Use of sepiapterin and metabolites thereof to treat radiation exposure

Abstract

To provide methods for treating a subject subjected to radiation exposure.SOLUTION: The invention provides a method for treating radiation exposure in a subject by administering an effective amount of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharmaceutically acceptable salt or cocrystal thereof to the subject.SELECTED DRAWING: None

Description

Background Art

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under grant number R01 AI133595 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0002] Background of the Invention Whether it is a terrorist act, a nuclear power accident, or a radiation disaster, the threat of radiation means a catastrophic public health emergency and emphasizes the need to develop medical measures to reduce whole-body radiation-induced injuries and deaths. The first health effects of whole-body irradiation (TBI) exposure are acute radiation syndrome (ARS) in the most radiation-sensitive organs, and survival from this exposure is determined by the degree of damage to hematopoietic stem and progenitor cells as well as the epithelium of the gastrointestinal tract. Hematopoietic growth factors, electrolytes and infusions, blood transfusions, and antibiotics have advanced as treatment methods for ARS and have brought about a significant improvement in survival rates. However, as seen in the survivors of Chernobyl, patients who have overcome ARS in the hematopoietic and gastrointestinal systems are often subject to late-stage disorders of the lungs and heart.

[0003] Therefore, there is a need for treatment methods to improve the mortality rate and organ function of radiation-exposed patients.

Summary of the Invention

[0004] The present invention features a method for treating radiation-exposed patients. For example, the present invention employs sepiapterin (SP) to mitigate the toxicity to the patient's heart, gastrointestinal tract, and / or lungs.

[0005] In one aspect, the present invention provides a method for treating a subject exposed to radiation by administering to the subject an effective amount of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharmaceutically acceptable salt or co-crystal thereof.

[0006] In certain embodiments, administration reduces or suppresses tissue and / or organ damage in a subject, reduces or suppresses cardiotoxicity, gastrointestinal toxicity, and / or pulmonary toxicity in a subject, reduces endothelial cell death in the gastrointestinal tract, heart, and / or lungs of a subject, and / or reduces radiation-induced inflammation in epithelial cells of the gastrointestinal tract, heart, and / or lungs of a subject.

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

[0008] In embodiments, the subject has acute radiation syndrome, and for example, administration is performed within 24 hours after exposure to radiation. In embodiments, the subject has chronic radiation syndrome. In embodiments, the subject has 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, for example, at least one week, at least 10 days (for example, at least 14 days), at least about one month, at least about three months, at least about six months, at least about nine months, or at least about one year.

[0010] In an embodiment, 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 (e.g., serum exosomes) (e.g., when BH4 is administered), and / or decreases the expression of IL-1β, IL-6, IL-17A, Spp2, and / or TGF-β1 in lung epithelial cells (e.g., when SP is administered). In an embodiment, 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 present invention features a method of reducing or suppressing damage to tissues and / or organs of a subject exposed to radiation by administering to the subject an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.

[0012] In another aspect, the present invention features a method of reducing or suppressing heart and / or lung toxicity in a subject exposed to radiation by administering to the subject an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.

[0013] In another aspect, the present invention features a method of decreasing heart and / or lung endothelial cell death in a subject exposed to radiation by administering to the subject an effective amount of sepiapterin or a pharmaceutically acceptable salt thereof.

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

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

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

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

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

[0019] In another embodiment of the four aspects, the effective amount of sepiapterin or a pharmaceutically acceptable salt thereof results in increasing 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.

[0020] Definitions In this application, unless otherwise apparent from the context, (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 itemized components or steps whether presented by themselves or in conjunction with one or more additional components or steps, and (iv) the terms "about" and "approximately" may be understood to allow for standard variations as would be understood by a person of ordinary skill in the art, and (v) when ranges are provided, endpoints are included.

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

[0022] References to "about" or "approximately" a value or parameter herein include (and describe) variations that are directed to that value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0023] As used herein, the term "administering" means administering a composition to a subject. Administration to an animal subject (e.g., administration to a human) may be by any suitable route. For example, in some embodiments, administration is by bronchus (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, trachea (including by tracheal instillation), transdermal, vaginal, or intravitreal.

[0024] The "effective amount" of a compound can vary depending on factors such as the condition of the individual, age, sex, weight, and the ability of the compound to elicit the desired response. A therapeutically effective amount encompasses an amount where the toxic or detrimental effects of the compound do not outweigh the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount sufficient to provide a benefit, e.g., a clinical benefit.

[0025] "Level" means the level of a compound when compared to a reference. The reference can be any useful reference as defined herein. A "decreased level" or "increased level" of a compound means a decrease or increase in the compound level when compared to the 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 400%, at least about 500%, or a greater percentage decrease or increase, a decrease or increase of less than about 0.01-fold, less than about 0.02-fold, less than about 0.1-fold, less than about 0.3-fold, less than about 0.5-fold, less than about 0.8-fold, or less than a smaller multiple, or an increase of greater than about 1.2-fold, greater than about 1.4-fold, greater than about 1.5-fold, greater than about 1.8-fold, greater than about 2.0-fold, greater than about 3.0-fold, greater than about 3.5-fold, greater than about 4.5-fold, greater than about 5.0-fold, greater than about 10-fold, greater than about 15-fold, greater than about 20-fold, greater than about 30-fold, greater than about 40-fold, greater than about 50-fold, greater than about 100-fold, greater than about 1000-fold, or a greater multiple). The level of a compound can be expressed as mass / volume (e.g., g / dL, mg / ml, μg / ml, ng / ml) or as a percentage of the total compound in a sample.

[0026] As used herein, the term "pharmaceutical composition" refers to a composition comprising a compound described herein formulated with a pharmaceutically acceptable excipient. The pharmaceutical composition may be produced or sold with the approval of a government regulatory agency as part of a treatment regimen for the treatment of diseases in mammals. The pharmaceutical composition can be formulated, for example, for oral administration in unit dosage form (e.g., tablets, capsules, caplets, gelcaps, powders for suspension, suspensions, solutions, or syrups), for topical administration (e.g., as creams, gels, lotions, or ointments), for intravenous administration (e.g., as a sterile solution in a solvent system suitable for intravenous use, free of particulate emboli), or as any other pharmaceutically acceptable formulation.

[0027] As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin. Pharmaceutically acceptable salts include ion pairs of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin in the solid state and / or in solution. Pharmaceutically acceptable co-crystals include the free base of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin in the solid state and an acid. A mixture of the salt form and the co-crystal form may be present in the same composition. For example, pharmaceutically acceptable salts of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin are within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic response, and include salts commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in the following reference: Remington: The Science and Practice of Pharmacy, (22nd ed.) ed. L.V. Allen, Jr., 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 group 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, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, maleate, 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 salts.

[0028] "Reference" means any useful criterion used to compare at the compound level or other symptoms, such as damage to tissues and / or organs, toxic cells, death, or inflammation. A reference can be any sample, standard, standard curve, or level used for comparison. A reference can be a normal reference sample or reference standard or level. A "reference sample" can be, for example, a control, such as a predetermined negative control value like a "normal control", or a prior sample taken from the same subject, a sample taken from a normal healthy subject such as normal cells or normal tissue, a sample from a subject without a disease (e.g., cells or tissue), a sample from a subject diagnosed with a disease but not yet treated with the compound of the present invention, a sample from a subject treated with the compound of the present invention, or a sample of a purified compound at a known normal concentration (e.g., any of those described herein). A "reference standard or level" means a value or numerical value derived from a reference sample. A "normal control value" is a predetermined value indicating a non-diseased state, for example, a value expected in a healthy control subject. Typically, 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 measurement within the normal control value for a particular biomarker is typically said to be "within the normal range" of that biomarker. A normal reference standard or level can be a value or numerical value derived from a normal subject without a disease or disorder (e.g., ARS). In a preferred embodiment, the reference sample, standard, or level is matched to the subject sample by at least one of the following criteria: age, weight, gender, disease stage, and general health status. A standard curve of the level of a purified compound (e.g., any of those described herein) within a normal reference range can also be used as a reference.

[0029] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the invention can be administered for, e.g., experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal, such as mammals including mice, rats, rabbits, non-human primates, and humans. A subject may be a human or an animal that seeks treatment, needs treatment, is undergoing treatment, will receive treatment in the future, or is receiving care by a professional trained in a particular disease or condition.

[0030] As used herein, the term "treat", "treated", or "treating" means both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (mitigate) an undesirable physiological state, disorder, or disease, or to obtain a beneficial or desirable clinical outcome. Beneficial or desired clinical outcomes include alleviation of symptoms, reduction in the degree of a state, disorder, or disease, stabilization (i.e., not worsening) of a state, disorder, or disease, delay in onset or retardation of progression of a state, disorder, or disease, improvement or remission (partial or total, whether detectable or not) of a state, disorder, or disease, improvement in at least one measurable physical parameter (not necessarily distinguishable by the patient), or promotion or improvement of a state, disorder, or disease, but are not limited thereto. Treatment includes eliciting a clinically significant response without undue levels of side effects. Treatment also includes prolonging survival as compared to the expected survival in the absence of treatment.

[0031] The descriptions of the compounds, compositions, formulations, and treatment methods described herein are to be understood to include "comprising," "consisting of," and "consisting essentially of" embodiments. In some embodiments, for all of the compositions described herein and all of the methods of using the compositions described herein, the composition can include the recited components or steps, or can "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" the recited components, the composition includes the recited components and can include other components that do not substantially affect the state being treated, but does not include other components that substantially affect the state being treated other than those explicitly recited, or, if the composition includes additional components other than the recited components that substantially affect the state being treated, the composition does not include the additional components in a concentration or amount sufficient to substantially affect the state being treated. When a method is described as "consisting essentially of" the recited steps, the method includes the recited steps and can include other steps that do not substantially affect the state being treated, but the method does not include other steps that substantially affect the state being treated other than those explicitly recited. By way of non-limiting specific example, when a composition is described as "consisting essentially of" a certain component, the composition can additionally include any amount of a pharmaceutically acceptable carrier, vehicle, or diluent, and other such components that do not substantially affect the state being treated.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials for use in the disclosure of this specification are described herein, other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and 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 case of conflict, the specification including definitions will control.

[0033] Details of one or more embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will become apparent from the description and claims.

Brief Description of the Drawings

[0034]

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[0035] The present invention features a method of using sepiapterin, tetrahydrobiopterin, dihydrobiopterin, or a pharmaceutically acceptable salt or co - crystal thereof for treating a subject exposed to radiation. Without wishing to be bound by theory, we believe that radiation - induced late - stage lung, gastrointestinal, and heart toxicities are the result of endothelial dysfunction defined as a decrease in uncoupled NOS activity and NO bioavailability, establishing a chronic inflammatory state that drives a persistent profibrotic process associated with abnormal wound repair and late - stage normal tissue damage. We show that sepiapterin and its metabolites (such as tetrahydrobiopterin and dihydrobiopterin) can be used as a radiation countermeasure to mitigate radiation - induced heart and lung damage and improve survival rates in a mouse model with radiation sensitivity similar to that of humans.

[0036] Active Compound The method of the present invention is characterized by using sepiapterin, tetrahydrobiopterin, or dihydrobiopterin, or salts and / or co-crystals thereof. Sepiapterin can be converted into dihydrobiopterin and tetrahydrobiopterin in vivo, and dihydrobiopterin and tetrahydrobiopterin can be interconverted in vivo.

[0037] Sepiapterin has the following structure. JPEG2025098011000001.jpg3460

[0038] Tetrahydrobiopterin has the following structure. JPEG2025098011000002.jpg3460

[0039] Dihydrobiopterin has the following structure. JPEG2025098011000003.jpg3362

[0040] The active compound can be in any suitable form, for example, free base, salt, or 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, gentisate, glucoheptonate, glycerophosphate, glycolate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, maleate, 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 salts. In some embodiments, the active compound or its salt is in crystalline form.

[0041] Exemplary salts, co-crystals, and crystalline forms of sepioptin are described in WO2018 / 102314, WO2018 / 102315, WO2019 / 046849, and WO2019 / 232120, and can include, for example, any of crystalline forms A, B, C, D, E, F, or G described in WO2018 / 102314 or WO2018 / 102315. Other salts or crystalline forms of sepiapterin, dihydrobiopterin, and / or tetrahydrobiopterin are known in the art.

[0042] Treatment method The present invention provides a method of treating a subject in the treatment of a subject after radiation exposure, such as acute radiation syndrome (ARS), cutaneous radiation syndrome, or chronic radiation syndrome. In particular, the present invention provides a method of treating a patient exposed to at least about 0.05 Gy in a period of less than 24 hours. For example, the patient may be 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, for example, between about 0.3 and about 6, between about 0.7 and about 6, between about 1 and about 2, between about 2 and about 6, between about 6 and about 20, between about 6 and about 10, between about 6 and about 8, between about 10 and about 20, between about 8 and about 12, or between about 20 and about 50 Gy, and the exposure period is less than about 18 hours, for example, less than about 12 hours, less than about 6 hours, less than about 3 hours, less than about 1 hour, less than about 0.5 hours, or less than about 0.1 hours, or less than about 5 minutes, less than about 3 minutes, less than about 2 minutes, 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 be 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 is 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 more than those periods, for example, about 1 week to 3 years, about 1 week to 1 year, about 1 week to 1 month, about 1 month to 2 years, about 1 month to 1 year, about 6 months to 2 years, about 9 months to 2 years, or about 9 months to 15 months. The radiation exposure may be whole body or local, for example, irradiation to the skin. In certain embodiments, the radiation is not due to radiotherapy.

[0043] The treatment according to the present invention results in reducing or suppressing tissue and / or organ damage in a subject exposed to radiation, thereby reducing or suppressing the toxicity of a tissue or organ (e.g., lung, heart, or gastrointestinal tract (e.g., lung or heart)) in a subject exposed to radiation, resulting in a decrease in endothelial cell death (e.g., lung, heart, or gastrointestinal tract (e.g., lung or heart)) in a subject exposed to radiation, resulting in a decrease in radiation-induced inflammation of epithelial cells (e.g., lung, heart, or gastrointestinal tract (e.g., lung or heart)) in a subject receiving radiation, resulting in an increase in 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, resulting in a decrease in the expression of IL-1β, IL-6, IL-17A, Spp2, and / or TGF-β1 (e.g., in lung epithelial cells), resulting in an increase in 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 resulting in a decrease in the expression of let-7g-5p, let-7i-5p, and / or miR-16-5p. The reduction in symptoms or cytokines may be, for example, 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% relative to a reference. The increase in oligonucleotide expression may be, for example, 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% relative to a reference.

[0044] The active compound can be administered at any suitable dosage. The actual dosage of the composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of the condition, previous or concurrent therapeutic interventions, the patient's characteristics, and the route of administration. Depending on the dosage and the route of administration, the preferred dosage and / or the number of administrations of the effective amount can be varied according to the response of the subject. The physician in charge of the administration can determine, in any case, the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject. The therapeutically effective amount can be, for example, from about 0.1 mg / kg / day to about 200 mg / kg / day, such as from about 0.1 to about 150 mg / kg / day, from about 0.1 to about 125 mg / kg / day, from about 0.1 to about 100 mg / kg / day, from about 0.1 to about 80 mg / kg / day, from about 0.1 to about 60 mg / kg / day, from about 0.1 to about 40 mg / kg / day, from about 0.1 to about 25 mg / kg / day, from about 0.1 to about 20 mg / kg / day, from about 0.1 to about 15 mg / kg / day, from about 0.1 to about 10 mg / kg / day, from about 0.1 to about 5 mg / kg / day, from about 0.1 to about 2.5 mg / kg / day, from about 0.1 to about 1 mg / kg / day, from about 0.1 to about 0.5 mg / kg / day, from about 0.5 to about 200 mg / kg / day, from about 1 to about 200 mg / kg / day, from about 2.5 to about 200 mg / kg / day, from about 5 to about 200 mg / kg / day, from about 10 to about 200 mg / kg / day, from about 15 to about 200 mg / kg / day, from about 20 to about 200 mg / kg / day, from about 25 to about 200 mg / kg / day, from about 40 to about 200 mg / kg / day, from about 60 to about 200 mg / kg / day, from about 80 to about 200 mg / kg / day, from about 100 to about 200 mg / kg / day, from about 120 to about 200 mg / kg / day, from about 140 to about 200 mg / kg / day, from about 160 to about 200 mg / kg / day, from about 180 to about 200 mg / kg / day, from about 5 to about 80 mg / kg / day, from about 10 to about 160 mg / kg / day, from about 20 to about 140 mg / kg / day, from about 40 to about 120 mg / kg / day, from about 60 to about 100 mg / kg / day, from about 5 to about 50 mg / kg / day, or from about 10 to about 20 mg / kg / day of the active compound. The administration can be carried out any suitable number of times, for example, once a day, twice a day or three times a day during the treatment.Administration may be continued for as long as necessary, for example, 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.

[0045] The active compound can be initially administered at any suitable time, for example, within about 1 hour, within about 2 hours, within about 6 hours, within about 12 hours, within about 18 hours, within about 24 hours, within about 2 days, or within about 1 week of radiation exposure.

[0046] Formulation The active compound can be formulated into a pharmaceutical composition in a manner known in the art. Such compositions may contain various components as are known in the art, see, for example, Remington (The Science and Practice of Pharmacy, (22nd ed.) ed. L.V. Allen, Jr., 2013, Pharmaceutical Press, Philadelphia, PA).

[0047] The composition may contain a pharmaceutically acceptable carrier, for example, any of those conventionally used, and is limited only by chemical-physical considerations such as solubility and lack of reactivity with the compound, and the route of administration. Pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, or diluents, are well known to those skilled in the art and are generally readily available. The pharmaceutically acceptable carrier is preferably chemically inert to the active compound and has no harmful side effects or toxicity under the conditions of use.

[0048] Antioxidant The pharmaceutical composition employed in the present method may or may not contain an antioxidant. The antioxidant can minimize the oxidative degradation of the active compound. Examples of antioxidants include, but are not limited to, 4-chloro-2,6-di-tert-butylphenol, tocopherol, α-tocopherol, alkylated diphenylamines, ascorbic acid, ascorbyl myristate, ascorbyl palmitate, ascorbyl stearate, β-carotene, butylated hydroxyanisole, butylated hydroxytoluene, citric acid, cysteine, D-α-tocopheryl polyethylene glycol 1000 succinate, deferoxamine mesylate, dodecyl gallate, ethyl paraben, folic acid, fumaric acid, gallic acid, glutathione, lecithin, 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 hydrosulfite, sodium isoascorbate, sodium metabisulfite, sodium sulfite, sodium thiosulfate, tartaric acid, tert-butylhydroquinone, tocopherol acetate, vitamin A, vitamin B6, vitamin B12, or vitamin E. In some embodiments, the method of the present invention can use a pharmaceutical composition containing, as an antioxidant, ascorbic acid, tocopherol, retinol, ascorbyl palmitate, N-acetylcysteine, glutathione, butylated hydroxytoluene, and / or butylated hydroxyanisole.

[0049] In some embodiments, the method comprises a pharmaceutical composition comprising less than about 10 wt% antioxidant, 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 the 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 80%, about 90%, or about 100% of the USP maximum daily dose of the antioxidant. In some embodiments, the ratio of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharmaceutically acceptable salt and / or cocrystal thereof, to the antioxidant is at least about 1:1, such as 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 the pharmaceutically acceptable salt and / or cocrystal of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin to the antioxidant is greater than about 4:1 (e.g., greater than about 5:1, greater than about 6:1, 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 ratio (e.g., the weight ratio of the salt to the antioxidant).

[0050] Dispersant In some embodiments, the method uses a pharmaceutical composition comprising at least one dispersant. The dispersant can separate the particles in the formulation and release the medicinal substance, for example, upon contact with moisture. Examples of dispersants include, but are not limited to, cross-linked polyvinylpyrrolidone, carboxymethylcellulose (e.g., croscarmellose salts, such as sodium croscarmellose), starch (e.g., sodium starch glycolate), or alginic acid. In some embodiments, the dispersant in the pharmaceutical composition is carboxymethylcellulose, such as a pharmaceutically acceptable salt of croscarmellose. In some embodiments, the method uses a pharmaceutical composition that can contain a dispersant in an amount of about 0.1 to 1.5% 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% dispersant, for example, less than about 1%, less than about 0.5%, or less than about 0.1% dispersant.

[0051] Anticaking agent Anticaking agents are often added to pharmaceutical compositions, for example, to prevent the formation of lumps in solution. Thus, in some embodiments, the pharmaceutical composition used in the method of the present invention comprises at least one anticaking agent. In some embodiments, the pharmaceutical composition used in the method of the present invention comprises at least two anticaking agents. Exemplary anticaking 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, talcum powder, sodium aluminosilicate, potassium alumina silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, and polydimethylsiloxane. In some embodiments, at least one anticaking agent is colloidal silicon dioxide or microcrystalline cellulose. In some embodiments, the pharmaceutical composition used in the method of the present invention may comprise from about 65% to 75% by total weight of an anticaking agent, for example, about 65%, about 67%, about 70%, about 73%, or about 75%. In some embodiments, the pharmaceutical composition used in the method of the present invention comprises both colloidal silicon dioxide and microcrystalline cellulose. In some embodiments, the pharmaceutical composition used in the method of the present invention comprises from about 60% to 65% by total weight of microcrystalline cellulose and from about 5% to 7% by total weight of colloidal silicon dioxide.

[0052] Dosage vehicle In some embodiments, the pharmaceutical composition used in the method of the present invention is combined with a dosage vehicle prior to administration. In some embodiments, the composition can be administered in a dosage vehicle having a viscosity of, for example, about 50 to 1750 centipoise (cP) to aid in the suspension and administration of the pharmaceutical composition. One type of suspending agent that can be used is a combination of glycerin and sucrose in water (e.g., MEDISCA having 2.5% glycerin and 27% sucrose in water) (R)Oral mix). An appropriate amount of the composition can be added to the vehicle mixture for administration and stirred to suspend the composition immediately before administration.

[0053] Other suspending agents can also be used as the vehicle for administration. Exemplary suspending agents include water, agar, alginic acid, sodium carboxymethyl cellulose, carrageenan, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, methyl cellulose, polyethylene glycol, povidone, tragacanth, xanthan gum, or other suspending agents known to those skilled in the art.

[0054] Route of administration Suitable formulations for use with the present invention are diverse. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.

[0055] The pharmaceutical composition may be a liquid formulation, such as in the form of a solution, suspension, or emulsion. Formulations suitable for oral administration may be (a) capsules, sachets, tablets, lozenges, and troches, where each may contain a predetermined amount of the active ingredient as a solid or granule, (b) powders, (c) liquid solutions, for example, those obtained by dissolving an effective amount of the compound in a diluent such as water, physiological saline, orange juice, etc., (d) suspensions in a suitable liquid, and (e) suitable emulsions. Preferably, they are solid oral dosage forms such as capsule form, tablet form, and powder form. The capsule form can be of the usual hard or soft shell gelatin type containing, for example, surfactants, lubricants, and inert fillers (such as lactose, sucrose, calcium phosphate, and corn starch). The tablet form may contain one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring agents, and pharmaceutically compatible carriers. The lozenge form usually consists of the active ingredient in sucrose and a flavor such as acacia or tragacanth, and pastilles consisting of the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia. In addition to the active ingredient, it may include emulsions, gels, etc. containing carriers as known in the art.

[0056] Formulations suitable for oral and / or parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which contain antioxidants, buffers, bacteriostats, and / or solutes that render the formulation isotonic with the blood of the intended patient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and / or preservatives. The above compounds can be administered in a pharmaceutical carrier, added to a physiologically acceptable diluent (e.g., a sterile liquid or a mixture of liquids). Such liquids include water, saline, aqueous dextrose solutions, and related sugar solutions, alcohols such as ethanol, benzyl alcohol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol (e.g., polyethylene glycol 400), and other polyethylene alcohols, glycerol ketals such as 2,2-dimethyl-1,3-dioxolan-4-methanol, ethers, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides. These may further contain, or may not contain, pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents, and other pharmaceutical adjuvants.

[0057] Examples of oils that can be used in parenteral dosage forms include petroleum, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Fatty acids suitable for use in parenteral dosage forms include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Soaps suitable for use in parenteral dosage forms include alkali metal salts, ammonium salts, and triethanolamine salts of fatty acids. Also, suitable surfactants include (a) cationic surfactants such as, for example, dimethyldialkylammonium halides and alkylpyridinium halides, (b) anionic surfactants such as, for example, alkyl-, aryl-, and olefin-sulfonates, alkyl-, olefin-, ether-, and monoglyceride-sulfates, and sulfosuccinate esters, (c) nonionic surfactants such as, for example, aliphatic amine oxides, fatty acid alkanolamides, and polyoxyethylene·polypropylene copolymers, (d) amphoteric surfactants such as, for example, alkyl β-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof, etc.

[0058] Parenteral dosage forms can typically contain from about 20% to about 25% by weight of the active compound in solution. Suitable preservatives and buffers can be used in such dosage forms. 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 from about 12 to about 17. The amount of surfactant in the above dosage form is 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 and propylene glycol. Parenteral formulations can be presented in unit dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a lyophilized (freeze-dried) state that requires only the addition of a sterile liquid carrier for injection, such as water, immediately prior to use. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the types described above.

[0059] The pharmaceutical composition may be an injectable formulation. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art. See Remington (The Science and Practice of Pharmacy, (22nd ed.) ed. L.V. Allen, Jr., 2013, Pharmaceutical Press, Philadelphia, PA).

[0060] Topical formulations containing those useful for transdermal drug delivery are well known to those skilled in the art and are suitable in the context of the present invention for application to the skin. Topical application compositions are typically in the form of liquids, creams, pastes, lotions, and gels. Topical administration includes application to the oral mucosa, including the oral cavity, oral epithelium, palate, gingiva, and nasal mucosa. In some embodiments, the compositions used in the methods of the present invention include sepiapterin, tetrahydrobiopterin, or dihydrobiopterin, as well as a suitable vehicle or carrier. It may also include other components such as anti-irritants. The carrier can be liquid, solid, or semi-solid. In some embodiments, the composition is an aqueous solution. Alternatively, the composition may be a dispersion, emulsion, gel, lotion, or cream-like vehicle for the various components. In one embodiment, the primary vehicle is water or a biocompatible solvent that is substantially neutral or has been made substantially neutral. The liquid vehicle may contain other materials such as buffers, alcohols, glycerin, and mineral oil, having various emulsifiers or dispersants known in the art to obtain the desired pH, consistency, and viscosity. The composition can be produced as a solid such as a powder or granule. The solid can be applied directly or dissolved in water or a biocompatible solvent before use to form a solution that is substantially neutral or has been made substantially neutral and can then be applied to the target site. In embodiments of the present invention, the vehicle for topical application to the skin can include water, buffer solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oil, phosphoglycerides, collagen, gelatin, and silicone-based materials.

[0061] The pharmaceutical composition may be an aerosol formulation administered via inhalation. Such aerosol formulations can be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, and nitrogen. They may also be formulated as pharmaceuticals for non-pressurized formulations such as nebulizers or atomizers.

[0062] Furthermore, the pharmaceutical composition may be a suppository. Formulations suitable for vaginal administration may, in addition to the active ingredient, be present as pessary, tampon, cream, gel, paste, foam, or spray formulations, and such carriers are known to be appropriate in the art.

[0063] Solid formulations for oral administration Formulations for oral use contain particles comprising the active compound in a mixture with non-toxic pharmaceutically acceptable excipients, such formulations being known to those skilled in the art (e.g., U.S. Patent 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, which are incorporated herein). Excipients are, 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, croscarmellose sodium, alginates or alginic acid), binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinyl pyrrolidone, or polyethylene glycol), and lubricants, glidants, antiadhesion agents (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc), and anticaking 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, talcum powder, sodium aluminosilicate, potassium alumina silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, polydimethylsiloxane) may also be. Other pharmaceutically acceptable excipients may be coloring agents, flavorings, plasticizers, humectants, and buffering agents. In some embodiments, the excipient (e.g., flavoring) is packaged with the composition.In some embodiments, the excipient (e.g., flavor) is packaged separately from the composition (e.g., combined with the composition prior to administration).

[0064] The solid composition used in the method of the present invention may include a coating adapted to protect the composition from unwanted chemical changes (e.g., chemical decomposition prior to release of the active substance). The coating can be applied onto the solid dosage form in a manner similar to that described in Remington (The Science and Practice of Pharmacy, (22nd ed.) ed. L.V. Allen, Jr., 2013, Pharmaceutical Press, Philadelphia, PA).

[0065] Powders and granules can be prepared by conventional methods using the above ingredients, for example, using a mixer, fluid bed apparatus, melt solidification apparatus, rotary granulator, extrusion / spheronizer, spray dryer, etc.

Examples

[0066] Certain features of the invention have been illustrated and described herein, but many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Accordingly, it is understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention. And the following examples are provided to teach various aspects of the invention. These examples represent individual embodiments of aspects of the invention, and those skilled in the art will recognize that additional examples can be generated to equally teach aspects of the invention.

[0067] Example 1. Treatment of C57L / J Mouse Groups Animals Both male and female C57L / J wild-type 6-8-week-old mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). The mice were housed on a 12:12-hour light / dark schedule and allowed free access to water and food. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (2011 revised edition) issued by the National Institutes of Health. The research protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

[0068] Experimental treatment plan Equal numbers of male and female mice were assigned to each group. Under ketamine / xylazine anesthesia (100 mg / kg and 10 mg / kg, respectively), mice were subjected to 5 Gy of total body irradiation (TBI) using a Varian 21EX LINAC (Palo Alto, Calif.), followed immediately by a 6.5 Gy top-up irradiation to the chest, for a total of 11.5 Gy of chest irradiation. Twenty-four hours after irradiation, the mice were treated orally once a day for 6 days with 1 mg / kg of sepiapterin or 5 mg / kg of BH4 dissolved in water (Figure 1).

[0069] Example 2. Evaluation of cardiac function Echocardiography At the baseline (before irradiation (IR)), and on days 8, 30, 60, 90, and 180, all mice were subjected to transthoracic echocardiography under light anesthesia (30 mg / kg pentobarbital sodium). Echocardiography was performed using a Vevo770 imaging system (VisualSonics, Toronto, Ontario, Canada) and a 30-MHz probe. The heart was imaged in B-mode from 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 Heart Association. LV ejection fraction (EF) and LV mass were calculated from the measured values of wall thickness and chamber diameter. LVEF was calculated as previously described using the Teicholz formula (LVEF = [LVEDD3 - LVESD3] / LVEDD3). Transmitral LV outflow tract Doppler spectra (E, A, ET) were recorded from the apical four-chamber view, and myocardial performance index (MPI) was calculated as the ratio of isovolumic contraction time (ICT) and isovolumic relaxation time (IRT) divided by ejection time (ET). The researchers who performed and interpreted the echocardiography were blinded to the treatment assignment. The cardiac contractility of irradiated mice not treated with SP was evaluated using 20 ng / mouse of the β-adrenergic agonist isoproterenol (Sigma Aldrich, St. Louis, MO, USA). Cardiac contractility (contractile reserve) was expressed as the rate of change in LVEF measured at rest (LVEFr) and 3 minutes after isoproterenol injection (LVEFi), and calculated as [(LVEFi - LVEFr) / LVEFr] * 100. Measurements of contractile reserve were performed at baseline, 8, 30, 60, 90, and 180 days after IR. Evaluation by echocardiography was performed by an operator blinded to the treatment assignment.

[0070] Effect of Sepiapetin on IR-Induced Cardiomyopathy Before receiving IR and before starting SP treatment, cardiac performance was evaluated at baseline in all mice. Subsequently, after receiving IR, the mice were randomly assigned to treatment groups (vehicle or SP). Thoracic top-up dosing was used to evaluate the impact of our TBI model on systolic, diastolic function, and contractile reserve to isoproterenol. Radiation blunted the response to isoproterenol (Figures 2, 7) and increased the myocardial performance index (Figures 3, 8) and isovolumic relaxation time. Daily oral administration of SP for 6 days after IR was able to restore the response to β-adrenergic stimulation by isoproterenol. Furthermore, SP administration suppressed the progression of systolic and diastolic dysfunction and improved the incremental increase in myocardial performance index and IRT. These results were further evaluated by animal gender. There was no significant difference in cardiac function response to radiation or 1 mg / kg SP with respect to systolic and diastolic function (Figures 14A-14B and Figures 15A-15B). However, in male mice, a further significant decrease in contractile reserve was seen compared to female mice at 90 and 180 days after radiation exposure (Figures 16A-16B). Importantly, in both males and females, SP was equally effective in reducing this loss of contractile reserve.

[0071] Example 3. Evaluation of Lung Function Evaluation of Lung Injury by Respiratory Rate Respiratory rate was measured biweekly from week 6 using a Mouse Ox system (STARR Life Sciences Corp., Allison Park, PA). Animals were placed under ketamine / xylazine anesthesia and the hair in the analysis area was shaved. Ten minutes after injection, the animals were placed in the supine position and the sensor was attached to the upper thigh. Respiratory rate was recorded for 3 minutes. Respiratory rate was calculated using an algorithm within the Mouse Ox software.

[0072] SP and BH4 Reduce Radiation-Induced Lung Function Loss Using the MouseOx system, the respiratory rate was measured as an index of respiratory function in mice irradiated with radiation, irradiated mice with SP (Figure 9), and irradiated mice with BH4 (Figure 5). Mice administered 1 mg / kg / day of SP or 5 mg / kg of BH4 for 6 days showed a marked delay in the onset of lung injury (14 weeks with SP administration and 10 weeks with vehicle administration), and it was confirmed that the impairment of lung function was significantly reduced compared to vehicle-treated mice at all measured points.

[0073] Example 4. Survival rate evaluation of mouse groups SP improves the survival rate after radiation exposure To determine whether the improvement in cardiopulmonary function affected overall survival, mice were followed for 180 days after IR. Figure 4 shows the Kaplan-Meier survival plots for irradiation of C57L / J mice with a total dose of 11.5 Gy to the chest (a dose of 6.5 Gy to the chest following 5 Gy of TBI) with or without administration of 1 mg / kg / day of SP for 6 days. Mice irradiated without treatment had a median survival period of 137 days and a survival rate of 40% at the end of the study, showing a significant decrease in the survival rate. On the other hand, mice administered 1 mg / kg / day of SP for 6 days had a significantly delayed survival period, and 71% survived at the 180-day time point at the end of the study, with an overall improvement in the survival rate.

[0074] Example 5. Expression of serum exosomal miRNA Blood was collected by cardiac puncture using an EDTA vial. The blood sample was centrifuged at 3,000 × g for 25 minutes to separate the plasma fraction, which was aliquoted and stored in the gas phase of liquid nitrogen. Exosomes were isolated from the plasma of mice at the time points shown in Figures 6A - 6C using an exosome isolation kit from 101Bio. The exosome concentration was quantified using the EXOCET Exosome Quantitation Kit (System Biosciences). Equal numbers of exosomes were combined from 5 animals at each time point, and total exosomal RNA was extracted using the miRNeasy Micro Kit (QIAGEN). The miRNA expression profile was analyzed using miRCURY LNATM The Universal RT microRNA PCR assay (QIAGEN) and ExiLENT SYBR (R) Green Maser Mix (QIAGEN) were used. Initial estimation of exosomal miRNA expression was performed with a QuantStudio 5 RT-PCR machine (Applied Biosystems) using a serum / plasma LNA TM miRNA PCR panel (Cat.# YAHS-106Y, QIAGEN).

[0075] The cycle threshold (Ct) values shown in FIGS. 6B-6C are the average values from 5 mice per group and are normalized to hsa-miR-145-5p and hsa-miR-221-3p, which were experimentally shown as high-quality normalization controls. At 30 days after IR, two different groups of exosomal miRNAs that showed significant differences between vehicle-administered animals and SP-administered animals were revealed. The first group included let-7a-5p, miR-1, miR-106b-3p, miR-106b-5p, miR-126-3p, miR-181a-5p, miR-335-3p, and miR-335-5p. The expression of miRNAs in this group was significantly decreased in vehicle-administered animals compared to SP-administered animals. In the second group, the expression of miRNAs was significantly higher in the vehicle-administered group compared to the SP-administered group. The second group included let-7g-5p, let-7i-5p, and miR-16-5p. No significant differences were observed between males and females in either the changes in exosomal miRNA expression due to IR or the effect of sepiapterin on miRNA expression. miRNAs that showed significant expression changes due to radiation and were regulated by sepiapterin were involved in inflammatory processes (e.g., let-7a-5p, miR-106b-3p, miR-181a-5p, let-7i-5p), angiogenesis and vascular homeostasis (miR-106b-3p, miR-126-3p), and cardiac inflammation and fibrosis (miR-335-5p, let-7i-5p, miR-126-3p, miR-16-5p). miR-16-5p inhibits the TGF-beta / VEGF signal. 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.

[0076] Example 6. Expression of cytokine mRNAs in heart and lung tissues Isolation of RNA and RT-PCR Total RNA was isolated from lung and heart tissue samples using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration was evaluated using a NanoDrop ND-1000 spectrometer (Thermo Scientific). RNA purity was evaluated by the ratios of A260 / A280 and A260 / A230. RNA integrity was evaluated by the ratio of 28S / 18S ribosomal RNA (rRNA) and the RNA Integrity Number (RIN) using an Agilent 2100 BioAnalyzer (Agilent Technologies). cDNA synthesis and genomic DNA removal were performed using the RT2 First Strand Kit (QIAGEN). Samples were amplified on a QuantStudio 5 RT-PCR machine (Applied Biosystems) using the QIAGEN RT2 SYBR (R) 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).

[0077] Changes in cytokine mRNA expression levels after radiation exposure Animals were irradiated with radiation and, 24 hours later, treated with SP or water for 6 days, after which the expression of various 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. The mRNA expression of tissue samples was tested at three different time points (8 days, 30 days, 16 weeks) after IR and compared with the mRNA expression of non-treated non-irradiated control animals (Figure 10). In heart tissue, no significant difference in expression levels was found between the vehicle-treated group and the SP-treated group for all cytokines. In lung tissue, cytokines IL-1β, IL-6, IL-17A, Spp2, and TGF-β1 showed significant differences in expression between the vehicle-treated group and the SP-treated group at different time points after IR. The vehicle-treated group had a significantly higher relative expression of IL-1β in lung tissue compared to the SP-treated group at 8 days and 30 days after IR (2.9 ± 0.649 vs 1.54 ± 0.825 and 3.39 ± 0.532 vs 1.66 ± 0.51, respectively). The relative expression of IL-6 was significantly higher in the vehicle-treated group than in the SP-treated group at all time points after IR (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). Along with IL-6, 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). Cytokines Spp2 and TGF-β1 showed a significant increase in lung tissue expression in the vehicle-treated group compared to the SP-treated group at 16 weeks after IR (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 the relative expression of lung tissue in all animals at 30 days, and no significant difference was found between the vehicle-treated group and the SP-treated group.

[0078] Example 7. IHC Analysis of Heart and Lung Tissues IHC analysis At 16 weeks after IR, the heart and right lung were harvested for histological analysis (Figure 11A - 11C). The heart was recovered in formalin, embedded in paraffin, and sliced into 5 mm sections. The right lung was perfused with OCT and sliced into 6 mm sections. To detect collagen fibers, Masson's trichrome staining was performed according to the instructions of the supplying company (Richard-Allan Scientific Masson's trichrome kit, Thermo Fisher Scientific, Waltham, MA, USA). The fibrotic areas of the myocardium and lung were calculated as the ratio of the collagen area to the total tissue area using computerized morphometric analysis (Image ProPlus 6.0 software, Media Cybernetics, Rockville, MD, USA). Inflammation, angiogenesis, and smooth muscle cell differentiation were measured by staining the heart and right lung with the following antibodies: Ly-6G / Ly-6C (Invitrogen, Carlsbad, CA, USA), F40 / 80 and alpha-SMA (Cell Signaling, Danvers, MA, USA), CD-31 (BD Pharmigen, San Jose, CA, USA). After detecting the primary antibody with the secondary antibody, staining was performed with Novared (Vector Laboratories, Burlingame, CA, USA) for antibody detection. The evaluation of the staining was performed by two investigators blinded to the treatment assignment using a dichotomous (positive / negative) method, and positive results were subsequently graded as mild (1), moderate (2), or intense (3) staining according to the intensity and spread of the staining.

[0079] Hydroxyproline The amount of hydroxyproline was measured as previously described using a hydroxyproline assay kit from Sigma Aldrich (St. Louis, MO).

[0080] Example 8. Proteins Measured by ELISA Plasma Analysis At the time of slaughter, blood was collected by cardiac puncture into EDTA-containing tubes and centrifuged at 3000 rpm for 25 minutes to isolate 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 according to the manufacturer's recommendations and used in the ELISA kits. All cytokines were reported in absolute units. Major molecular players of the coagulation cascade such as tissue factor, thrombin, fibrinogen are epidemiologically and mechanistically associated with inflammatory components. Genetic and pharmacological studies have revealed that fibrinogen plays a crucial role in determining the degree of local or systemic inflammation. Neutrophil elastase is a serine protease present in the azurophilic granules of neutrophils, is released in inflammatory conditions, and due to its low substrate specificity, it disrupts the extracellular matrix and causes tissue damage. Neutrophil elastase is one of the most destructive enzymes in vivo. Once this enzyme gets out of control, it disturbs the function of the lung's permeability barrier and induces the release of inflammatory cytokines along with the stimulation of acute lung injury. The inflammatory cytokine IL-6 can increase in expression as early as 6 hours after radiation in lung tissue and blood. High IL-6 concentration in plasma worsens the inflammatory response of lung tissue, and ultimately, leakage of IL-6 into the bronchoalveolar space may occur, further progressing lung damage. High concentration of IL-6 in lung tissue attracts inflammatory cells such as neutrophils, monocytes, macrophages to the damaged lung, and ultimately may cause severe damage to the lung and lead to chronic fibrosis. A sudden increase in plasma IL-6 can result in an increase in C-reactive protein (CRP). C-reactive protein (CRP) is produced in the liver and is a biomarker of the general stress response to inflammation and infectious agents. Serum CRP value has been shown to be an effective indicator for predicting the occurrence of radiation pneumonitis and can be a valuable factor for evaluating the degree of lung damage caused by radiation.

[0081] Changes in cytokine concentrations in plasma after IR Animals in the vehicle treatment group showed significantly higher plasma levels of IL-6 at 4 days, 8 days, and 180 days after IR compared to the SP treatment 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)) (Figure 13C). Animals in the vehicle treatment group showed significantly higher plasma levels of fibrinogen at 8 days after IR compared to the SP treatment group (948.77 ± 98.54 g / ml vs 332.38 ± 109.02 g / ml (p < 0.001)) (Figure 13B). Animals in the vehicle treatment group showed significantly higher plasma levels of neutrophil elastase at 180 days after IR compared to the SP treatment group (25.21 ± 6.54 ng / ml vs 12.15 ± 2.87 ng / ml (p = 0.034)) (Figure 13D). C-reactive protein did not show a significant difference between the vehicle-treated and SP-treated animal groups at any time point after IR (Figure 13A).

[0082] Example 9. Spearman's correlation between fibrosis and cytokine mRNA expression Statistical analysis The two-sided Student's t-test was used to examine the statistical significance of groups with a single independent variable. P < 0.05 was considered significant. Spearman's rank correlation was used to evaluate the relationship between specific mRNA expression and the degree of pulmonary fibrosis (Figure 12). Log-rank analysis was used to compare the survival rates between groups.

[0083] Conclusion Oral administration of BH4 or its metabolic precursor SP from 24 hours after radiation exposure enhances lung and heart function, as evidenced by a decrease in respiratory rate, an increase in contractile reserve, improvement in systolic and diastolic function, and an increase in survival rate.

Claims

1. 20. A method of treating a subject exposed to radiation, comprising administering to the subject an effective amount of sepiapterin, tetrahydrobiopterin, or dihydrobiopterin, or a pharma- ceutically acceptable salt or cocrystal thereof.

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

3. 13. The method of claim 1, wherein the administering step reduces or inhibits cardiac and / or pulmonary toxicity in the subject.

4. 10. The method of claim 1, wherein the administering step reduces endothelial cell death in the gastrointestinal tract, heart, and / or lungs in the subject.

5. 10. The method of claim 1, wherein the administering step reduces radiation-induced inflammation in epithelial cells of the gastrointestinal tract, heart, and / or lungs in the subject.

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

7. The method of any one of claims 1 to 6, wherein the subject has acute radiation syndrome.

8. 10. The method of claim 1, wherein the administering step occurs within 24 hours after exposure to radiation.

9. 8. The method of claim 7, wherein the administering step occurs at least 24 hours after exposure to radiation.

10. The method of any one of claims 1 to 6, wherein the subject has chronic radiation syndrome.

11. The method of any one of claims 1 to 6, wherein the subject has cutaneous radiation syndrome.

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

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

14. The method of any one of claims 1 to 13, wherein the administering step occurs at least once daily for at least six days.

15. 15. The method of any one of claims 1 to 14, wherein the administering step 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.

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

17. The method according to any one of claims 1 to 16, wherein sepiapterin or a pharma- ceutically acceptable salt or cocrystal thereof is administered.

18. 18. The method of any one of claims 1 to 17, wherein the administering step increases 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 to 18, wherein the administering step reduces expression of let-7g-5p, let-7i-5p, and / or miR-16-5p.

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

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