Use of ink pterin and metabolites thereof to treat radiation exposure

Abstract

The present invention relates to the use of Inopterin and its metabolites to treat radiation exposure. The present invention provides methods for treating radiation exposure in a subject by administering Inopterin, Tetrahydrobiopterin, or Dihydrobiopterin.

Description

This application is a divisional application. The application date of the original application is August 5, 2020, the application number is 202080069308.5 (PCT / US2020 / 045026), and the name of the invention is "The use of septopterin and its metabolites for the treatment of radiation exposure."

[0001] Statement Regarding Federally Funded Research This invention was made with government support under Grant No. R01 AI133595 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. Background of the Invention The threat of radiation disasters represents a potentially catastrophic public health emergency, which emphasizes the need to develop medical countermeasures to mitigate radiation-induced damage and death throughout the body. The initial health consequence of total body radiation (TBI) exposure is the development of acute radiation syndrome (ARS) in the most radiation-sensitive organs, and the survival rate of this exposure depends on the degree of damage to stem cells and progenitor cells in the hematopoietic system and epithelial cells in the gastrointestinal system. Hematopoietic growth factors, electrolytes and fluids, blood transfusions and antibiotics are all advances in therapies for the treatment of ARS, which have led to significant improvements in survival rates. However, patients who survive ARS of the hematopoietic and GI systems usually die from advanced damage to the lungs and heart.

[0003] Therefore, there is a need for treatments that improve mortality and organ function in patients exposed to radiation. SUMMARY OF THE INVENTION The invention features methods for treating patients suffering from radiation exposure. For example, the invention uses sepiopterin (SP) to reduce toxicity to the patient's heart, gastrointestinal tract, and / or lungs.

[0005] In one aspect, the invention provides a method of treating a subject exposed to radiation by administering to the subject an effective amount of sepapterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharmaceutically acceptable salt or cocrystal thereof.

[0006] In certain embodiments, administration reduces or inhibits tissue and / or organ damage in a subject; reduces or inhibits cardiac, gastrointestinal, and / or pulmonary toxicity in a subject; reduces gastrointestinal, cardiac, and / or pulmonary endothelial cell death in a subject; and / or reduces radiation-induced inflammation in gastrointestinal, cardiac, and / or pulmonary epithelial cells in a subject.

[0007] An effective amount is, for example, about 0.1 to about 200 mg / kg / day, such as about 5 mg / kg / day to about 35 mg / kg / day.

[0008] In embodiments, the subject suffers from acute radiation syndrome, e.g., where administration is performed within 24 hours after exposure to radiation. In embodiments, the subject suffers from chronic radiation syndrome. In embodiments, the subject suffers from cutaneous radiation syndrome. In embodiments, the subject is exposed to at least 0.3 Gy in less than one day. In embodiments, the subject is exposed to at least 0.7 Gy over a period of more than one day.

[0009] In embodiments, administration is performed at least once a day for at least 6 days, e.g., at least 1 week, at least 10 days (e.g., at least 14 days), at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, or at least about 1 year.

[0010] In embodiments, administration increases expression of miR-15b-3p, miR-106a-5p, miR-133b, miR-136-5p, miR-451a, miR-1, miR-335-3p, let-7d-3p and / or let-7c-5p, e.g., in serum exosomes (e.g., when BH4 is administered), and / or decreases expression of IL-1β, IL-6, IL-17A, Spp2 and / or TGF-β1 in lung epithelial cells (e.g., when SP is administered). In embodiments, administration increases the expression of let-7a-5p, miR-1, miR-106b-3p, miR-106b-5p, miR-126-3p, miR-181a-5p, miR-335-3p and / or miR-335-5p and / or decreases the expression of let-7g-5p, let-7i-5p and / or miR-16-5p, e.g., serum exosomes (e.g., when SP is administered).

[0011] In one aspect, the invention features a method of reducing or inhibiting tissue and / or organ damage in a subject that has been exposed to radiation by administering to the subject an effective amount of septerin or a pharmaceutically acceptable salt thereof.

[0012] In another aspect, the invention features a method of reducing or inhibiting cardio- and / or pulmonary toxicity in a subject that has been exposed to radiation by administering to the subject an effective amount of septerin or a pharmaceutically acceptable salt thereof.

[0013] In another aspect, the invention features a method of reducing cardiac and / or pulmonary endothelial cell death in a subject that has been exposed to radiation by administering to the subject an effective amount of septerin or a pharmaceutically acceptable salt thereof.

[0014] In another aspect, the invention features a method of reducing radiation-induced inflammation in cardiac and / or pulmonary epithelial cells in a subject by administering to the subject an effective amount of septerin or a pharmaceutically acceptable salt thereof.

[0015] In one embodiment of the aforementioned four aspects, the effective amount of septerin or a pharmaceutically acceptable salt thereof is less than 1 mg / kg.

[0016] In another embodiment of the four preceding aspects, septerin or a pharmaceutically acceptable salt thereof is administered about 24 hours after exposure to radiation.

[0017] In another embodiment of the four preceding aspects, the method comprises administering septerin or a pharmaceutically acceptable salt thereof in multiple doses.

[0018] In another embodiment of the four preceding aspects, the method comprises administering septerin or a pharmaceutically acceptable salt thereof daily for at least 6 days.

[0019] In another embodiment of the aforementioned four aspects, an effective amount of septerin or a pharmaceutically acceptable salt thereof results in increased expression of miR-15b-3p, miR-106a-5p, miR-133b, miR-136-5p, miR-451a, miR-1, miR-335-3p, let-7d-3p and / or let-7c-5p.

[0020] definition In this application, unless the context clearly indicates otherwise, (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to include the listed components or steps, whether presented alone or with one or more additional components or steps; and (iv) the terms "about" and "approximately" may be understood to allow for standard deviations as would be understood by a person of ordinary skill in the art; and (v) where a range is provided, the endpoints are included.

[0021] In some embodiments, the term "about" as used herein means ± 10% of a specified value.

[0022] Reference herein to "about" or "approximately" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, description referring to "about X" includes description of "X".

[0023] As used herein, the term "administering" refers to administering a composition to a subject. An animal subject (e.g., a human) may be administered by any appropriate route. For example, in some embodiments, administration may be intrabronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, or vitreous.

[0024] An "effective amount" of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the compound to elicit a desired response. A therapeutically effective amount includes an amount in which any toxic or deleterious effects of the compound are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also includes an amount sufficient to confer a benefit (e.g., a clinical benefit).

[0025] "Level" means the level of a compound compared to a reference. A reference can be any useful reference as defined herein. "Reduced level" or "increased level" of a compound means a decrease or increase in the level of the compound compared to a reference (e.g., a decrease or increase of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 350%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about The level of a compound can be expressed as a mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or as a percentage of the total compound in the sample.

[0026] As used herein, the term "pharmaceutical composition" means a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient. A pharmaceutical composition may be a composition that is manufactured or sold as part of a therapeutic regimen for treating a mammalian disease with approval by a governmental regulatory agency. For example, a pharmaceutical composition may be formulated in unit dosage form for oral administration (e.g., tablets, capsules, caplets, caplets, powders for suspensions, suspensions, solutions, or syrups); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or formulated into any other pharmaceutically acceptable formulation.

[0027] As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of septopterin, tetrahydrobiopterin or dihydrobiopterin. Pharmaceutically acceptable salts include ion pairs of septopterin, tetrahydrobiopterin or dihydrobiopterin in solid state and / or solution. Pharmaceutically acceptable co-crystals include free base septopterin, tetrahydrobiopterin or dihydrobiopterin and solid acid. A mixture of salt form and co-crystal form may be present in the same composition. For example, pharmaceutically acceptable salts of septopterin, tetrahydrobiopterin or dihydrobiopterin include those salts that are within the scope of reasonable medical judgment, suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Remington (Remington: The Science and Practice of Pharmacy, (22nd Edition) LV Allen, Jr. Edited, 2013, Pharmaceutical Press, Philadelphia, PA). The salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate.

[0028] "Reference" means any useful reference for comparing compound levels or other symptoms, such as tissue and / or organ damage, toxic cells, death or inflammation. The reference may be any sample, standard, standard curve or level for comparison purposes. The reference may be a normal reference sample or reference standard or level. A "reference sample" may be, for example, a control, such as a predetermined negative control value, such as a "normal control" or a previous sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (such as a cell or tissue) from a subject without a disease; a sample from a subject diagnosed with a disease but not yet treated with the compounds of the invention; a sample from a subject treated with the compounds of the invention; or a sample of a purified compound (such as any compound described herein) at a known normal concentration. "Reference standard or level" means a value or quantity derived from a reference sample. A "normal control value" is a predetermined value indicating a non-disease state, such as an expected value in a healthy control subject. Generally, a normal control value is expressed as a range ("between X and Y"), a high threshold ("not higher than X"), or a low threshold ("not lower than X"). A subject having a measured value within the normal control value for a particular biomarker is generally referred to as being "within normal limits" for that biomarker. A normal reference standard or level may be a value or quantity derived from a normal subject without a disease or disorder (e.g., ARS). In a preferred embodiment, a reference sample, standard, or level is matched to a subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of levels of a purified compound (e.g., any compound described herein) within a normal reference range may also be used as a reference.

[0029] As used herein, the term "subject" or "patient" refers to any organism to which the compositions of the invention may be administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may be seeking or requiring treatment, in need of treatment, currently receiving treatment, being treated in the future, or a human or animal being cared for by a trained professional for a particular disease or condition.

[0030] As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and prophylactic or preventative measures, wherein the purpose is to prevent or slow down (mitigate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, relief of symptoms; reduction of the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; delaying the onset of a condition, disorder, or disease progression or slowing the progression of a condition, disorder, or disease; improving the condition, disorder, or disease state or alleviation (whether partial or complete), whether detectable or undetectable; improving at least one measurable physical parameter (not necessarily discernible by the patient); or enhancing or ameliorating a condition, disorder, or disease. Treatment includes inducing a clinically significant response without an excessive level of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment.

[0031] It should be understood that descriptions of compounds, compositions, formulations, and methods of treatment described herein include embodiments of "comprising," "consisting of," and "consisting essentially of." In some embodiments, for all compositions described herein and all methods of using the compositions described herein, the composition may comprise the listed components or steps, or may "consist essentially of the listed components or steps." When a composition is described as "consisting essentially of the listed components," the composition contains the listed components and may contain other components that do not significantly affect the condition being treated, but does not contain any other components that significantly affect the condition being treated other than those components explicitly listed; or, if the composition does contain additional components that significantly affect the condition being treated other than those listed, the composition does not contain the additional components in sufficient concentration or amount to significantly affect the condition being treated. When a method is described as "consisting essentially of the listed steps," the method contains the listed steps and may contain other steps that do not significantly affect the condition being treated, but does not contain any other steps that significantly affect the condition being treated other than those steps explicitly listed. As a non-limiting specific example, when a composition is described as "consisting essentially of one component," the composition may additionally contain any amount of pharmaceutically acceptable carriers, vehicles or diluents and other such components that do not significantly affect the condition being treated.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. Materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail.

[0033] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The experimental protocol used in the present invention is described. Experimental groups received radiation treatment followed by tetrahydrobiopterin (BH4), sepiopterin (SP) or vehicle treatment and were evaluated at 10, 30, 60, 90 and 180 days after radiation treatment.

[0035] Figure 2 Isoproterenol responses of vehicle-, BH4-, and SP-treated groups of the irradiated group are illustrated. Evaluation of contractile reserve was measured at 10, 30, 60, 90, and 180 days.

[0036] Figure 3 Myocardial performance index for vehicle-, BH4- and SP-treated groups are illustrated for radiation-treated groups. Evaluation of myocardial performance index was measured at 10, 30, 60, 90 and 180 days.

[0037] Figure 4 Shown are the survival rates of experimental groups treated with radiation followed by vehicle or SP.

[0038] Figure 5 Illustrated is the respiratory rate of the irradiated experimental group relative to the control group.

[0039] Figures 6A-6C The relative expression of serum exosomal miRNAs after radiation treatment normalized to the untreated control group is shown. Data are provided for experimental groups treated with radiation, BH4, or both (6A) and radiation, septopterin, or both (6B (inhibits expression) and 6C (induces expression)).

[0040] Figure 7Illustrate the isoproterenol response of the vehicle- or SP-treated group of the radiation-treated group. The evaluation of contractile reserve was measured at 0, 30, 60, 90 and 180 days. Compared with the vehicle baseline at 10, 30, 60, 90 and 180 days after irradiation, the contractile reserve of the irradiated vehicle group was reduced by radiation (*p<0.05 relative to baseline). Giving SP for 6 days slowed down the decline of contractile reserve at all time points (#p<0.05 relative to the same time point of vehicle). Data are expressed as mean ± SEM.

[0041] Figure 8 The systolic and diastolic function of the vehicle- or SP-treated groups at 10, 30, 60, 90 and 180 days after radiation treatment are illustrated. The data are plotted as Tei index and isovolumetric relaxation time (IRT). Sepiampterin alleviates the inhibition of systolic (Tei index) and diastolic (Tei index and IRT) function caused by radiation. From 10 days and until 180 days, the irradiated mice receiving vehicle treatment showed a significant gradual increase in Tei and IRT (*p<0.05 relative to baseline). Giving SP for 6 days slowed down the increase in Tei index and IRT at all time points (#p<0.05 relative to the same time point of vehicle). Data are expressed as mean ± SEM.

[0042] Fig. 9 Lung function of irradiated experimental groups receiving vehicle or SP treatment is illustrated. Respiratory rate was recorded every 2 weeks for 5 minutes. SP treated mice had significant improvement in lung function at all measurement points. For irradiation + vehicle, n = 7-12, and for irradiation + SP, n = 8-14. *p < 0.05.

[0043] Fig.10 Figure 3. Changes in cytokine mRNA expression in heart and lung tissues. Plotted data represent radiation-treated experimental groups that received vehicle or SP treatment. Post-irradiation changes in cytokine mRNA expression in lung and heart tissues of animals that received vehicle or SP treatment. Lung and heart tissues were collected from animals at different times after irradiation. Total RNA was extracted from all tissue samples and qPCR was performed for cytokine mRNA. Data are expressed as mean ± SD of 5-6 animals in each group. P values ​​were calculated using Student's t-test and are shown as: **-p<0.05; ***-p<0.001.

[0044] Figures 11A-11C Illustrated are immunohistochemistry (IHC) (11A-11B) and hydroxyproline (11C) analyses of the control group and irradiated experimental groups receiving vehicle or SP treatment measured at 16 weeks.

[0045] Fig.12Illustrate the Spearman correlation between fibrosis and relative expression of cytokine mRNA in the lungs of vehicle- or SP-treated mice at 16 weeks after radiation. Distribution plot of significant Spearman correlations between fibrosis and relative expression of cytokine mRNA in the lungs of vehicle- or SP-treated mice at 16 weeks after radiation. The darkest shades represent mice with higher inflammation / fibrosis. IL = Interleukin, Ccl2 = CC Motif Chemokine Ligand 2, Runx2 = Runt-related transcription factor 2, Ccl2 = CC Motif Chemokine Ligand 2, Runx2 = Runt-related transcription factor 2, Ctgf = Connective Tissue Growth Factor, Spp2 = Secreted Phosphoprotein 2, TGF-β1 = Tissue Growth Factor β1, BMP2 = Bone Morphogenetic Protein 2, Trim72 = Tripartite Motif Containing 72, IR = Radiation, M = Male, F = Female, r = Spearman Rank Correlation Coefficient.

[0046] Figures 13A-13D Proteins measured by ELISA as potential biomarkers are illustrated: CRP (13A), fibrinogen (13B), IL-6 (13C) and neutrophil elastase (13D). Measurements were made at 4, 8, 16 and 180 days in the irradiated experimental groups that received vehicle or SP treatment and compared to the control group.

[0047] Figures 14A-14B Illustrated are the effects of vehicle (14A) and SP (14B) on systolic function after radiation as assessed by sex.

[0048] Figures 15A-15B Illustration of the effects of vehicle (15A) and SP (15B) on diastolic function after radiation assessed by gender.

[0049] Figures 16A-16B The effects of vehicle (16A) and SP (16B) on contractile reserve assessed by sex are illustrated. DETAILED DESCRIPTION OF THE INVENTION The invention features methods of treating a subject exposed to radiation using sepipterin, tetrahydrobiopterin, dihydrobiopterin, or pharmaceutically acceptable salts or cocrystals thereof. Without wishing to be bound by theory, we believe that radiation-induced late pulmonary, gastrointestinal, and cardiac toxicity is a consequence of endothelial dysfunction, defined as uncoupled NOS activity and reduced NO bioavailability, which establishes a chronic inflammatory state that drives a persistent profibrotic process associated with aberrant wound repair and late normal tissue damage. In a murine model with radiation sensitivity similar to that of humans, we show that sepipterin and its metabolites, such as tetrahydrobiopterin and dihydrobiopterin, can be used as radiation countermeasures to mitigate radiation-induced cardiac and pulmonary damage and improve survival.

[0051] Active compounds The method of the present invention is characterized by using sepipterin, tetrahydrobiopterin or dihydrobiopterin or a salt and / or co-crystal thereof. Sepipterin can be converted into dihydrobiopterin and tetrahydrobiopterin in vivo, and dihydrobiopterin and tetrahydrobiopterin are mutually converted in vivo.

[0052] Sepipterin has the following structure:

[0053] Tetrahydrobiopterin has the following structure:

[0054] Dihydrobiopterin has the following structure:

[0055] The active compound may be used in any suitable form, for example as a free base, a salt or a co-crystal. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gentisinate, glucoheptonate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. In some embodiments, the active compound or salt thereof is in crystalline form.

[0056] Exemplary salts, co-crystals and crystalline forms of septopterin are described in WO 2018 / 102314, WO 2018 / 102315, WO 2019 / 046849 and WO 2019 / 232120, for example any of crystalline forms A, B, C, D, E, F or G described in WO 2018 / 102314 or WO 2018 / 102315. Other salts or crystalline forms of septopterin, dihydrobiopterin and / or tetrahydrobiopterin are known in the art.

[0057] Treatment The present invention provides methods for treating a subject after radiation exposure, for example in the treatment of acute radiation syndrome (ARS), cutaneous radiation syndrome or chronic radiation syndrome. In particular, the present invention provides methods for treating a patient exposed to at least about 0.05 Gy in a period of less than 24 hours. For example, the patient may have been exposed to at least about 0.3, at least about 0.7, at least about 1, at least about 3, at least about 5, at least about 6, at least about 8, at least about 10, at least about 15, at least about 20, at least about 30, or at least about 50 Gy, such as between about 0.3-about 6, between about 0.7-about 6, between about 1-about 2, between about 2-about 6, between about 6-about 20, between about 6-about 10, between about 6-about 8, between about 10-about 20, between about 8-about 12, or between about 20-about 50 Gy, and the exposure period may be less than about 18 hours, such as less than about 12, less than about 6, less than about 3, less than about 1, less than about 0.5, or less than about 0.1 hours, or less than about 5, less than about 3, less than about 2, or less than about 1 minute. Alternatively, the present invention provides a method of treating a patient exposed to at least about 0.7 Gy over a period of more than 24 hours. For example, the patient may have been exposed to at least about 0.7, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, or at least about 8 Gy, and the exposure period may be at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years, at least about 3 years, or longer, such as about 1 week-3 years, about 1 week-1 year, about 1 week-1 month, about 1 month-2 years, about 1 month-1 year, about 6 months-2 years, about 9 months-2 years, or about 9 months-15 months. The radiation exposure may be systemic or localized, for example, to the skin. In certain embodiments, the radiation is not produced by radiation therapy.

[0058] Treatment according to the invention can result in reducing or inhibiting tissue and / or organ damage in a subject that has been exposed to radiation; reducing or inhibiting tissue or organ toxicity (e.g., lung, heart, or gastrointestinal (such as lung or heart)) in a subject that has been exposed to radiation; reducing endothelial cell death (e.g., lung, heart, or gastrointestinal (such as lung or heart)) in a subject that has been exposed to radiation; reducing radiation-induced inflammation in epithelial cells (e.g., lung, heart, or gastrointestinal (such as lung or heart)) of a subject; increasing miR-15b-3p, miR-106a-5p, miR-133b, miR-136-5p, miR-451a , miR-1, miR-335-3p, let-7d-3p and / or let-7c-5p; reduce the expression of IL-1β, IL-6, IL-17A, Spp2 and / or TGF-β1 in, for example, lung epithelial cells; increase the expression of let-7a-5p, miR-1, miR-106b-3p, miR-106b-5p, miR-126-3p, miR-181a-5p, miR-335-3p and / or miR-335-5p; and / or reduce the expression of let-7g-5p, let-7i-5p and / or miR-16-5p. The reduction in symptoms or cytokines can be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, for example relative to a reference. The increase in oligonucleotide expression can be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, or at least about 500%, for example relative to a reference.

[0059] The active compound can be administered in any suitable dose. The actual dose of the composition of the present invention administered to the patient can be determined according to the patient's physical and physiological factors such as body weight, severity of the disease, previous or simultaneous therapeutic interventions, idiopathic diseases, and according to the route of administration. Depending on the dose and route of administration, the number of administrations of the preferred dose and / or effective amount can vary according to the subject's response. In any case, the practitioner responsible for administration will determine the concentration and appropriate dose of the active ingredient in the composition for the individual subject. The therapeutically effective amount can be about 0.1 mg / kg / day to about 200 mg / kg / day, for example, about 0.1-about 150 mg / kg / day, about 0.1-about 125 mg / kg / day, about 0.1-about 100 mg / kg / day, about 0.1-about 80 mg / kg / day, about 0.1-about 60 mg / kg / day, about 0.1-about 40 mg / kg / day, about 0.1-about 25 mg / kg / day, about 0.1-about 20 mg / kg / day, about 0.1-about 15mg / kg / day, about 0.1-about 10mg / kg / day, about 0.1-about 5mg / kg / day, about 0.1-about 2.5mg / kg / day, about 0.1-about 1mg / kg / day, about 0.1-about 0.5mg / kg / day, about 0.5-about 200mg / kg / day, about 1-about 200mg / kg / day, about 2.5-about 200mg / kg / day, about 5-about 200m g / kg / day, about 10-about 200 mg / kg / day, about 15-about 200 mg / kg / day, about 20-about 200 mg / kg / day, about 25-about 200 mg / kg / day, about 40-about 200 mg / kg / day, about 60-about 200 mg / kg / day, about 80-about 200 mg / kg / day, about 100-about 200 mg / kg / day, about 120-about 200 mg / kg / day, about 140-about The active compound of about 200mg / kg / day, about 160-about 200mg / kg / day, about 180-about 200mg / kg / day, about 5-about 180mg / kg / day, about 10-about 160mg / kg / day, about 20-about 140mg / kg / day, about 40-about 120mg / kg / day, about 60-about 100mg / kg / day, about 5-about 50mg / kg / day or about 10-about 20mg / kg / day can be administered at any suitable number of times, such as once a day, twice a day or three times a day during the treatment period. Administration can be continuous as long as necessary, such as from 1 day to about 1 year or at least 6 days, at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 3 months, at least about 6 months or at least about 9 months.

[0060] The active compound can be first administered at any suitable time, for example within about 1 hour, about 2 hours, about 6 hours, about 12 hours, or about 18 hours, about 24 hours, about 2 days, or about 1 week of radiation exposure.

[0061] preparation The active compound can be formulated into a pharmaceutical composition as known in the art. Such compositions may include various components as known in the art, for example, see Remington: The Science and Practice of Pharmacy, (22nd Edition) LV Allen, Jr. Editor, 2013, Pharmaceutical Press, Philadelphia, PA.

[0062] The composition may include a pharmaceutically acceptable carrier, such as any of those carriers that are conventionally used and limited only by chemico-physical considerations (such as solubility and lack of reactivity with the compound) and the route of administration. Pharmaceutically acceptable carriers, such as vehicles, adjuvants, excipients or diluents, as described herein are well known to those skilled in the art and are readily available to the public. It is preferred that a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active compound and a carrier that has no harmful side effects or toxicity under the conditions of use.

[0063] Antioxidants The pharmaceutical composition used in the method may or may not include an antioxidant. Antioxidants can minimize oxidative degradation of the active compound. Examples of antioxidants include, but are not limited to, 4-chloro-2,6-di-tert-butylphenol, tocopherol, alpha-tocopherol, alkylated diphenylamines, ascorbic acid, ascorbyl myristate, ascorbyl palmitate, ascorbyl stearate, beta-carotene, butylated hydroxyanisole, butylated hydroxytoluene, citric acid, cysteine, D-alpha-tocopheryl polyethylene glycol 1000 succinate, deferoxamine mesylate, dodecyl gallate, ethyl paraben, folic acid, fumaric acid, gallic acid, glutathione, egg Phospholipids, malic acid, methyl paraben, monothioglycerol, N-acetylcysteine, nordihydroguaiaretic acid, octyl gallate, p-phenylenediamine, potassium ascorbate, potassium metabisulfite, potassium sorbate, propionic acid, propyl gallate, retinol, sorbic acid, sodium ascorbate, sodium bisulfite, sodium dithionite, sodium isoascorbate, sodium metabisulfite, sodium sulfite, sodium thiosulfate, tartaric acid, tert-butylhydroquinone, tocopheryl acetate, vitamin A, vitamin B6, vitamin B12, or vitamin E. In some embodiments, the methods of the present invention can use a pharmaceutical composition comprising ascorbic acid, tocopherol, retinol, ascorbyl palmitate, N-acetylcysteine, glutathione, butylhydroxytoluene and / or butylhydroxyanisole as an antioxidant.

[0064] In some embodiments, the method includes a pharmaceutical composition comprising less than about 10% antioxidant by weight, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In some embodiments, the method uses a pharmaceutical composition comprising about 2-9% antioxidant by total weight, such as about 2-4%, about 3-5%, about 4-6%, about 5-7%, about 6-8%, or about 7-9%. In some embodiments, the method uses a pharmaceutical composition comprising about 5-100% of the USP maximum daily dose of antioxidant, for example, in some embodiments, the method uses a pharmaceutical composition comprising about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the USP maximum daily dose of antioxidant. In some embodiments, the ratio of sepiaterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharmaceutically acceptable salt and / or co-crystal thereof to the antioxidant is at least about 1:1, e.g., at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, or at least about 10:1 wt / wt. In some embodiments of the compositions described herein, the composition comprises an antioxidant (e.g., ascorbic acid), wherein the ratio of a pharmaceutically acceptable salt and / or co-crystal of sepiaterin, tetrahydrobiopterin, or dihydrobiopterin to the antioxidant is greater than about 4:1 (e.g., greater than about 5:1, greater than about 6:1, greater than about 7:1, greater than about 8:1, greater than about 9:1, greater than about 10:1, greater than about 15:1, or greater than about 20:1) by weight (e.g., weight of salt to weight of antioxidant).

[0065] Dispersants In some embodiments, the method uses a pharmaceutical composition comprising at least one dispersant. The dispersant can cause the particles in the formulation to separate, for example, releasing its medicinal substance when in contact with moisture. Examples of dispersants include, but are not limited to, cross-linked polyvinyl pyrrolidone, carboxymethyl cellulose (e.g., cross-linked carboxymethyl cellulose salts, such as cross-linked carboxymethyl cellulose sodium), starch (e.g., sodium starch glycolate), or alginic acid. In some embodiments, the dispersant in the pharmaceutical composition is carboxymethyl cellulose, such as a pharmaceutically acceptable salt of cross-linked carboxymethyl cellulose. In some embodiments, the method uses a pharmaceutical composition that may contain about 0.1-1.5% of a dispersant by total weight, for example, about 0.1%, about 0.5%, about 1%, or about 1.5%. In some embodiments, the method uses a pharmaceutical composition that contains less than about 1.5% of a dispersant, for example, less than about 1%, less than about 0.5%, or less than about 0.1%.

[0066] Anti-caking agent Anti-caking agents are usually added to pharmaceutical compositions to prevent, for example, the formation of lumps in a solution. Therefore, in some embodiments, the pharmaceutical composition for the method of the present invention comprises at least one anti-caking agent. In some embodiments, the pharmaceutical composition for the method of the present invention comprises at least two anti-caking agents. Exemplary anti-caking agents include colloidal silicon dioxide, microcrystalline cellulose, tricalcium phosphate, microcrystalline cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, calcium phosphate, sodium silicate, colloidal silicon dioxide, calcium silicate, magnesium trisilicate, talc, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid and polydimethylsiloxane. In some embodiments, at least one anti-caking agent is colloidal silicon dioxide or microcrystalline cellulose. In some embodiments, the pharmaceutical composition for use in the methods of the present invention may comprise about 65-75% of an anti-caking agent by total weight, e.g., about 65%, about 67%, about 70%, about 73%, or about 75%. In some embodiments, the pharmaceutical composition for use in the methods of the present invention comprises both colloidal silicon dioxide and microcrystalline cellulose. In some embodiments, the pharmaceutical composition for use in the methods of the present invention comprises about 60-65% microcrystalline cellulose by total weight and about 5-7% colloidal silicon dioxide by total weight.

[0067] Drug delivery vehicle In some embodiments, the pharmaceutical composition used in the methods of the present invention is combined with a dosing vehicle prior to administration. In some embodiments, the composition can be administered in a dosing vehicle having a viscosity of about 50-1750 centipoise (cP), for example, to aid in the suspension and administration of the pharmaceutical composition. One type of suspending agent that can be used is a combination of glycerol and sucrose in water (e.g., a 2.5% glycerol and 27% sucrose in water). Oral mixture). An appropriate amount of the composition can be added to the administration vehicle mixture, and the mixture is stirred to suspend the composition just prior to administration.

[0068] Other suspending agents may also be used as administration vehicles. Exemplary suspending agents include water, agar, alginic acid, sodium carboxymethylcellulose, carrageenan, dextrin, gelatin, guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hypromellose, methylcellulose, polyethylene glycol, povidone, tragacanth gum, xanthan gum, or other suspending agents known in the art.

[0069] Ways to Give There is a wide variety of formulations suitable for use with the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal, and vaginal administration are exemplary only and are in no way limiting.

[0070] The pharmaceutical composition may be a liquid preparation, such as in the form of a solution, suspension or emulsion. Formulations suitable for oral administration may consist of: (a) capsules, sachets, tablets, lozenges and lozenges, each containing a predetermined amount of the active ingredient as a solid or granules; (b) powders; (c) liquid solutions, such as an effective amount of the compound dissolved in a diluent such as water, saline or orange juice; (d) suspensions in appropriate liquids; and (e) suitable emulsions. Preferred are solid oral dosage forms, such as capsule forms, tablet forms and powder forms. Capsule forms may be of the ordinary hard or soft shell gelatin type containing, for example, surfactants, lubricants and inert fillers such as lactose, sucrose, calcium phosphate and corn starch. Tablet forms may contain one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, gum arabic, gelatin, guar gum, colloidal silicon dioxide, crosslinked sodium carboxymethylcellulose, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid and other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring agents and pharmacologically compatible carriers. Lozenge forms may contain the active ingredient in a flavoring agent, usually sucrose and gum arabic or tragacanth, and pastilles contain the active ingredient in an inert matrix, such as gelatin and glycerin, or sucrose and gum arabic, emulsions, gels, etc. contain in addition to the active ingredient such carriers as are known in the art.

[0071] Formulations suitable for oral and / or parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostats and / or solutes that render the formulation isotonic with the blood of the intended recipient) and aqueous and non-aqueous sterile suspensions (which may include suspending agents, solubilizers, thickening agents, stabilizers and / or preservatives). The compounds can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or liquid mixture, including water, saline, aqueous dextrose and related sugar solutions, alcohols (such as ethanol, benzyl alcohol or hexadecanol), glycols (such as propylene glycol or polyethylene glycol and other polyethylene glycols), glycerol ketals (such as 2,2-dimethyl-1,3-dioxolane-4-methanol), ethers (such as poly(ethylene glycol) 400), oils, fatty acids, fatty acid esters or glycerides or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants (such as soaps or detergents), suspending agents (such as pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose) or emulsifiers and other pharmaceutical adjuvants.

[0072] Oils useful for parenteral preparations include petroleum, animal, vegetable or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, vaseline and mineral oil. Suitable fatty acids for parenteral preparations include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for parenteral preparations include fatty alkali metal, ammonium and triethanolamine salts, and suitable detergents include: (a) cationic detergents, such as dimethyl dialkyl ammonium halides and alkyl pyridines halides, (b) anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as alkyl-β-aminopropionates and 2-alkyl-imidazopeak quaternary ammonium salts, and (3) mixtures thereof.

[0073] Parenteral formulations generally contain about 20 to about 30% active compound by weight in solution. Suitable preservatives and buffers can be used in such formulations. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters (such as sorbitan monooleate) and high molecular weight adducts of ethylene oxide with hydrophobic bases (formed by the condensation of propylene oxide with propylene glycol). Parenteral formulations may be present in unit dose or multi-dose sealed containers (such as ampoules and vials) and may be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier for injection, such as water, before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described previously.

[0074] The pharmaceutical composition may be an injectable formulation. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Remington: The Science and Practice of Pharmacy, (22nd edition) LV Allen, Jr. ed., 2013, Pharmaceutical Press, Philadelphia, PA.

[0075] Topical preparations, including those that can be used for transdermal drug delivery, are well known to those skilled in the art and are suitable for application to the skin in the context of the present invention. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to oral mucosa, including oral cavity, oral epithelium, palate, gums and nasal mucosa. In some embodiments, the composition used for the method of the present invention contains septopterin, tetrahydrobiopterin or dihydrobiopterin and a suitable vehicle or carrier. It may also contain other components, such as anti-irritants. The carrier may be a liquid, solid or semisolid. In some embodiments, the composition is an aqueous solution. Alternatively, the composition may be a dispersion, emulsion, gel, lotion or cream vehicle of various components. In one embodiment, the primary vehicle is water or a biocompatible solvent that is substantially neutral or has become substantially neutral. The liquid vehicle may include other materials, such as buffers, alcohols, glycerol and mineral oils, together with various emulsifiers or dispersants as known in the art, to obtain the desired pH, consistency and viscosity. It is possible that the composition can be prepared as a solid, such as a powder or granules. The solid can be applied directly, or dissolved in water or a biocompatible solvent before use to form a solution, which is substantially neutral or has become substantially neutral, and can then be applied to the target site. In embodiments of the present invention, vehicles for topical application to the skin may include water, buffered solutions, various alcohols, glycols (such as glycerol), lipid materials (such as fatty acids), mineral oils, phosphoglycerides, collagen, gelatin, and silicone-based materials.

[0076] The pharmaceutical composition may be an aerosol preparation for administration by inhalation. These aerosol preparations may be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, etc. They may also be formulated as medicaments for non-pressurized preparations, such as in a nebulizer or atomizer.

[0077] Additionally, the pharmaceutical composition may be a suppository. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0078] Solid dosage forms for oral administration Formulations for oral use include granules containing the active compound mixed with nontoxic pharmaceutically acceptable excipients, and such formulations are known to the skilled artisan (e.g., U.S. Pat. Nos. 5,817,307, 5,824,300, 5,830,456, 5,846,526, 5,882,640, 5,910,304, 6,036,949, 6,036,949, 6,372,218, hereby incorporated by reference). The excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives (including microcrystalline cellulose), starch (including potato starch), cross-linked sodium carboxymethyl cellulose, alginates or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose); , hydroxypropyl methylcellulose, ethyl cellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants, anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oils or talc) and anti-caking agents (e.g., colloidal silicon dioxide, microcrystalline cellulose, tricalcium phosphate, microcrystalline cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, calcium phosphate, sodium silicate, colloidal silicon dioxide, calcium silicate, magnesium trisilicate, talc, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, polydimethylsiloxane). Other pharmaceutically acceptable excipients may be colorants, flavoring agents, plasticizers, wetting agents and buffers. In some embodiments, excipients (e.g., flavoring agents) are packaged together with the composition. In some embodiments, excipients (eg, flavoring agents) are packaged separately from the composition (eg, combined with the composition prior to administration).

[0079] The solid composition used in the method of the present invention may include a coating suitable for protecting the composition from undesirable chemical changes (e.g., chemical degradation prior to release of the active substance). The coating may be applied to the solid dosage form in a manner similar to that described in Remington: The Science and Practice of Pharmacy, (22nd Edition) LV Allen, Jr., ed., 2013, Pharmaceutical Press, Philadelphia, PA.

[0080] Powders and granules can be prepared using the above-mentioned ingredients in a customary manner using, for example, mixers, fluidized bed apparatus, melt-congealing apparatus, rotor granulators, extruders / spheronizers or spray-drying apparatus. Example

[0081] Although certain features of the present invention have been illustrated and described herein, those of ordinary skill in the art will now expect many modifications, substitutions, variations and equivalents. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations that fall within the true spirit of the present invention. Therefore, the following examples are provided to teach various aspects of the present invention. These examples represent separate embodiments of various aspects of the present invention, and those of ordinary skill in the art will recognize that additional examples can be produced to teach various aspects of the present invention in the same manner.

[0082] Example 1. Treatment of C57L / J mouse group animal Both male and female C57L / J wild-type 6-8 week old mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed on a 12:12 hour light-dark schedule with free access to water and food. These experiments were performed in accordance with the Guide for Laboratory Animals in Biomedical Research (2011 Revised Edition) published by the National Institutes of Health. The study protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

[0083] Experimental treatment plan An equal number of male and female mice were assigned to each group. Mice received 5 Gy total body irradiation (TBI) using a Varian 21EX LINAC (Palo Alto, CA) while under ketamine / xylazine anesthesia (100 mg / kg and 10 mg / kg, respectively), followed immediately by a 6.5 Gy supplemental dose to the thorax for a total thorax dose of 11.5 Gy. 24 hours after irradiation, mice were treated once daily by oral gavage with 1 mg / kg septopterin or 5 mg / kg BH4 dissolved in water for 6 days ( Figure 1 ).

[0084] Example 2. Assessment of cardiac function Echocardiography All mice underwent transthoracic echocardiography at baseline (before irradiation (IR)), 8, 30, 60, 90, and 180 days under light anesthesia (30 mg / kg sodium pentobarbital). Echocardiography was performed with a Vevo 770 imaging system (VisualSonics, Toronto, Ontario, Canada) and a 30 MHz probe. The heart was displayed in B-mode from the parasternal short-axis and apical views. Left ventricular (LV) end-diastolic diameter (EDD), LV end-systolic diameter (ESD), LV anterior wall diastolic thickness (AWDT) and LV posterior wall diastolic thickness (PWDT), LV anterior wall systolic thickness (AWST), and LV posterior wall systolic thickness (PWST) were measured in M-mode according to the recommendations of the American Society of Echocardiography. LV ejection fraction (EF) and LV mass were calculated based on the measurements of wall thickness and chamber diameter. As previously described, LVEF was derived using the Teicholz formula (LVEF = [LVEDD3 - LVESD3] / LVEDD3). Doppler spectra (E, A, ET) of the transmitral LV outflow tract were recorded from the apical four-chamber view, and the myocardial performance index (MPI) was calculated as the ratio of isovolumetric contraction time (ICT) and isovolumetric relaxation time (IRT) divided by ejection time (ET). The investigators who performed and read the echocardiograms were blinded to the treatment allocation. The β-adrenergic agonist isoproterenol (Sigma Aldrich, St. Louis, MO, USA) 20 ng / mouse was used to evaluate myocardial contractility in irradiated mice not treated with SP. Myocardial contractility (contractile reserve) was expressed as the percentage change in LVEF measured at rest (LVEFr) and 3 minutes after isoproterenol injection (LVEFi), and was calculated as [(LVEFi-LVEFr) / LVEFr]*100. Contractile reserve was measured at baseline and at 8, 30, 60, 90, and 180 days after IR.Echocardiographic evaluations were performed by operators blinded to treatment assignment.

[0085] Effects of septopterin on cardiomyopathy induced by IR Cardiac performance was assessed at baseline in all mice before IR and before starting SP treatment. Mice were then randomly assigned to treatment groups (vehicle or SP) after IR. The effects of our TBI model were assessed using a chest supplemental dose on systolic and diastolic function and contractile reserve in response to isoproterenol. Radiation resulted in a slowed response to isoproterenol ( Figure 2 and 7 ) and myocardial performance index ( Figure 3 and 8) and an increase in isovolumetric relaxation time. Daily oral administration of SP for 6 days after IR was able to restore the response to β-adrenergic stimulation with isoproterenol. In addition, SP treatment reduced the progression of systolic and diastolic dysfunction and improved the myocardial performance index and the gradual increase in IRT. These results were further evaluated according to the sex of the animals. There were no significant differences in the cardiac functional response to irradiation or 1 mg / kg SP in terms of systolic and diastolic function ( Figures 14A-14B and 15A-15B). However, male mice had an additional significant loss of contractile reserve at 90 and 180 days post-IR compared with female mice ( Figures 16A-16B Importantly, SP was equally effective in attenuating this loss of contractile reserve in both sexes.

[0086] Example 3. Assessment of lung function Evaluation of lung injury by respiratory rate Starting at week 6, respiratory rate was measured every other week using the Mouse Ox system (STARR Life Sciences Corp., Allison Park, PA). Animals were placed under ketamine / xylazine anesthesia and shaved in the analysis area. Ten minutes after injection, the animals were placed in the supine position and the sensor was clamped to the upper thigh. Respiratory rate was recorded for 3 minutes. Respiratory rate was calculated using the algorithm in the Mouse Ox software.

[0087] SP and BH4 reduce radiation-induced loss of lung function The respiratory rate of mice was measured using the MouseOx system as a function of irradiation, irradiation and SP ( Fig. 9 ) and irradiation with BH4( Figure 5 ) mice. Mice receiving 1 mg / kg / day SP or 5 mg / kg BH4 for 6 days had a significant delay in the development of lung lesions compared to vehicle-treated mice (14 weeks for SP and 10 weeks for vehicle) and significantly reduced lung function impairment at all measured points.

[0088] Example 4. Evaluation of the survival rate of mouse groups SP improves survival after radiation To determine whether improved cardiopulmonary function affects overall survival, mice were followed for 180 days after IR. Figure 4Kaplan Meyer survival graph showing C57L / J mice after a total chest dose of 11.5Gy (5GyTBI followed by a 6.5Gy chest dose) with and without 1mg / kg / day SP for 6 days. Untreated mice exposed to radiation resulted in a significant decrease in survival, with a median survival of 137 days and a survival rate of 40% at the end of the study. In contrast, mice receiving 1mg / kg / day SP treatment for 6 days had a significantly delayed and overall improved survival rate, with 71% of mice surviving at 180 days (i.e., the end of the study).

[0089] Example 5. Expression of serum exosome miRNA Blood was collected by cardiac puncture using EDTA vials. Blood samples were centrifuged at 3,000 x g for 25 minutes, and the plasma fraction was aliquoted and stored in the vapor phase of liquid nitrogen. Exosome isolation kit from 101Bio was used in Figures 6A-6C Exosomes were isolated from mouse plasma at the indicated time points. Exosome concentrations were quantified by the EXOCET Exosome Quantification Kit (SystemBiosciences). Equal exosome numbers from 5 animals were pooled for each time point, and total exosomal RNA was extracted by using the miRNeasy Micro Kit (QIAGEN). For miRNA expression profiling, miRCURY LNA TM Universal RT microRNA PCR assay (QIAGEN) and ExiLENT GreenMaser Mix (QIAGEN). Serum / Plasma LNA was used on a QuantStudio 5 RT-PCR machine (Applied Biosystems). TM The miRNA PCR Panel (Cat.#YAHS-106Y, QIAGEN) was used for initial estimation of exosomal miRNA expression.

[0090] Figure 6B-6CThe cycle threshold values ​​(Ct values) shown in are the average values ​​from 5 mice per group and are normalized for hsa-miR-145-5p and hsa-miR-221-3p, which have been experimentally proven to be high-quality normalized controls. Two different groups of exosomal miRNAs showing significant differences between vehicle-treated and SP-treated animals are shown at the 30-day time point after IR. The first group includes let-7a-5p, miR-1, miR-106b-3p, miR-106b-5p, miR-126-3p, miR-181a-5p, miR-335-3p, and miR-335-5p. Compared with animals treated with SP, the expression of miRNAs in this group was significantly lower in animals treated with vehicle. In the second group, the expression of miRNAs was significantly higher in vehicle-treated animals compared to SP-treated animals. The second group included let-7g-5p, let-7i-5p, and miR-16-5p. There were no significant differences between males and females in terms of IR-altered exosomal miRNA expression or the effect of septopterin on miRNA expression. The miRNAs that showed significant changes in expression due to radiation and were regulated by septopterin are involved in inflammatory processes (e.g., let-7a-5p, miR-106b-3p, miR-181a-5p, let-7i-5p), angiogenesis and vascular integrity (miR-106b-3p, miR-126-3p), cardiac inflammation and fibrosis (miR-335-5p, let-7i-5p, miR-126-3p, miR-16-5p). miR-16-5p inhibits TGF-β / VEGF signaling. Treatment with BH4 ( Fig. 6A ) showed increased expression of miR-15b-3p, miR-106a-5p, miR-133b, miR-136-5p, miR-451a, miR-1, miR-335-3p, let-7d-3p and / or let-7c-5p.

[0091] Example 6. Cytokine mRNA Expression in Heart and Lung Tissues RNA isolation and RT-PCR Total RNA was isolated from lung and heart tissue samples using RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA concentration was assessed by using NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA purity was evaluated by the ratio of A260 / A280 and A260 / A230. RNA integrity was assessed by the ratio of 28S / 18S ribosomal RNA (rRNA) and RNA integrity number (RIN) using Agilent 2100 BioAnalyzer (Agilent Technologies). cDNA synthesis and genomic DNA elimination were performed by using RT2 First Strand Kit (QIAGEN). RT2 from QIAGEN was used to analyze RNA. Samples were amplified using the QuantStudio 5 RT-PCR machine (Applied Biosystems) using the Green qPCR Mastermix probe. The following RT2 qPCR Primer Assays (QIAGEN) were used: mouse Actin-β (NM_007393), mouse Bmp2 (NM_007553), mouse Ccl2 (NM_011333), mouse Ctgf (NM_010217), mouse IL-1β (NM_008361), mouse IL-6 (NM_031168), mouse IL-10 (NM_010548), mouse IL-17α (NM_010552), mouse Runx2 (NM_001145920), mouse Spp1 (NM_001204201), mouse TGF-β1 (NM_011577), mouse Trim72 (NM_001079932).

[0092] Changes in cytokine mRNA expression after radiation The expression of different cytokines (Bmp2, Ccl2, Ctgf, IL-1β, IL-6, IL-10, IL-17A, IL-33, Runx2, Spp2, TGF-β1, and Trim72) in lung and heart tissues was evaluated 6 days after animals were irradiated and 24 hours later received SP or water treatment. The mRNA expression in tissue samples was tested at 3 different time points after IR (8 days, 30 days, 16 weeks) and compared with that in non-treated non-irradiated control animals ( Fig.10). There were no significant differences in the expression of all evaluated cytokines in cardiac tissue between vehicle-treated and SP-treated animals. In lung tissue, the cytokines IL-1β, IL-6, IL-17A, Spp2, and TGF-β1 showed significant differences in expression between vehicle-treated and SP-treated animals at different time points after IR. At the 8-day and 30-day time points, the relative expression of IL-1β in lung tissue was significantly higher in vehicle-treated animals compared to the SP-treated group (2.9±0.649 vs. 1.54±0.825 and 3.39±0.532 vs. 1.66±0.51, respectively). At all time points after IR, the relative expression of IL-6 was significantly higher in the vehicle-treated group compared to the SP-treated group (8 days: 16.88±4.91 vs. 1.36±0.509; 30 days: 9.09±0.533 vs. 4.96±0.905; 16 weeks: 28.85±5.987 vs. 17.05±4.12). In addition to IL-6, the cytokine IL-17A also showed significantly higher expression in the vehicle-treated group compared to the SP-treated group at all time points after IR (8 days: 9.19±2.783 vs. 2.71±1.874; 30 days: 5.65±2.263 vs. 1.49±0.379; 16 weeks: 4.5±0.824 vs. 2.12±1.173). At 16 weeks after IR, the vehicle-treated group showed significantly higher lung tissue expression of cytokines Spp2 and TGF-β1 compared to the SP-treated group (4.25±0.537 vs. 2.38±0.211 and 3.26±0.339 vs. 1.53±0.367). Cytokine Ccl2 showed a significant increase in relative expression in lung tissue for all animals at the 30-day time point, with no significant differences between vehicle-treated and SP-treated animal groups.

[0093] Example 7. IHC analysis of heart and lung tissues IHC analysis 16 weeks after IR, hearts and right lungs were collected for histological analysis ( Figures 11A-11C). The heart was collected in formalin, embedded in paraffin, and cut into 5-mm slices. The right lung was perfused with OCT and cut into 6-mm slices. Masson trichrome staining was performed to detect collagen fibers according to the supplier's instructions (Richard-Allan Scientific Masson's trichrome kit, Thermo Fisher Scientific, Waltham, MA, USA). The area of ​​myocardial and pulmonary fibrosis was calculated as the percentage of collagen area to total tissue area using computer morphometric analysis (ImageProPlus 6.0 software, Media Cybernetics, Rockville, MD, USA). Inflammation, angiogenesis, and smooth muscle cell differentiation were measured, and the heart and right lung were stained with the following antibodies: Ly-6G / Ly-6C (Invitrogen, Carlsbad, CA, USA), F40 / 80 and α-sma (CellSignaling, Danvers, MA, USA), CD-31 (BD Pharmigen, San Jose, CA, USA). The primary antibody was detected by the secondary antibody, followed by staining with Novared (Vector Laboratories, Burlingame, CA, USA) for antibody detection. Staining was evaluated on a dichotomous basis (positive / negative) by two investigators blinded to treatment allocation, and positive results were subsequently graded as mild (1), moderate (2), or strong (3) staining based on the intensity and extension of staining.

[0094] Hydroxyproline Hydroxyproline content in the left upper lobe of the lung was measured using the Hydroxyproline Assay Kit from Sigma Aldrich (St. Louis, MO) as previously described.

[0095] Example 8. Proteins measured by ELISA Plasma analysis At the time of execution, blood was obtained by cardiac puncture into a tube containing EDTA and centrifuged at 3000rpm for 25 minutes to separate plasma. The following ELISA plates from Abcam were used for analysis: fibrinogen (ab213478), neutrophil elastase (ab252356), C-reactive protein (ab157712) and IL-6 (ab100713). Plasma samples were processed and ELISA kits were run according to the manufacturer's recommendations. All cytokines were reported in absolute units. Key molecular participants of the coagulation cascade (such as tissue factor, thrombin and fibrinogen) are epidemiologically and mechanistically related to inflammatory components. Genetic and pharmacological studies have revealed the key role of fibrinogen in determining the degree of local or systemic inflammation. Neutrophil elastase is a serine protease present in the azure granules of neutrophils, which is released under inflammatory conditions and decomposes the extracellular matrix due to its low substrate specificity, resulting in tissue damage. Neutrophil elastase is one of the most destructive enzymes in vivo. Once unregulated, this enzyme disrupts the function of the lung permeability barrier and induces the release of proinflammatory cytokines, accompanied by the stimulation of acute lung injury. The inflammatory cytokine IL-6 can be upregulated in lung tissue and blood as early as 6 hours after radiation. High levels of IL-6 in plasma can aggravate the inflammatory response in lung tissue, ultimately leading to IL-6 leakage into the bronchoalveoli and further lung damage. High levels of IL-6 in lung tissue can attract more inflammatory cells (such as neutrophils, monocytes, macrophages) to the damaged local lung, ultimately leading to severe lung damage and chronic fibrosis. Acute increases in IL-6 in plasma can lead to an increase in C-reactive protein (CRP). C-reactive protein (CRP) is produced by the liver and is a biomarker for general stress responses to inflammation or infection. It shows that serum CRP levels can be an effective indicator for predicting the occurrence of radiation pneumonitis and a valuable factor for assessing the extent of radiation-induced lung damage.

[0096] Changes in plasma cytokine concentrations after IR At 4, 8, and 180 days after IR, animals in the vehicle-treated group exhibited significantly higher plasma IL-6 levels compared to the SP-treated group (304.05±153.23 pg / ml vs. 97.00±45.71 pg / ml (p=0.02); 606.09±72.72 pg / ml vs. 49.85±39.36 pg / ml (p<0.001); and 197.22±55.55 pg / ml vs. 40.06±43.26 pg / ml (p=0.018), respectively) ( Fig. 13CAt the 8-day time point after IR, animals in the vehicle-treated group exhibited significantly higher plasma fibrinogen levels compared to the SP-treated group (948.77±98.54 μg / ml vs. 332.38±109.02 μg / ml (p<0.001)) ( Fig. 13B At the 180-day time point after IR, animals in the vehicle-treated group exhibited significantly higher plasma neutrophil elastase levels compared to the SP-treated group (25.21±6.54 ng / ml vs. 12.15±2.87 ng / ml (p=0.034)) ( Fig.13D C-reactive protein showed no significant difference between vehicle-treated and SP-treated animal groups at any time point after IR ( Fig.13A ).

[0097] Example 9. Spearman correlation between fibrosis and cytokine mRNA expression Statistical analysis Two-tailed Student's T test was used to examine statistical significance in groups with a single independent variable. P < 0.05 was considered significant. Spearman rank correlation was used to evaluate the relationship between specific mRNA expression and the degree of lung fibrosis ( Fig.12 ). Log-rank analysis was used to compare survival rates between groups.

[0098] Conclusion. Oral administration of BH4 or its metabolic precursor SP starting 24 hours after radiation exposure can enhance lung and cardiac function as demonstrated by decreased respiratory rate, enhanced contractile reserve, improved systolic and diastolic function, and increased survival.

Claims

1. A method of treating a subject exposed to radiation comprising administering to the subject an effective amount of septopterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharmaceutically acceptable salt or cocrystal thereof.

2. The method of claim 1, wherein said administering reduces or inhibits tissue and / or organ damage in said subject.

3. The method of claim 1, wherein said administering reduces or inhibits cardio- and / or pulmonary toxicity in said subject.

4. The method of claim 1, wherein said administering reduces gastrointestinal, cardiac and / or pulmonary endothelial cell death in said subject.

5. The method of claim 1, wherein said administering reduces radiation-induced inflammation in gastrointestinal, cardiac, and / or pulmonary epithelial cells in said subject.

6. The method of any one of claims 1-5, wherein the effective amount is about 0.1 to about 200 mg / kg / day.

7. The method of any one of claims 1-6, wherein the subject suffers from acute radiation syndrome.

8. The method of claim 7, wherein said administering is performed within 24 hours after exposure to radiation.

9. The method of claim 7, wherein said administering is performed at least 24 hours after exposure to radiation.

10. The method of any one of claims 1-6, wherein the subject suffers from chronic radiation syndrome.

11. The method of any one of claims 1-6, wherein the subject suffers from cutaneous radiation syndrome.

12. The method of any one of claims 1-6, wherein the subject is exposed to at least 0.3 Gy in less than one day.

13. The method of any one of claims 1-6, wherein the subject is exposed to at least 0.7 Gy over a period of more than one day.

14. The method of any one of claims 1-13, wherein the administering is performed at least once a day for at least 6 days.

15. The method of any one of claims 1-14, wherein the administration increases the expression of miR-15b-3p, miR-106a-5p, miR-133b, miR-136-5p, miR-451a, miR-1, miR-335-3p, let-7d-3p and / or let-7c-5p.

16. The method of any one of claims 1-15, wherein the administration reduces the expression of IL-1β, IL-6, IL-17A, Spp2 and / or TGF-β1 in lung epithelial cells.

17. The method of any one of claims 1-16, wherein septerin or a pharmaceutically acceptable salt or cocrystal thereof is administered.

18. The method of any one of claims 1-17, wherein the administration increases the expression of let-7a-5p, miR-1, miR-106b-3p, miR-106b-5p, miR-126-3p, miR-181a-5p, miR-335-3p and / or miR-335-5p.

19. The method of any one of claims 1-18, wherein the administering reduces the expression of let-7g-5p, let-7i-5p and / or miR-16-5p.

20. The method of any one of claims 1-19, wherein the administering occurs at least daily for at least 14 days.

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