radiation or after exposure to radiation has ceased. In certain embodiments, the first dose of the therapeutic agent is administered to the subject during exposure to radiation. In certain other embodiments, the first dose of the therapeutic agent is administered to the subject after exposure to radiation has ceased. In certain embodiments, the first dose of the therapeutic agent is administered to the subject within 1 day after exposure to radiation has ceased. In certain embodiments, the first dose of the therapeutic agent is administered to the subject within 48, 24, 12, 6, 3, or 2 hours after exposure to radiation has ceased. In certain embodiments, the first dose of the therapeutic agent is administered to the subject within 1 hour after exposure to radiation has ceased.

[0071] In certain other embodiments, a dose of the therapeutic agent is administered to the subject (i) prior to exposure to radiation and (ii) during exposure to radiation. In certain other embodiments, a dose of the therapeutic agent is administered to the subject (i) prior to exposure to radiation and (ii) after exposure to radiation. In certain other embodiments, a dose of the therapeutic agent is administered to the subject (i) prior to exposure to radiation, (ii) during exposure to radiation, and (iii) after exposure to radiation.

[0072] The therapeutic method can be characterized by the frequency with which the therapeutic agent is administered. For example, in certain embodiments, the therapeutic agent is administered to the subject no more than once a week. In certain embodiments, the therapeutic agent is administered to the subject once a week for at least two weeks. In certain other embodiments, the therapeutic agent is administered to the subject at least once a week. In certain embodiments, the therapeutic agent is administered to the subject at least twice a week. In certain embodiments, the therapeutic agent is administered to the subject at least once every two days. In certain embodiments, the therapeutic agent is administered to the subject at least once a day. In certain embodiments, the therapeutic agent is administered to the subject at least twice a day.

[0073] The therapeutic method can be characterized according to the dosage of the therapeutic agent. For example, in certain embodiments, the therapeutic agent is given in a certain dosage, and the dosage provides RRx-001 in an amount ranging from about 0.01 mg to about 1000 mg RRx-001 per day when the therapeutic agent is given to the subject. For example, in certain embodiments, the therapeutic agent is given in a certain dosage, and the dosage provides RRx-001 in an amount ranging from about 0.05 mg to about 500 mg RRx-001 per day when the therapeutic agent is given to the subject. For example, in certain embodiments, the therapeutic agent is given in a certain dosage, and the dosage provides RRx-001 in an amount ranging from about 0.1 mg to about 200 mg RRx-001 per day when the therapeutic agent is given to the subject. For example, in certain embodiments, the therapeutic agent is given in a certain dosage, and the dosage provides RRx-001 in an amount ranging from about 0.5 mg to about 150 mg RRx-001 per day when the therapeutic agent is given to the subject. For example, in certain embodiments, the therapeutic agent is administered at a dose that provides RRx-001 in an amount ranging from about 1 mg to about 100 mg of RRx-001 on each day the therapeutic agent is administered to the subject. For example, in certain embodiments, the therapeutic agent is administered at a dose that provides RRx-001 in an amount ranging from about 5 mg to about 50 mg of RRx-001 on each day the therapeutic agent is administered to the subject. For example, in certain embodiments, the therapeutic agent is administered at a dose that provides RRx-001 in an amount ranging from about 0.5 mg to about 166 mg of RRx-001 on each day the therapeutic agent is administered to the subject.

[0074] In certain embodiments, the therapeutic agent is administered at a dose that ranges from about 0.005 mg / m2 to about 0.005 mg / m2 on each day the therapeutic agent is administered to the subject. 2 to about 500 mg / m 2 In certain embodiments, the therapeutic agent is administered at a dose ranging from about 0.025 mg / m2 per day of administration of the therapeutic agent to the subject. 2 Up to about 250 mg / m 2 In certain embodiments, the therapeutic agent is administered at a dose ranging from about 0.05 mg / m2 to about 0.05 mg / m2 per day of administration of the therapeutic agent to the subject. 2 to about 100 mg / m 2 In certain embodiments, the therapeutic agent is administered at a dose ranging from about 0.25 mg / m2 per day of administration of the therapeutic agent to the subject. 2 Up to 75 mg / m2 In certain embodiments, the therapeutic agent is administered at a dose ranging from about 0.5 mg / m2 to about 0.5 mg / m2 per day of administration of the therapeutic agent to the subject. 2 Up to 50 mg / m 2 In certain embodiments, the therapeutic agent is administered at a dose ranging from about 2.5 mg / m2 to about 1.5 mg / m2 per day of administration of the therapeutic agent to the subject. 2 Up to 25 mg / m 2 In certain embodiments, the therapeutic agent is administered at a dose ranging from about 0.25 mg / m2 per day of administration of the therapeutic agent to the subject. 2 to about 83 mg / m 2 The amount of RRx-001 provides RRx-001.

[0075] The method of treatment can be characterized by the route of administering the therapeutic agent, and can be further characterized by the duration of administration. For example, in certain embodiments, the therapeutic agent is administered intravenously to the subject. In certain embodiments, the therapeutic agent is administered intravenously to the subject over a duration of at least thirty minutes. In certain embodiments, the therapeutic agent is administered intravenously to the subject over a duration of at least sixty minutes. In certain embodiments, the therapeutic agent is administered intravenously to the subject over a duration ranging from 30 minutes to 90 minutes.

[0076] In certain other embodiments, the therapeutic agent (e.g., RRx-001) is administered by intravenous injection of a mixture of blood and a composition comprising a therapeutic agent. In some embodiments, a certain amount of blood (e.g., about 1 to about 50 ml of blood) is taken out from the subject and mixed with a composition comprising a therapeutic agent (e.g., RRx-001). The mixture containing the therapeutic agent (e.g., RRx-001) is then administered intravenously to the patient. In some embodiments, before mixing with the therapeutic agent, blood is mixed with an anticoagulant (e.g., using a syringe preloaded with an anticoagulant). In some embodiments, the method of taking out blood from the subject, mixing with the therapeutic agent and administering the mixture to the subject is performed in a sterile closed system (e.g., a connection system of sterile tubing, syringes, containers, etc.), wherein the blood is not exposed to the environment. In some embodiments, the closed system is rinsed with sterile saline, wherein the saline rinse is administered to the patient to ensure the complete delivery of the therapeutic agent.

[0077] In certain other embodiments, the therapeutic agent is administered to the subject by intraperitoneal injection.In certain embodiments, the therapeutic agent is administered to the subject by intraperitoneal injection over a duration of at least thirty minutes.

[0078] In certain other embodiments, the therapeutic agent is administered by subcutaneous injection. In certain embodiments, the therapeutic agent is administered to the subject by intravenous injection over a duration of at least 5 minutes. In certain other embodiments, the therapeutic agent is administered subcutaneously to the subject via a pump device implanted in the subject containing the therapeutic agent. In certain embodiments, when the therapeutic agent is administered subcutaneously to the subject via a pump device implanted in the subject containing the therapeutic agent, the pump device is an osmotic pump.

[0079] In certain other embodiments, the therapeutic agent is administered topically. The topical administration can be, for example, a topical gel containing the first therapeutic agent that is applied to the subject's skin. The topical gel can be a sustained-release gel that slowly releases the first therapeutic agent over time.

[0080] In certain other embodiments, the therapeutic agent is administered by oral administration (eg, a pill, capsule, sublingual tablet, sustained release formulation, delayed release formulation, liquid, or aerosol).

[0081] The therapeutic method can be characterized by the location at which the therapeutic agent is administered. For example, in certain embodiments, the therapeutic agent is administered near a tissue that needs to be protected from radiation. In certain embodiments, the tissue that needs to be protected from radiation is bone marrow, skin, lung tissue, thyroid tissue, gonadal tissue, gastrointestinal tissue, bone tissue, fetal tissue, or a combination thereof.

[0082] Subjects for treatment

[0083] The therapeutic method can be further characterized based on the subject to be treated. In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult. In certain embodiments, the subject is an adult at risk of exposure to radiation from a nuclear emergency. In certain other embodiments, the subject is a child. In certain other embodiments, the subject is an animal, such as a domesticated animal (e.g., a dog, cat, or livestock).

[0084] In certain other embodiments, the subject is at risk of exposure to radiation from a nuclear emergency or from space travel. In certain embodiments, the subject is at risk of exposure to radiation from a nuclear emergency. In certain other embodiments, the subject is at risk of exposure to radiation from space travel. In certain other embodiments, the subject is an astronaut.

[0085] In certain other embodiments, the subject is at risk for radiation-induced damage due to radiation therapy used to treat cancer.

[0086] In certain other embodiments, the subject has a suppressed immune system. In certain embodiments, the suppressed immune system is caused by immunosuppressive drugs. In certain embodiments, the immunosuppressive drugs are steroids, calcineurin inhibitors, interleukin receptor inhibitory antibodies or interferons. In certain embodiments, the immunosuppressive drugs are steroids. In certain other embodiments, the suppressed immune system is caused by immunodeficiency syndrome (e.g., human immunodeficiency virus). In certain other embodiments, the subject with a suppressed immune system is a subject with a history of hematopoietic stem cell transplantation to help alleviate suppressed immune system symptoms.

[0087] Administration of additional therapeutic agents

[0088] In certain embodiments, the methods provided herein further comprise administering a therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof) in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are administered before, simultaneously with, or after administration of the therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof).

[0089] analgesics

[0090] In certain embodiments, the methods provided herein further comprise administering a therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof) in combination with an analgesic. In some embodiments, the analgesic is administered before, simultaneously with, or after the administration of the therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof). Exemplary analgesics include topical analgesics, aspirin, corticosteroids, and nonsteroidal anti-inflammatory agents. In certain embodiments, the analgesic is aspirin, a corticosteroid, or a nonsteroidal anti-inflammatory agent.

[0091] In certain embodiments, the method further comprises administering a local analgesic to a tissue of the subject proximal to the site of administration of the therapeutic agent prior to administering the therapeutic agent. In certain embodiments, the local analgesic is a caine analgesic. In certain embodiments, the local analgesic comprises lidocaine. In certain embodiments, the local analgesic is lidocaine hydrochloride. In certain other embodiments, the local analgesic is VanPen cream. In certain other embodiments, the local analgesic is an NSAID. In certain other embodiments, the local analgesic is acetaminophen. In certain other embodiments, the local analgesic is VanPen cream, an NSAID, or acetaminophen.

[0092] In certain embodiments, the topical analgesic is a formulation comprising: i) a single active ingredient selected from lecithin, isopropyl palmitate, isopropyl myristate, and combinations thereof; and ii) an excipient to form an ointment, cream, gel, lotion, spray, foam, paste, suspension, or dispersion for topical application to the skin. In certain embodiments, the single active ingredient is a combination of lecithin, isopropyl palmitate, and isopropyl myristate. In certain embodiments, the formulation comprises soy lecithin, isopropyl palmitate, stearic acid, glycerin, monostearate, isopropyl myristate, and polyoxyl 40 stearate.

[0093] In certain embodiments, the topical analgesic is a formulation consisting of: (i) lecithin and optionally one or two penetration enhancer fatty acid ester compounds (as the sole active ingredients), and (ii) an excipient to form an ointment, cream, gel, lotion, spray, foam, paste, suspension, or dispersion for topical application to a subject's skin. In certain embodiments, the formulation has one or two penetration enhancer fatty acid ester compounds.

[0094] In certain embodiments, the formulation has a penetration enhancer fatty acid ester compound selected from isopropyl palmitate and isopropyl laurate. In certain embodiments, the penetration enhancer fatty acid ester compound is isopropyl palmitate. In certain embodiments, one of the excipients is an emulsifier. In certain embodiments, the emulsifier is a poloxamer, a polyoxyethylene alkyl ether, a polyoxyethylene sorbitan fatty acid ester, or polyoxyethylene stearate. In certain embodiments, the emulsifier is polyoxyethylene stearate. In certain embodiments, another of the excipients is a surfactant selected from glyceryl monostearate and glyceryl monooleate. In certain embodiments, the formulation is in the form of a gel.

[0095] anticancer agents

[0096] In certain embodiments, the method further comprises administering an anticancer agent in combination with a therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof) to a subject. In certain embodiments, the anticancer agent is a chemotherapeutic agent (also referred to as an antitumor agent or antiproliferative agent). Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents, antibiotics, antimetabolites, antidotes, interferons, polyclonal or monoclonal antibodies, EGFR inhibitors, HER2 inhibitors, histone deacetylase inhibitors, hormones, mitotic inhibitors, MTOR inhibitors, multikinase inhibitors, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, VEGF / VEGFR inhibitors, taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule-targeted drugs, topoisomerase poison drugs, inhibitors of molecular targets or enzymes (e.g., kinase inhibitors), cytidine analogs, or any chemotherapeutic agent, antitumor agent, or antiproliferative agent known in the art.

[0097] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); nitrogen mustard (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozotocin (Zanosar).

[0098] Exemplary antibiotics include, but are not limited to, Adriamycin; Doxil; Novantrone; Blenoxane; Cerubidine; Daunorubicin; Cosmegen; Ellence; Idamycin; Mithracin; Mutamycin; Nipent; or Valstar.

[0099] Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); liposomal cytarabine (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

[0100] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0101] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0102] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab / iodine-131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab tiuxetan (Zevalin; In-I 11; Y-90Zevalin); gemtuzumab tuzumab (Mylotarg); eculizumab (Soliris); denosumab; ramucirumab (Cyramza), and olaratumab (Lartruvo).

[0103] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (Emd7200); or EKB-569.

[0104] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb); or AC-480.

[0105] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0106] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol acetate (Megace); leuprorelin (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

[0107] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamelliposide D (LAM-D).

[0108] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); sirolimus, riboside; or AP23573.

[0109] Exemplary multi-kinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0110] Exemplary serine / threonine kinase inhibitors include, but are not limited to, rubustard; efavirenz / fasudil hydrochloride; flavopiridol; seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412; bryostatin; KAI-9803; SF1126; VX-680; Azd1 152; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0111] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath); Gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0112] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; ramucirumab (Cyramza); pegaptanib; or vandetanib.

[0113] Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilone, and navelbine.

[0114] Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, actinomycin D, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0115] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

[0116] Exemplary general chemotherapeutic agents, anti-tumor agents, and anti-proliferative agents include, but are not limited to, Hexalen; Accutane; Amnesteem; Clara vis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisol (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase (Oncaspar); denileukin (Ontak); porflin sodium (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (leucenol); (1 M fluazifop-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 M potassium oxazolidinone) or lovastatin.

[0117] In another aspect, the additional therapeutic agent can be a cytokine, such as G-CSF (granulocyte colony stimulating factor). In another aspect, the composition of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog, or derivative thereof, can be administered in combination with radiation therapy. Radiation therapy can also be administered in combination with a composition of the present disclosure and another chemotherapeutic agent described herein as part of a multi-agent therapy. In yet another aspect, the compositions of the present disclosure, or pharmaceutically acceptable salts, prodrugs, metabolites, mimetics, analogs or derivatives thereof, can be administered in combination with standard chemotherapy such as, but not limited to, CMF (cyclophosphamide, methotrexate and 5-fluorouracil), CAF (cyclophosphamide, doxorubicin and 5-fluorouracil), AC (doxorubicin and cyclophosphamide), FEC (5-fluorouracil, epirubicin and cyclophosphamide), ACT or ATC (doxorubicin, cyclophosphamide and paclitaxel), rituximab, capecitabine, cisplatin (CDDP), carboplatin, TS-1 (fluidoxime, gimeracil and otastat potassium in a molar ratio of 1:0.4:1), camptothecin-11 (CPT-11, irinotecan or Camptosar), or cisplatin (CDDP). TM ) or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone).

[0118] Exemplary kinase inhibitors include, but are not limited to, bevacizumab (targeting VEGF), BIBW 2992 (targeting EGFR and Erb2), cetuximab / Erbitux (targeting Erb1), imatinib / Gleevic (targeting Bcr-Abl), trastuzumab (targeting Erb2), gefitinib / Iressa (targeting EGFR), ranibizumab (targeting VEGF), pegaptanib (targeting VEGF), erlotinib / Tarceva (targeting Erb1), nilotinib (targeting Bcr-Abl), lapatinib (targeting Erb1 and Erb2 / Her2), GW-572016 / lapatinib ditosylate (targeting HER2 / Erb2), panitumumab / Victoza (targeting VEGF), selegiline / selegiline (targeting HER2 / Erb2 ... Tibipro (targeting EGFR), vandetanib (targeting RET / VEGFR), E7080 (multiple targets, including RET and VEGFR), Herceptin (targeting HER2 / Erb2), PKI-166 (targeting EGFR), canertinib / CI-1033 (targeting EGFR), sunitinib / SU-11464 / Sutent (targeting EGFR and FLT3), matuzumab / Emd7200 (targeting EGFR), EKB-569 (targeting EGFR), Zd6474 (targeting EGFR and VEGFR), PKC-412 (targeting VEGR and FLT3), Vatalanib / Ptk787 / ZK222584 (targeting VEGR), CEP-701 (targeting FLT3), SU5614 (targeting FLT3), MLN518 (targeting FLT3), XL999 (targeting FLT3), VX-322 (targeting FLT3), Azd0530 (targeting SRC), BMS-354825 (targeting SRC), SKI-606 (targeting SRC), CP-690 (targeting JAK), AG-490 (targeting JAK), WHI-P154 (targeting JAK), WHI-P131 (targeting JAK), sorafenib / Nexavar (targets RAF kinases, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3, and RET), dasatinib / Sprycel (BCR / ABL and Src), AC-220 (targets Flt3), AC-480 (targets all HER proteins, “panHER”), motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), denosumab (targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets, including Flt3).

[0119] Exemplary serine / threonine kinase inhibitors include, but are not limited to, sirolimus (targeting mTOR / FRAP1), rapamycin (targeting mTOR), Certican / everolimus (targeting mTOR / FRAP1), AP23573 (targeting mTOR / FRAP1), Eril / fasudil hydrochloride (targeting RHO), Flavopiridol (targeting CDK), Seliciclib / CYC202 / Roscovitrine (targeting CDK), SNS-032 / BMS-387032 (targeting CDK), Rubustadine (targeting PKC), Pkc412 (targeting PKC), Bryostatin (targeting PKC), KAI-9803 (targeting PKC), SF1126 (targeting PI3K), VX-680 (targeting Aurora kinase), Azd1 l52 (targeting Aurora kinase), Arry-142886 / AZD-6244 (targeting MAP / MEK), SCIO-469 (targeting MAP / MEK), GW681323 (targeting MAP / MEK), CC-401 (targeting JNK), CEP-1347 (targeting JNK) and PD 332991 (targeting CDK).

[0120] In certain embodiments, the method further comprises administering to the subject an EGFR inhibitor in combination with a therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof). In certain embodiments, the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof. In certain embodiments, the EGFR inhibitor is erlotinib hydrochloride. In certain embodiments, the EGFR inhibitor comprises erlotinib.

[0121] The therapeutic method can be characterized according to the dose of erlotinib administered to the subject. For example, in certain embodiments, on any day that erlotinib is administered to the subject, a daily dose of at least 500 mg of erlotinib is administered to the subject. In certain embodiments, on any day that erlotinib is administered to the subject, a daily dose of at least 1000 mg of erlotinib is administered to the subject. In certain embodiments, on any day that erlotinib is administered to the subject, a daily dose of at least 2000 mg of erlotinib is administered to the subject. In certain other embodiments, on any day that erlotinib is administered to the subject, a daily dose of erlotinib in the range of about 1,000 mg to about 3,000 mg is administered to the subject. In certain embodiments, on any day that erlotinib is administered to the subject, a daily dose of erlotinib in the range of about 1,500 mg to about 2,500 mg is administered to the subject. In certain embodiments, on any day that erlotinib is administered to the subject, a daily dose of erlotinib in the range of about 1,800 mg to about 2,200 mg is administered to the subject. In certain embodiments, on any day that erlotinib is administered to a subject, a daily dose of erlotinib of about 2,000 mg is administered to the subject.

[0122] Inorganic nitrite

[0123] In certain embodiments, the methods provided herein further include administering an inorganic nitrite in combination with a therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof) to a subject. In certain embodiments, the inorganic nitrite is an alkali metal nitrite. In certain embodiments, the inorganic nitrite is sodium nitrite.

[0124] In certain embodiments, the inorganic nitrite is administered to the subject prior to administration of the first therapeutic agent, concurrently with administration of the first therapeutic agent to the subject, after administration of the first therapeutic agent to the subject, and / or each of the foregoing.

[0125] In certain embodiments, the inorganic nitrite is administered before the subject is exposed to radiation, simultaneously with the subject's exposure to radiation, and / or after the subject has been exposed to radiation.

[0126] Treating biomaterials with therapeutic agents

[0127] In certain embodiments, the method includes treating a biomaterial, such as isolated cells (e.g., blood cells), tissues, and organs, with a therapeutic agent (e.g., RRx-001 or a pharmaceutically acceptable salt thereof). In some embodiments, the method can be characterized according to the time over which the biomaterial is exposed to the therapeutic agent. For example, in certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 1 day prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 12 hours prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 6 hours prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 3 hours prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 2 hours prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 1 hour prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 30 minutes prior to exposure to radiation. In certain embodiments, the biomaterial is exposed to at least one dose of the therapeutic agent within 15 minutes prior to exposure to radiation.

[0128] In certain other embodiments, the biological material is exposed to a dose of the therapeutic agent for the first time during exposure to radiation or after exposure to radiation has ceased.

[0129] In certain embodiments, the biological material is first exposed to a dose of the therapeutic agent during exposure to radiation. In certain other embodiments, the biological material is first exposed to a dose of the therapeutic agent after exposure to radiation has ceased. In certain embodiments, the biological material is first exposed to a dose of the therapeutic agent within 1 day after exposure to radiation has ceased. In certain embodiments, the biological material is first exposed to a dose of the therapeutic agent within 12, 6, 3, or 2 hours after exposure to radiation has ceased. In certain embodiments, the biological material is first exposed to a dose of the therapeutic agent within 1 hour after exposure to radiation has ceased.

[0130] The methods can be characterized based on the frequency with which the biomaterial is exposed to the therapeutic agent. For example, in certain embodiments, the biomaterial is exposed to the therapeutic agent no more than once a week. In certain embodiments, the biomaterial is exposed to the therapeutic agent once a week for at least two weeks. In certain other embodiments, the biomaterial is exposed to the therapeutic agent at least once a week. In certain embodiments, the biomaterial is exposed to the therapeutic agent at least twice a week. In certain embodiments, the biomaterial is exposed to the therapeutic agent at least once every two days. In certain embodiments, the biomaterial is exposed to the therapeutic agent at least once a day. In certain embodiments, the biomaterial is exposed to the therapeutic agent at least twice a day.

[0131] In certain embodiments, the method further comprises exposing the biomaterial to an inorganic nitrite. In certain embodiments, the inorganic nitrite is an alkali metal nitrite. In certain embodiments, the inorganic nitrite is sodium nitrite. In certain embodiments, the inorganic nitrite is administered before, while, or after the administration of the first therapeutic agent, and / or in each of the foregoing.

[0132] In certain embodiments, the inorganic nitrite is administered prior to exposing the biological material to radiation, concurrently with exposing the biological material to radiation, and / or after the biological material has been exposed to radiation.

[0133] Pharmaceutical compositions

[0134] As noted above, the present disclosure provides pharmaceutical compositions comprising an amount of one or more of the compounds described above formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical compositions can be specifically formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (such as those for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, such as sterile solutions or suspensions or sustained-release formulations; (3) topical administration, such as application to the skin as a cream, ointment, or controlled-release patch or spray; (4) intravaginally or intrarectally, such as as a pessary, cream, or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

[0135] As used herein, the phrase "therapeutically effective amount" means a specific amount of a compound, material, or composition comprising a compound of the present disclosure that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0136] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and with a reasonable benefit / risk ratio commensurate.

[0137] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0138] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0139] The formulations of the present disclosure include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form will vary according to the host being treated, the specific mode of administration. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form is typically the amount of the compound that produces the therapeutic effect. Typically, in one hundred parts, this amount will be about 0.1% to about 99% active ingredient, preferably about 5% to about 70%, most preferably about 10% to about 30%.

[0140] In certain embodiments, the formulations of the present disclosure comprise an excipient selected from cyclodextrins, celluloses, liposomes, micelle formers (e.g., bile acids), and polymeric carriers (e.g., polyesters and polyanhydrides); and a compound of the present disclosure. In certain embodiments, the foregoing formulations render the compound of the present disclosure bioavailable upon oral administration.

[0141] The method for preparing these formulations or compositions comprises the step of combining a compound of the present disclosure with a carrier and optionally one or more auxiliary ingredients. In general, the formulations are prepared by uniformly and intimately combining a compound of the present disclosure with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0142] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of a compound of the present disclosure as the active ingredient. The compounds of the present disclosure may also be administered as a bolus, electuary, or paste.

[0143] In solid dosage forms of the present disclosure for oral administration (capsules, tablets, pills, lozenges, powders, granules, lozenges, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato starch, etc. or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarder such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds and surfactants such as poloxamers and sodium lauryl sulfate; (7) wetting agents such as, for example, cetyl alcohol, glyceryl monostearate, and nonionic surfactants; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (11) controlled release agents such as crospovidone or ethylcellulose.

[0144] In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions may also be used as fillers in soft and hard shell gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

[0145] Tablets can be manufactured by compression or molding (optionally with one or more auxiliary ingredients). Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant, or a dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0146] Tablets and other solid dosage forms of the pharmaceutical compositions of the present disclosure (e.g., dragees, capsules, pills, and granules) can optionally be scored or prepared with coatings and shells (e.g., enteric coatings and other coatings well known in the pharmaceutical formulation art). They can also be formulated to provide slow or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in varying proportions to provide a desired release profile. They can be formulated for rapid release, for example, by freeze drying.

[0147] They can be sterilized by, for example, filtering through a filter that retains bacteria or by incorporating a sterilizing agent into the form of a sterile solid composition, which can be dissolved in sterile water or some other sterile injectable medium just before use. These compositions can also optionally contain an opacifying agent and can have a composition such that they only release one or more active ingredients, or preferentially release in a certain part of the gastrointestinal tract, preferably in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. If appropriate, the active ingredient can also be in the form of microcapsules using one or more of the above-mentioned excipients.

[0148] Liquid dosage forms for oral administration of the compounds of the present disclosure may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate,

[0149] Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuranol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0150] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0151] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0152] Formulations of the pharmaceutical compositions of the present disclosure for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more compounds of the present disclosure with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0153] Formulations of the present disclosure suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0154] Dosage forms for topical or transdermal administration of the compounds of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0155] Ointments, pastes, creams and gels may contain, in addition to the active compounds of this disclosure, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0156] In addition to the compounds of this disclosure, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (e.g., butane or propane).

[0157] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present disclosure into the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0158] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated as being within the scope of this invention.

[0159] Pharmaceutical compositions of the present disclosure suitable for parenteral administration comprise a combination of one or more compounds of the present disclosure with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just before use, which combination may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0160] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0161] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersants. Can ensure to prevent the effect of microorganisms on the subject compound by comprising various antibacterials and antifungals (such as parabens, chlorobutanol, phenol sorbic acid etc.). In the composition, including isotonic agents, such as sugar, sodium chloride etc. also may be desirable. In addition, the extended absorption of injectable drug form can be realized by comprising the reagent (such as aluminum monostearate and gelatin) that delays absorption.

[0162] In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the drug injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenteral drug form can be achieved by dissolving or suspending the drug in an oily vehicle.

[0163] Injectable depot forms are prepared by forming a microcapsule matrix of the subject compound in a biodegradable polymer (such as polylactide-polyglycolide). Depending on the ratio of drug to polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by embedding the drug in liposomes or microemulsions compatible with body tissues.

[0164] When the compounds of the present disclosure are administered to humans and animals as medicines, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99% (more preferably 10% to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0165] The formulations of the present disclosure can be administered orally, parenterally, topically, or rectally. The formulations are, of course, administered in a form suitable for each route of administration. For example, they can be administered in tablet or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc., by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository. Oral administration is preferred.

[0166] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0167] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "peripherally administered" mean administration of a compound, drug, or other material other than directly into the central nervous system, such that the compound, drug, or other material enters the patient's system and thereby undergoes metabolism and other similar processes, such as subcutaneous administration.

[0168] The compounds can be administered to humans and other animals for therapeutic use by any suitable route of administration, including oral, nasal (e.g., as a spray), rectal, vaginal, parenteral, intracerebral, and topical (e.g., by powders, ointments, or drops, including buccal and sublingual).

[0169] Regardless of the route of administration chosen, the compounds of the disclosure (which may be used in suitable hydrated form and / or pharmaceutical compositions of the disclosure) can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0170] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0171] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the disclosure employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0172] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can initiate the dosage of a compound of the present disclosure employed in the pharmaceutical composition at a level that is lower than the level required to achieve the desired therapeutic effect, and then gradually increase the dosage until the desired effect is achieved.

[0173] In general, a suitable daily dose of a compound of the present disclosure is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors mentioned above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably at about 0.1 mg / kg to about 100 mg / kg, and even more preferably at about 0.5 mg / kg to about 50 mg / kg.

[0174] When a compound described herein is co-administered with another agent (eg, an additional radioprotectant), the effective amount may be less than when that agent is used alone.

[0175] If desired, the effective daily dose of active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form.Preferred administration is once daily.

[0176] Kits for protection against radiation and reducing the effects of radiation

[0177] Another aspect of the present disclosure provides a kit for protecting against or reducing the effects of radiation. The kit comprises (i) a therapeutic agent selected from RRx-001 or a pharmaceutically acceptable salt thereof, and (ii) instructions for protecting against or reducing the effects of radiation according to the procedures described herein. In certain embodiments, the kit further comprises (iii) a local analgesic, such as lidocaine hydrochloride.

[0178] Example

[0179] Example 1 - Effect of RRx-001 on survival after lethal irradiation

[0180] In this example, the effect of systemic administration of RRx-001 on survival in response to a lethal radiation dose was determined in mice. 9.5-11 week old CD2F1 male mice were given a single dose of RRx-001 by intraperitoneal (IP) injection 24 hours prior to the lethal radiation dose. 24 mice received 10 mg / kg RRx-001 (formulated in 5% DMSO in sterile H2O) and 24 mice received vehicle control (5% DMSO in sterile H2O alone).

[0181] Mice were subjected to total body irradiation (TBI) of 9.35 Gy (LD70 / 30) using high levels of cobalt-60 at 0.6 Gy / min. Unanesthetized mice were placed in well-ventilated Plexiglas restrainers and bilaterally irradiated. Sham-operated (sham) irradiated mice were also placed in the same Plexiglas restrainer and simultaneously kept in a non-irradiated room. In each experiment, a dose of approximately 0.6 Gy / min was delivered to the abdominal core of the animal. Before the animals were irradiated, a highly accurate alanine / electron spin resonance (ESR) dosimetry system (American Society for Testing and Materials, Standard E 1607) was used to measure the dose rate (to water) in the acrylic mouse model core located in the exposure rack compartment. The dose was measured using a calibration curve based on a standard alanine calibrated dosimeter provided by the National Institute of Standards and Technology (NIST, Gaithersburg, Maryland). The accuracy of the dose rate calibration was verified several times using the services of the National Physical Laboratory (UK National Standards Laboratory, London, UK) and the MD Anderson Cancer Center (Houston, Texas). The dose rates measured in the phantom were corrected for small differences in Co-60 energy between the mass energy absorption coefficients of soft tissue and water, as well as for source attenuation. The radiation field was uniform within ±1.2%.

[0182] Mice were monitored at least twice daily for 30 days after irradiation. During the critical period (days 10-20), mice were monitored at least three times daily with observation intervals of no more than 10 hours. Mice showing any signs of discomfort were given wet chow in their cages. Mice showing obvious dyspnea, weight loss, lethargy or other signs of moribundity and appearing distressed were humanely euthanized using carbon dioxide gas in a separate room, followed by cervical dislocation after respiratory arrest as a confirmation method for euthanasia. This experiment was repeated for a total of n=24 mice per group.

[0183] Survival curves were estimated using the Kaplan-Meier method and compared using a two-way log-rank test at a significance level of 0.05. P values were considered statistically significant if they were less than 0.05.

[0184] The improvement in survival with pretreatment with a dose of 10 mg / kg RRx-001 relative to vehicle control irradiated mice was highly significant, with a 33.4% reduction in the risk of 30-day mortality ( Figure 1A). Time-to-death data depicting 30-day survival are in Figure 1B The scatter plot of survival time by treatment group shows that the mean ± standard error values for the vehicle and RRx-001 groups were 20.2 ± 1.6 and 27.2 ± 1.1, respectively. Therefore, compared to the vehicle control, administration of 10 mg / kg RRx-001 24 hours prior to the lethal TBI dose not only significantly increased survival by 33.4%, but also significantly extended mean survival by 7 days.

[0185] Example 2 - Effect of RRx-001 on Hematopoietic Recovery after Irradiation

[0186] To determine the pathophysiological role of RRx-001 in hematopoietic protection in mice, CD2F1 male mice were treated with 10 mg / kg RRx-001 or vehicle control 24 hours prior to sublethal dose TBI (7 Gy at 0.6 Gy / min using high levels of cobalt-60) or sham irradiation (day 0) according to the table below.

[0187]

[0188] CD2F1 male mice (n = 3 / group) were divided into 4 experimental groups: 1) irradiation + vehicle, 2) irradiation + RRx-001, 3) sham irradiation + vehicle, and 4) sham irradiation + RRx-001. 10 mg / kg RRx-001 or vehicle control was injected intraperitoneally 24 hours before irradiation or sham irradiation (day 0). Mice were humanely euthanized on days 2, 7, 14, 21, and 28 after irradiation (day 0). Blood, bone marrow, and sternum were then collected. This experiment was performed in duplicate for a total of n = 6 mice per group at each time point.

[0189] Irradiated whole blood was obtained by terminal cardiac puncture. Blood samples were immediately transferred to EDTA tubes (Sarstedt Inc., Newton, North Carolina) and gently spun until analysis time. Complete blood cell counts were performed on the tubes using an ADVIA 2120 (Siemens Medical Solutions Diagnostics, Dublin, Ireland) and Microsoft software version 5.9 (Microsoft Corp., Redmond, Washington) using differential and reticulocyte counts to generate data.

[0190] The sternum of euthanized mice (n=6 per group at each time point) was collected on the 2nd, 7th, 14th, 21st and 28th day after irradiation. The sternum was fixed in 10% zinc-buffered formalin for at least 24 hours and for a maximum of 7 days. The fixed sternum was decalcified in 12%-18% sodium EDTA (pH 7.4-7.5) for 3 hours, and the sample was dehydrated and embedded in paraffin using graded ethanol concentrations. The longitudinal 4 μm sections were stained with conventional hematoxylin and eosin. Two pathologists, both certified by their profession, evaluated the samples for histopathology. One of the pathologists scored all samples using a blinded method. For total cell structure, bone marrow was evaluated in situ in the sternum and graded (grade 1: <10%; grade 2: 11%-30%; grade 3: 31%-60%; grade 4: 61%-89%; grade 5:>90%). Megakaryocytes were also quantified based on the average of 10 high-power fields (HPF) at 400× using a BX43 or BX53 microscope (Olympus, Minneapolis, MN). Images were captured with an Olympus DP22 camera and imported into Olympus Cellsens Standard software for viewing.

[0191] Analysis of variance (ANOVA) was used to compare blood parameters between treatment groups. The Wilcoxon test was also used for sensitivity and to potentially address the problem of data deviation from normality. A longitudinal mixed model repeated measures was also implemented to provide a more complete data analysis based on the overall time distribution mean differences of the sham-irradiated treatment group for blood parameters. Statistical analysis of bone marrow data (megakaryocytes and grade) was performed using parametric tests composed of general linear model analysis of variance (ANOVA, with factors composed of treatment group and pathologist) and Kruskal-Wallis nonparametric tests. Statistical data were analyzed using R software (version 3.4.3, 2016), and graphics were produced using GraphPad Prism version 7.03 (GraphPad Software, La Jolla, California).

[0192] To determine the effect of RRx-001 on the bone marrow, histopathological analysis of bone marrow sternums was performed, and cellularity, as reported by grade (grade 1: ≤10%; grade 2: 11%-30%; grade 3: 31%-60%; grade 4: 61%-89%; grade 5: ≥90% cellularity) and number of megakaryocytes (averaged per 10 high-power fields; HPF), was determined by two pathologists, one of whom scored all samples in a blinded manner (TAS, WEC). There were no significant differences in the significance of cellularity grade and the mean number of megakaryocytes per 10 HPF, and no significant interaction between pathologists or treatment and pathologist.

[0193] During the study, the total cellularity of the bone marrow never dropped below 90% in both the sham-irradiated RRx-001 and vehicle-treated groups, thus maintaining grade 5 ( Figure 2 ). Figure 3 A- Figure 3 B shows representative normal bone marrow morphology and cellularity. As expected after irradiation, both the RRx-001 and vehicle-treated groups experienced a substantial loss of bone marrow cellularity (Grade 1). By day 7, pathologists observed a slight increase in cellularity in RRx-001-treated mice compared to vehicle controls. Figure 2 As shown, pretreatment with RRx-001 significantly accelerated hematopoietic recovery, as determined by the grade of bone marrow cellularity, compared to controls at day 14. Compared to the irradiated RRx-001 treated group at day 14, where a significant recovery of bone marrow cellularity was observed, the irradiated vehicle treated group showed a significant loss of bone marrow cellularity and increased adipocyte infiltration ( Figure 5 ).

[0194] On day 14, the number of megakaryocytes in the sham-irradiated RRx-001 group was significantly higher than that in the vehicle control ( Figure 3 In both irradiated groups, the number of megakaryocytes decreased on day 2 and became severely depleted on day 7. The irradiated RRx-001 group showed a steady increase in megakaryocyte numbers between days 7 and 28. Interestingly, the irradiated vehicle-treated group experienced a significant jump in megakaryocyte numbers between days 14 and 21, which then decreased and returned to the same number of megakaryocytes as the irradiated RRx-001-treated group by day 28 ( Figure 3 ).

[0195] RRx-001 treatment also significantly increased the production of white and red blood cells in irradiated mice compared to irradiated vehicle controls ( Figure 4 ). Longitudinal mixed model repeated measures analysis comparing differences in total platelet means over time revealed a statistically significant difference in least squares means in favor of RRx-001 treatment (p=0.01). The standard errors of the means for sham-irradiated RRx-001-treated platelets and vehicle-treated platelets were 1067.73 (32.26) and 913.66 (52.22), respectively. When RRx-001-treated mice were compared to vehicle controls, no significant differences were observed in white blood cells (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), platelets (PLT), hematocrit percentage (HCT%), and reticulocyte percentage (RETIC%) at days 2 and 21 after irradiation ( Figure 4Irradiated RRx-001-treated and vehicle-treated groups showed reductions in both erythrocytes and hemoglobin below sham-irradiated controls on days 7 and 14 before returning to control levels; however, the irradiated groups were not significantly different when compared to each other.

[0196] In both experimental groups, acute irradiation treatment with sublethal doses induced severe reticulocytopenia and leukocytopenia. Reticulocytopenia persisted up to day 7 after irradiation. On day 14 after irradiation, RETIC% in RRx-001 pre-treated mice increased significantly compared to control mice and returned to baseline levels. Although both irradiated groups returned to or were above baseline levels on day 28, RRx-001-treated mice still had significantly increased RETIC%. In both irradiated groups, WBC and ALC reached their lowest points on day 7; however, by day 14, WBC and ALC counts were also significantly increased in RRx-001-treated mice compared to controls. In both irradiated RRx-001- and control-treated mice, ANC and PLT reached their lowest points on day 7 and remained so until day 14. However, for both ANC and PLT, irradiated mice pretreated with RRx-001 had significant increases compared to controls on both days 7 and 14. Although HCT% reached its nadir on day 14, irradiated RRx-001 mice had significantly elevated levels on day 14 compared to irradiated controls.

[0197] Blood and bone marrow obtained from sublethal irradiation studies showed that bone marrow cellularity and the production of white and red blood cells were significantly increased at day 14 in the RRx-001-irradiated group compared to irradiated vehicle controls. This may provide sufficient protection to allow recovery during a critical period when infection and sepsis may occur. Taken together, these experiments demonstrate that systemic administration of RRx-001 prior to whole-body irradiation significantly improves overall survival and bone marrow regeneration.

[0198] Example 3 - Characterization of the Radioprotective Effect of RRx-001

[0199] Antioxidant response element (ARE) genes (such as heme oxygenase 1 (HO-1), NAD(P)H dehydrogenase [quinone] 1 (NQO-1), and superoxide dismutase (SOD)) are involved in the detoxification and elimination of reactive oxidants. This example is designed to show that in vitro treatment with RRx-001 induces mild oxidative stress, which increases antioxidant response element (ARE) proteins in normal human bone marrow mesenchymal stem cells (hMSCs), macrophages, and their precursor monocytes. The exemplary treatment regimen is Figure 7 Available in.

[0200] The expression of ARE proteins in hMSCs, macrophages, and monocytes was determined following in vitro irradiation of cells pre-treated with RRx-001 or vehicle. Protein expression was determined by Western blotting and protein quantification.

[0201] hMSCs were treated with 5mM RRx-001 or vehicle control (0.05% DMSO) for 16 hours, irradiated with 10Gy or sham-operated, and protein was collected 8 or 24 hours after sham-operated or irradiated. 24 hours before treatment, human monocytic leukemia THP-1 and U937 cells were differentiated into macrophages with 50nM phorbol 12-myristate 13-acetate (PMA). Before radiation, differentiated and undifferentiated cells were treated with 3mM RRx-001 or 0.05% DMSO (vehicle control) for 16 hours. Cells were irradiated with 5Gy or sham-operated, and collected 4-8 hours later to perform two analyses. For each experiment, each sample was run in duplicate.

[0202] The activity of superoxide dismutase (SOD) was also determined in hMSCs. hMSCs were treated with 5mM RRx-001 or vehicle for 16 hours, irradiated with 10 Gy or sham-operated, and whole cell homogenates were collected according to the manufacturer's protocol. The assay measured the activity of all three forms of SOD. The amount of SOD activity (U / mL) was normalized to protein levels.

[0203] RRx-001 showed a significant increase (22-26 fold) in HO-1 expression in both sham-operated and irradiated hMSC groups 8 hours after sham operation or irradiation ( Figure 8 At 24 hours, the RRx-001 treated group still had an increase in HO-1 expression; however, the increase decreased to 2-3.5 fold. RRx-001 treatment slightly decreased NQO-1 in all groups. SOD1 and 2 showed a slight increase 8 hours after RRx-001 treatment and 10 Gy irradiation. In the RRx-001 treated group, there was no increase in superoxide dismutase activity 8 hours after 10 Gy irradiation ( Figure 9 ).

[0204] In U937 macrophages, increased HO-1 production was observed in both sham and irradiated groups following RRx-001 treatment; however, no changes in SOD-1 or NQO-1 were observed ( Figure 10 The results in U937 monocytes were similar to those seen in U937 macrophages. Overall, a significant increase in HO-1 was seen in both the U937 macrophage and monocyte fractions. A similar trend was observed in THP-1 monocytes; however, no significant increase in HO-1 was seen in THP-1 macrophages ( Figure 11).

[0205] After irradiating human monocytic leukemia THP-1 cells in vitro, cytokine arrays were used to examine cytokine expression. 24 hours before treatment, THP-1 cells were differentiated into macrophages with 50nM phorbol 12-myristate 13-acetate (PMA). Before radiation, differentiated and undifferentiated cells were treated for 16 hours with 3mM RRx-001 or 0.05% DMSO (vehicle control). Cells were irradiated with 5Gy or sham-operated, and collected after 4-8 hours to perform two analyses. For cytokine analysis, cell culture medium was collected and blotted onto a proteome analyzer human cytokine array (Proteome Profiler Human Cytokine Arrays, R&D Systems, Inc.) according to the manufacturer's protocol. The cytokine arrays showed that the cytokines involved in inflammation in both the sham-operated and irradiated macrophage fractions of drug treatments were reduced, as well as the CCL5 / RANTES induction ( Figure 12 ).

[0206] Data suggest that RRx-001 may provide cellular protection against oxidative damage by increasing HO-1 production in macrophages, monocytes, and mesenchymal stem cells. One potential mechanism is through reducing the pro-inflammatory chemokine IL-8 in macrophages and upregulating CCL5 / RANTES in monocytes, which may enhance immune cell reprogramming. Without wishing to be bound by theory, the significant increase in HO-1 may protect cells from apoptosis and DNA damage and improve their survival compared to cells not pretreated with RRx-001.

[0207] Example 4 - Evaluation of RRx-001 for the Treatment of Oral Mucositis

[0208] In this example, the ability of RRx-001 to treat acute radiation-induced oral mucositis was evaluated in hamsters.

[0209] Fifty-six (56) male Syrian golden hamsters were used in this study. Mucositis was induced by an acute radiation dose of 40 Gy delivered to the left cheek pouch at a rate of 2-2.5 Gy / min on day 0. Clinical evaluation of mucositis began on day 6 and continued every other day until day 28. Hamsters with a mucositis severity score of 4 or greater received buprenorphine (0.5 mg / kg) subcutaneously (SC) twice daily for 48 hours or until the score dropped below 4.

[0210] The dosing schedule was as follows: for animals in Groups 1-4, RRx-001 (1, 3, or 10 mg / kg) or vehicle (1:2 DMA:PEG400 volume:volume ratio) was administered once daily (QD) via intraperitoneal (IP) administration on days -4, -1, 1, 4, 7, 11, 14, 18, 21, and 25; and for animals in Groups 5-7, RRx-001 (1, 3, or 10 mg / kg) was administered once daily on days -4, -1, 1, 8, 15, and 22.

[0211]

[0212] Due to adverse side effects following administration of the highest dose of RRx-001 (10 mg / kg, Groups 2 and 5), dosing with this compound was discontinued for the remainder of the study after Day 1; however, animals continued to be monitored and scored during the study. Dosing for all other groups continued as planned. Following the completion of the study, on Day 28, animals were euthanized by CO2 inhalation and death was confirmed by monitoring heartbeat according to USDA guidelines. Throughout the study, the animals steadily gained weight, except for animals in Groups 2 and 5, which were characterized by lower body weight than all other groups. For Groups 2 and 5, body weight slowly recovered after dosing was stopped, and by the time the study was terminated, body weight had rebounded to be consistent with the other groups in the study.

[0213] The mean daily mucositis scores are shown in Figure 13. In Group 2, animals treated with 10 mg / kg exhibited a modest but significant increase in disease healing, but dosing was terminated after Day 1. All other treatment groups had mucositis scores that were fairly close to each other, and vehicle-dosed controls were used.

[0214] By using chi-square analysis to compare the days of mucositis score ≥3 and <3 between control group and treatment group, the significance of the difference observed between the two groups was evaluated. Compared with the vehicle control group, the animals administered with 10mg / kg and 1mg / kg RRx-001 showed significant improvement (Figure 14) in terms of mucositis score for many days. In the vehicle group, the percentage of animal days with score ≥3 was 57.29%. Compared with the vehicle group, the percentage of days with score ≥3 of the animals in group 2 (administered with 10mg / kg on days -4, -1, 1) was statistically lower (p < 0.01). There were many days with the following situation: compared with the animals administered with vehicle, the animals administered with RRx-001 had a lower ulcer days percentage (this can be interpreted as the improvement of disease severity). For the concentration of 3 and 1mg / kg, administration twice a week seemed to provide a more beneficial effect (group 4 is relative to group 7) on the ulcer days percentage than administration once a week. Interestingly, in both Groups 2 and 5, animals dosed at the 10 mg / kg concentration (dosed only on days -4, -1, and 1) had the best response in terms of reduction in the percentage of ulcers.

[0215] Analysis of mucositis severity was performed using Mann-Whitney rank sum analysis to compare visual mucositis scores between the treatment groups and the vehicle control group on each day of evaluation. The results of this analysis are presented in Figure 5 and Figure 6. In this analysis, a significant decrease in mucositis scores typically requires 2 days to be considered significant. Animals dosed with 10 mg / kg and 1 mg / kg RX-001 demonstrated significant improvement in mucositis scores for multiple days compared to the vehicle control group. Figure 15 As shown, similar effects were observed for animals dosed 1X week.

[0216] On each day of evaluation, the percentage of animals in each group with ulcerative mucositis was Figure 16 . This evaluation is intended to clarify which days of treatment have the greatest impact on the course of ulcerative mucositis. There are many days where the animals administered with RRx-001 have a lower percentage of ulcer days (which can be interpreted as an improvement in disease severity) compared to the animals administered with the vehicle. For a concentration of 1 mg / kg, dosing twice a week appears to provide a more beneficial effect on the percentage of ulcer days than dosing once a week (Group 4 versus Group 7). Interestingly, in both Groups 2 and 5, animals administered with a concentration of 10 mg / kg (only administered on days -4, -1, and 1) had the best response in reducing the percentage of ulcers.

[0217] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes. The present disclosure is not limited to the specific embodiments described in this application, and it is intended that the embodiments be used as a single illustration of the separate aspects of the present disclosure. All different embodiments of the present disclosure will not be described herein. As will be apparent to those skilled in the art, various modifications and changes can be made to it without departing from the spirit and scope of the present disclosure. Based on the foregoing description, functionally equivalent methods and devices within the scope of the present disclosure, in addition to the methods and devices listed herein, are obvious to those skilled in the art. Such modifications and changes are intended to fall within the scope of the appended claims. The present disclosure is only limited to the terms of the appended claims and the full range of equivalents granted by these claims. It should be understood that the present disclosure is not limited to specific uses, methods, reagents, compounds, compositions or biological systems, but is of course subject to change. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments and are not intended to be restrictive.

[0218] In summary, the present invention includes but is not limited to the following:

[0219] 1. A method for treating a subject in need of protection against radiation, comprising administering to the subject in need thereof an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof, thereby protecting the subject from radiation.

[0220] 2. The method of claim 1 , wherein the administration achieves protection against radiation for a duration of at least 6 hours, at least 12 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

[0221] 3. The method of claim 1 , wherein the administration achieves protection against radiation for a duration of at least 48 hours.

[0222] 4. A method for reducing radiation exposure damage to a subject, the method comprising administering to the subject in need thereof an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof, thereby reducing radiation exposure damage to the subject.

[0223] 5. The method of claim 4, wherein the administering reduces radiation exposure damage to the subject for a duration of at least 6 hours, at least 12 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least

[0224] 4 weeks or more.

[0225] 6. The method of any one of items 1-5, wherein at least one dose of the therapeutic agent is administered to the subject prior to exposure to the radiation.

[0226] 7. The method of any one of items 1-6, wherein at least one dose of the therapeutic agent is administered to the subject within 48 hours prior to exposure to radiation.

[0227] 8. The method of any one of items 1-6, wherein the subject is administered at least one dose of the therapeutic agent within 24 hours prior to exposure to radiation.

[0228] 9. The method of any one of items 1-8, wherein the therapeutic agent is administered in a dose that provides RRx-001 in an amount ranging from about 0.01 mg to about 500 mg of RRx-001 on each day the therapeutic agent is administered to the subject.

[0229] 10. The method of any one of items 1-7, wherein the therapeutic agent is administered in a dose that provides RRx-001 in an amount ranging from about 0.1 mg to about 200 mg of RRx-001 on each day the therapeutic agent is administered to the subject.

[0230] 11. The method of any one of items 1-7, wherein the therapeutic agent is administered in a dose that provides RRx-001 in an amount ranging from about 0.5 mg to about 150 mg of RRx-001 on each day the therapeutic agent is administered to the subject.

[0231] 12. The method of any one of items 1 to 11, wherein the therapeutic agent is administered by a route selected from parenteral administration, oral administration, and topical administration.

[0232] 13. The method of any one of items 1-11, wherein the therapeutic agent is administered intravenously to the subject.

[0233] 14. The method of any one of items 1-11, wherein the therapeutic agent is administered intravenously to the subject as a single bolus injection, multiple injections, or infusion over time.

[0234] 15. The method of any one of items 1-11, wherein the therapeutic agent is administered intravenously to the subject over a duration of at least thirty minutes.

[0235] 16. The method of any one of items 1-11, wherein the therapeutic agent is administered to the subject by intraperitoneal injection.

[0236] 17. The method of any one of items 1-11, wherein the therapeutic agent is administered to the subject by intraperitoneal injection in the form of a single bolus injection, multiple injections, or infusion over time.

[0237] 18. The method of any one of items 1-11, wherein the therapeutic agent is administered to the subject by intraperitoneal injection over a duration of at least thirty minutes.

[0238] 19. The method of any one of items 1-11, wherein the therapeutic agent is administered by subcutaneous injection.

[0239] 20. The method of any one of items 1-11, wherein the therapeutic agent is administered to the subject by subcutaneous injection in the form of a single bolus injection, multiple injections, or infusion over time.

[0240] 21. The method of any one of items 1-11, wherein the therapeutic agent is administered to the subject by subcutaneous injection over a duration of at least 5 minutes.

[0241] 22. The method of any one of items 1-11, wherein the therapeutic agent is administered subcutaneously to the subject via a pump device containing the therapeutic agent implanted in the subject.

[0242] 23. The method of claim 22, wherein the pump device is an osmotic pump.

[0243] 24. The method of any one of items 1-23, wherein the therapeutic agent is administered to the subject once a week.

[0244] 25. The method of any one of items 1-23, wherein the therapeutic agent is administered to the subject once a week for at least two weeks.

[0245] 26. The method of any one of items 1-23, wherein the therapeutic agent is administered to the subject twice per week.

[0246] 27. A method according to any one of items 1-23, wherein the therapeutic agent is administered to the subject twice a week for at least two weeks.

[0247] 28. The method of any one of items 1-27, wherein the method further comprises administering an analgesic to the subject prior to administering the therapeutic agent.

[0248] 29. The method of claim 28, wherein the analgesic is aspirin, a corticosteroid, or a nonsteroidal anti-inflammatory agent.

[0249] 30. The method of any one of items 1-29, further comprising administering a local analgesic to tissue of the subject proximate to the site of administration of the first therapeutic agent prior to administering the therapeutic agent.

[0250] 31. The method of claim 30, wherein the local analgesic is a caine analgesic.

[0251] 32. The method of claim 30, wherein the topical analgesic comprises lidocaine.

[0252] 33. The method of claim 30, wherein the topical analgesic is lidocaine hydrochloride.

[0253] 34. The method of claim 30, wherein the topical analgesic is VanPen cream, an NSAID, or acetaminophen.

[0254] 35. A method according to any one of items 1-34, wherein the therapeutic agent is administered in the vicinity of tissue that is desired to be protected from radiation.

[0255] 36. The method of claim 35, wherein the tissue is bone marrow, skin, lung tissue, thyroid tissue, gonadal tissue, gastrointestinal tissue, bone tissue, fetal tissue, or a combination thereof.

[0256] 37. A method according to any one of items 1-36, wherein the subject is an adult.

[0257] 38. A method according to any one of items 1-36, wherein the subject is a child.

[0258] 39. The method of any one of items 1-36, wherein the subject is an animal.

[0259] 40. A method according to any one of items 1-39, wherein the subject is at risk of exposure to radiation from a nuclear emergency.

[0260] 41. A method according to any one of items 1-39, wherein the subject is receiving radiation therapy.

[0261] 42. A method according to claim 41, wherein the radiation therapy is used to treat cancer.

[0262] 43. The method of any one of items 1-42, further comprising administering an EGFR inhibitor to the subject.

[0263] 44. A method according to claim 43, wherein the EGFR inhibitor is administered to the subject according to a pulse dosing regimen.

[0264] 45. The method of item 43 or 44, wherein the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof.

[0265] 46. The method of any one of items 1-45, further comprising administering an inorganic nitrite to the subject.

[0266] 47. A method according to claim 46, wherein the inorganic nitrite is an alkali metal nitrite.

[0267] 48. A method according to claim 46, wherein the inorganic nitrite is sodium nitrite.

[0268] 49. A method of protecting a biological material from the damaging effects of radiation, the method comprising exposing the biological material to an effective amount of a therapeutic agent selected from RRx-001 and pharmaceutically acceptable salts thereof, thereby protecting the biological material from the damaging effects of radiation.

[0269] 50. The method of claim 49, wherein the biological material is protected from the damaging effects of radiation for a duration of at least 6 hours, at least 12 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least

[0270] 4 weeks or more.

[0271] 51. A method according to item 49 or 50, wherein the biological material is exposed to at least one dose of the therapeutic agent prior to exposure to the radiation.

[0272] 52. A method according to item 49 or 50, wherein the biological material is exposed to at least one dose of the therapeutic agent within 24 hours prior to exposure to the radiation.

[0273] 53. A method according to any one of items 49-52, wherein the biological material is exposed to the therapeutic agent once a week.

[0274] 54. A method according to any one of items 49-52, wherein the biological material is exposed to the therapeutic agent once a week for at least two weeks.

[0275] 55. A method according to any one of items 49-54, further comprising exposing the biological material to an inorganic nitrite.

[0276] 56. A method according to claim 55, wherein the inorganic nitrite is an alkali metal nitrite.

[0277] 57. A method according to claim 55, wherein the inorganic nitrite is sodium nitrite.

[0278] 58. The method according to any one of items 49-57, wherein the biological material comprises isolated cells,

[0279] Isolated tissue or isolated organ.

[0280] 59. A method according to claim 58, wherein the biological material comprises blood cells.

[0281] 60. A method according to any one of items 1-59, wherein the radiation is ionizing radiation.

[0282] 61. A method according to any one of items 1-60, wherein the radiation comprises alpha rays, beta rays, gamma rays, neutron radiation or a combination thereof.

[0283] 62. A method according to any one of items 1-60, wherein the radiation comprises x-rays.

[0284] 63. A method according to any one of items 1-62, wherein the radiation is ionizing radiation from sunlight.

[0285] 64. A method according to any one of items 1-62, wherein the radiation is ionizing radiation from a radioactive nucleus.

[0286] 65. A method according to any one of items 1 to 62, wherein the radiation is ionizing radiation from an explosive device.

[0287] 66. A method according to any one of items 1-65, wherein the therapeutic agent is RRx-001.

Claims

1. A method for treating a subject in need of protection against radiation, comprising administering to the subject in need thereof an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof, thereby protecting the subject from radiation.

2. The method of claim 1, wherein the administration achieves protection against radiation for a duration of at least 6 hours, at least 12 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

3. The method of claim 1, wherein the administration achieves protection against radiation for a duration of at least 48 hours.

4. A method for reducing radiation exposure damage to a subject, the method comprising administering to the subject in need thereof an effective amount of a therapeutic agent selected from RRx-001 and a pharmaceutically acceptable salt thereof, thereby reducing radiation exposure damage to the subject.

5. The method of claim 4, wherein the administration reduces radiation exposure damage to the subject for a duration of at least 6 hours, at least 12 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or longer.

6. The method of any one of claims 1-5, wherein at least one dose of the therapeutic agent is administered to the subject prior to exposure to the radiation.

7. The method of any one of claims 1-6, wherein the subject is administered at least one dose of the therapeutic agent within 48 hours prior to exposure to radiation.

8. The method of any one of claims 1-6, wherein the subject is administered at least one dose of the therapeutic agent within 24 hours prior to exposure to radiation.

9. The method of any one of claims 1-8, wherein the therapeutic agent is administered in a dose that provides RRx-001 in an amount ranging from about 0.01 mg to about 500 mg of RRx-001 on each day the therapeutic agent is administered to the subject.

10. The method of any one of claims 1-7, wherein the therapeutic agent is administered in a dose that provides RRx-001 in an amount ranging from about 0.1 mg to about 200 mg of RRx-001 on each day the therapeutic agent is administered to the subject.

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