Radiation protection by inhibition of superoxide dismutase 1

Abstract

The present invention relates to an inhibitor of intracellular copper-zinc superoxide dismutase for use in protecting subjects from radiation burst injury. The present invention also relates to a kit comprising an SOD1 inhibitor and a radiosensitizer for cancer cells, the in vitro use of an SOD1 inhibitor for protecting non-cancer cells from ionizing radiation, and methods, compositions, and uses relating thereto.

Description

[Technical Field]

[0001] This invention relates to an inhibitor of intracellular copper-zinc superoxide dismutase for use in protecting subjects from radiation burst injury. The invention also relates to a kit comprising an SOD1 inhibitor and a radiosensitizer for cancer cells, the in vitro use of an SOD1 inhibitor for protecting non-cancer cells from ionizing radiation, and related methods, compositions, and uses.

[0002] Ionizing radiation can have serious effects on living cells, depending on the dose. Direct effects are caused by radiation directly interacting with genetic material, such as DNA, or some other cellular components that are important for cell survival. However, since genetic material represents only a small proportion of the biomolecules that make up a cell, the probability of radiation interacting with genetic material is low. In contrast, indirect effects are mainly caused by the interaction of radiation with water, which generates radicals such as hydroxyl radicals and superoxide ions. These radicals cause the degradation of biomolecules, and depending on the intensity of the degradation, cause cell destruction and symptoms of radiation damage known in the art.

[0003] Since free radicals are also produced as an inevitable byproduct of oxidative metabolism, protective enzymes that catalyze the breakdown of radicals into non-toxic or at least less toxic products are needed, such as catalase, which catalyzes the conversion of hydrogen peroxide (H2O2) to water and oxygen, as well as superoxide ions (O2) to oxygen and hydrogen peroxide. -Superoxide dismutase (SOD), which disproportionates ) has evolved. In mammals, three types of SOD are known: SOD1 (Pfam PF00080), also known as copper-zinc SOD, is mostly found in the cytoplasm; SOD2 (PFAM PF02777), also known as manganese SOD, is localized in mitochondria; and SOD3, which is also a copper-zinc enzyme, is localized extracellularly. SOD inhibitors, such as compounds containing tetrathiomolybdate, have been proposed as anticancer agents (Donate et al. (2008), Br J Cancer 98:776).

[0004] High doses of radiation can kill cancer cells, and ionizing radiation has long been an important method in cancer treatment; however, it simultaneously induces toxicity in surrounding non-malignant tissue, which can cause adverse events and may affect patients' adherence to recommended protocols.

[0005] U.S. Patent No. 10,722,526B2 taught the radiation protection or radiation mitigation of radiation-induced damage by compounds such as 2-methoxyestradiol. Milas et al. (1984), Int J Rad Oncol Biol Phys 10(12):2335 reported the radiation protective activity of diethyldithiocarbamate (DDC), suggesting inhibition of superoxide dismutase as the interpretation; however, this was in direct contradiction with previous findings that DDC inhibits superoxide dismutase only weakly and with very slow kinetics (Misra et al. (1979), JBC 254(22):11623). Evans et al. (1985), Int J Rad Oncol Biol Phys 11(6):1163 reported tumor radiosensitization with concomitant myeloprotection in mice by DDC, suggesting a direct radical scavenging effect mediated by thiol compounds. Mashiba et al. (1991), Life Sciences 49:1419 described enhanced radiosensitization in tumor cells by the combination of DDC and RK28. Trapp et al. (2009), Melanoma Res 19(6):350 described the antimelanoma activity of ATN-224.

[0006] Following the role of antioxidant enzymes in protecting cells from ionizing radiation, one suggestion for improving radiotherapy was to inhibit antioxidant enzymes to increase radiation damage to cancer cells (Jiang et al. (2018), Cancer Lett 438:154, Che et al. (2016), Drug Discovery Today 21(1), 143).

[0007] The standard dose rate delivered during radiotherapy procedures ranges from 0.5 to 20 Gy / min, depending on the irradiation technique used. In recent years, protocols have been developed that provide ultra-high dose rate (FLASH) irradiation at several times the magnitude of that used in conventional clinical radiotherapy. Such treatments have been found to be remarkably effective against the tumor itself, as effectively as conventional radiotherapy, while significantly reducing adverse effects and effects on surrounding normal tissue (see, e.g., Vozenin et al. (2022), Nat Rev Clin Oncol 19:791). Therefore, FLASH irradiation has been considered a significant improvement in radiotherapy, as it enables the treatment of cancerous tumors with remarkably low radiotoxicity. Limitations to the widespread implementation of FLASH radiotherapy include the need for high doses per ray (at least 6 Gy) and very high dose rates (at least 40 Gy / s) to achieve FLASH protection, requiring specialized equipment that is not widely available. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 10,722,526 B2 [Non-patent literature]

[0009] [Non-Patent Document 1] Donate et al. (2008), Br J Cancer 98:776 [Non-Patent Document 2] Milas et al. (1984), Int J Rad Oncol Biol Phys 10(12):2335 [Non-Patent Document 3] Misra et al. (1979), JBC 254(22):11623 [Non-Patent Document 4] Evans et al. (1985), Int J Rad Oncol Biol Phys 11(6):1163

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, there is still a need for improved cancer treatment, particularly by radiotherapy. The technical problem underlying the present invention can be regarded as providing means and methods to meet the above need. This technical problem is solved by the embodiments characterized in the claims and the following description of the present specification.

Means for Solving the Problems

[0011] Therefore, the present invention relates to an inhibitor of intracellular copper-zinc superoxide dismutase (SOD1 inhibitor) for use in protecting a subject from injury by radiation bursts (radiation burst damage). [[ID=3,8]]

Brief Description of the Drawings

[0012] [Figure 1]A graph showing the percentage of cells that survived at the indicated doses under conventional irradiation and FLASH irradiation, with or without the presence of ATN-224 or NaCN. Controls: non-irradiated cells (survival = 1); control conventional: cells after irradiation at a conventional dose rate; control Flash: cells after irradiation at a high dose rate; ATN-224: (non-irradiated) cells in the presence of ATN-224; ATN conventional: cells after irradiation at a conventional dose rate in the presence of ATN-224; ATN Flash: cells after irradiation at a high dose rate in the presence of ATN-224; NaCN: (non-irradiated) cells in the presence of NaCN; NaCN conventional: cells after irradiation at a conventional dose rate in the presence of NaCN; cells after irradiation at a high dose rate in the presence of NaCN. Y-axis: percentage of surviving cells relative to non-irradiated cells.

Modes for Carrying Out the Invention

[0013] In general, terms used herein should be given their usual, customary meanings to those skilled in the art, and not limited to any special or customized meanings unless otherwise indicated. Where used below, the terms “have,” “comprise,” or “include,” or any grammatical variation thereof, are used non-exclusively. That is, these terms can mean both a situation in which no further features exist in the entity described herein other than those introduced by these terms, and a situation in which one or more further features exist. For example, the expressions “A has B,” “A contains B,” and “A includes B” can mean both a situation in which no other elements exist in A other than B (i.e., A consists solely and exclusively of B), and a situation in which one or more further elements exist in entity A other than B (such as element C, elements C and D, or further elements). Furthermore, as those skilled in the art will understand, the expressions "comprising a" and "comprising an" preferably mean "comprising one or more," that is, equivalent to "comprising at least one." Therefore, expressions relating to one of several items preferably relate to at least one such item, more preferably to multiple such items, unless otherwise indicated; that is, for example, identifying a cell relates to identifying at least one type of cell, preferably to identifying multiple cells.

[0014] Furthermore, where used below, the terms “preferably,” “more preferably,” “most preferably,” “particularly,” “more specifically,” “specifically,” “more specifically,” or similar terms are used with optional features without limiting further possibilities. In other words, the features introduced by these terms are optional features and are not intended in any way to limit the scope of the claims. The present invention may be implemented by using alternative features, as those skilled in the art will recognize. Similarly, features introduced by “in one embodiment” or similar expressions are intended to be optional features, without any limitation on further embodiments of the present invention, without any limitation on the scope of the present invention, and without any limitation on the possibility of combining such features with other optional or non-optional features of the present invention.

[0015] The methods specified herein are preferably in vitro methods. The steps of the method may, in principle, be carried out in any order deemed suitable by those skilled in the art, but preferably in the order shown; and one or more, preferably all, of the steps may be assisted in or carried out by automated equipment. Furthermore, the method may include steps in addition to those expressly mentioned above.

[0016] Where used herein, unless otherwise specified, the term “about” means with respect to the indicated value, preferably ±20%, more preferably ±10%, and most preferably ±5%, with respect to the indicated value, with respect to the technical precision generally accepted in the art. Furthermore, the term “essentially” means that there is no deviation that would have an effect on the indicated results or use, i.e., any possible deviation would not result in a deviation of more than ±20%, more preferably ±10%, and most preferably ±5% of the indicated results. In other words, “essentially consisting of” means including the identified components but excluding other components, except for substances present as impurities, unavoidable substances present as a result of processes used to provide the components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase “essentially consisting of” includes any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of one set of components would contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of the unspecified components.

[0017] The degree of identity (e.g., expressed as "identity %") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences across a comparison window, where the sequence fragments within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the sequence being compared for optimal alignment. The percentage is calculated by determining the number of positions where identical residues exist in both sequences, preferably across the entire length of the polynucleotide or polypeptide, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. The optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), by computer implementations of these algorithms (e.g., BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Given that two sequences are identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment, i.e., the degree of identity. Preferably, default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used. More preferably, a Basic Local Alignment Search Tool (BLAST) implementation is used with default parameter values ​​for alignment. In the context of biological sequences as referred to herein, the term “essentially identical” means an identity percentage value of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%.As will be understood, the term "essentially identical" includes 100% identity. The same applies to the term "essentially complementary."

[0018] As used herein, the term "polypeptide" means a molecule consisting of several amino acids, typically at least 20, that are covalently linked to one another by peptide bonds. Molecules consisting of fewer than 20 amino acids covalently linked by peptide bonds are generally considered "peptides." Preferably, polypeptides consist of 50 to 1000, more preferably 75 to 1000, even more preferably 100 to 500, and most preferably 110 to 400 amino acids. Preferably, polypeptides are included in fusion polypeptides and / or polypeptide complexes.

[0019] The term “intracellular copper-zinc superoxide dismutase,” which may also be called “superoxide dismutase 1” or “SOD1,” is known to those skilled in the art. Preferably, this term relates to cytoplasmic superoxide dismutase in eukaryotic cells, preferably mammals, more preferably humans. SOD1 is preferably a homodimeric enzyme and contains and requires copper and zinc ions for its activity. Preferably, SOD1 comprises the amino acid sequence shown in Sequence ID No. 1 (Genbank registration number AAB05662.1) or a sequence at least 70% identical thereto, more preferably at least 90% identical thereto. That is, SOD1 is preferably mammalian SOD1, more preferably human SOD1. The activity of SOD1 can be measured by methods known in the art, for example, from Vonk et al, (2010), JBC 285(37):2891; corresponding kits are commercially available.

[0020] The term "copper-zinc superoxide dismutase inhibitor," also called "SOD1 inhibitor," will be understood by those skilled in the art to relate to compounds that inhibit SOD1 activity. Preferably, an SOD1 inhibitor is a direct SOD1 inhibitor, i.e., a compound that preferably directly interacts with SOD1 or at least one of its components, thereby inhibiting SOD1 activity. An SOD1 inhibitor may be a nonspecific superoxide dismutase inhibitor, but is preferably a specific superoxide dismutase inhibitor, and more preferably a specific SOD1 inhibitor. Methods for identifying SOD inhibitors, particularly SOD1 inhibitors, are known to those skilled in the art. Preferably, an SOD1 inhibitor is a compound that inhibits SOD1 activity in cultured host cells by at least 20%, preferably at least 30%, more preferably at least 40%, and more preferably at least 50%, at a concentration of up to 1 mM, preferably up to 0.1 mM, more preferably up to 0.01 mM, and even more preferably up to 0.001 mM. Preferably, the SOD1 inhibitor is a compound that inhibits SOD1 activity in cultured host cells by at least 20%, preferably at least 30%, more preferably at least 40%, and more preferably at least 50%, at a concentration that causes cell death in up to 25%, preferably at least 10%, more preferably at least 5%, and even more preferably at least 1% of the cultured cells. Also preferably, the SOD1 inhibitor is a compound that inhibits SOD1 activity in vivo in the subject by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%, at a dose that causes up to a Grade 1 (mild) or Grade 2 (moderate) adverse event as specified in the Common Terminology Criteria for Adverse Events v3.0 (CTCAE, August 9, 2006).

[0021] Preferably, the SOD1 inhibitor is a low molecular weight compound having a molecular weight of preferably up to 1 kDa, more preferably up to 0.5 kDa. Preferably, the SOD1 inhibitor contains a copper complexant. More preferably, the SOD1 inhibitor contains a tetrathiomolybdate ion, a 4,5-dihalogeno-2-arylpyridazine-3(2H)-one, a 2-methoxyestradiol, and / or a cyanide ion.

[0022] Preferably, the SOD1 inhibitor contains a tetrathiomolybdate ion. The terms "tetrathiomolybdate" and "tetrathiomolybdate ion" are known to those skilled in the art. The molecular formula of tetrathiomolybdate is MoS4. 2- (CAS number 16330-92-0). The tetrathiomolybdate ion can be provided by any compound deemed suitable by those skilled in the art, for example, its acid H2MoS4, or a preferably pharmaceutically acceptable salt thereof, for example, bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0), diammonium tetrathiomolybdate, disodium tetrathiomolybdate, or dipotassium tetrathiomolybdate. Preferably, the SOD1 inhibitor is bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0).

[0023] Preferably, the SOD1 inhibitor is 4,5-dihalogeno-2-arylpyridazine-3(2H)-one. That is, preferably, the SOD1 inhibitor has the chemical structure of formula (I):

[0024] [ka] (In the formula, R 1 and R 2 These are independently selected from -Cl, -Br, -I, and -F, preferably R 1 and R 2 is either -Cl or -Br; R 3 ~R7 is independently selected from -H, -CH3, -CH2CH3, -Cl, -Br, -I, and -F, and preferably, R 4 is -CH3, and R 3 and R 5 ~R 7 is -H; or R 5 and R 7 is -Cl, and R 3 , R 4 , and R 6 is -H). That is, preferably, 4,5-dihalogeno-2-aryl-pyridazin-3(2H)-one is 4,5-dichloro-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1, CAS No. 41931-13-9), 4,5-dibromo-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1.28, CAS No. 1035450-90-8), or 4,5-dichloro-2(2,4-dichlorophenyl)pyridazin-3(2H)-one (LCS-1.34, CAS No. 24725-65-3).

[0025] Preferably, the SOD1 inhibitor is 2-methoxyestradiol ((8R,9S,13S,14S,17S)-2-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol, CAS number 362-07-2) or a derivative or prodrug. Where used herein, a “prodrug” of 2-methoxyestradiol refers to a compound having a different chemical structure from 2-methoxyestradiol but being metabolized to 2-methoxyestradiol in the body of the subject. Where used herein, a “derivative” of 2-methoxyestradiol refers to a compound having a structure similar to but not identical to 2-methoxyestradiol and possessing the property of being an SOD1 inhibitor. Preferably, the derivative is a compound obtainable from 2-methoxyestradiol by up to three, preferably up to two, more preferably one, derivatization steps known to those skilled in the art. More preferably, the derivative is a compound obtainable from 2-methoxyestradiol by up to three, preferably up to two, more preferably one, derivatization steps selected from (i) alkylation, preferably O-alkylation, preferably methylation, ethylation, propylation, or isopropylation; (ii) esterification, preferably -OSO2NH, -COOH and / or -OPO3H2 groups, preferably sulfamoylation, acetylation, propionylation, isopropionylation, or succinylation; (iii) reduction, preferably C=C and / or hydroxyl; and (iv) oxidation, preferably hydroxyl, CH, and / or CC groups. Preferably, the 2-methoxyestradiol derivative or prodrug has a structure according to formula (ii):

[0026] [ka] (In the formula, R 10 and R 11It is independently selected from -OSO2NH, -OH, -OSO3, OCH3, -OCH2CH3, -NH2, however (with the provision), R 10 and R 11 Both are not -OH; preferably, R 10 and R 11 (is -OSO2NH). In other words, the 2-methoxyestradiol derivative is preferably 2-methoxyestradiol disulfamate ([(8R,9S,13S,14S,17S)-2-methoxy-13-methyl-3-sulfamoyloxy-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]sulfamate, CAS number 401600-86-0).

[0027] Preferably, the SOD1 inhibitor is one of N,N'-bis(2-aminoethyl)-2ethanediamine (triethylenetetramine, CAS number 112-24-3) or a salt thereof, preferably its dihydrochloride (trientine hydrochloride, CAS number 38260-01-4).

[0028] More preferably, the SOD1 inhibitor contains a cyanide ion (CAS number 57-12-5). That is, the SOD1 inhibitor is preferably hydrogen cyanide or a salt thereof deemed suitable by those skilled in the art, such as sodium cyanide or potassium cyanide. Given the well known toxicity of cyanide to living subjects, they are preferably used in vitro, for example, as SOD1 inhibitors against cultured cells, and / or also preferably not used in vivo in subjects, especially when the subjects are human.

[0029] As used herein, the term “subject” refers to an animal, preferably a vertebrate, more preferably a mammal, and most preferably a human. Preferably, the subject is known or presumed to have experienced a radiation burst, preferably radiotherapy. Preferably, the subject has cancer. As used herein, the term “host cell” refers to any cell containing SOD, preferably SOD1. Preferably, the cell is a eukaryotic cell, preferably a yeast cell, e.g., a cell of a strain of baker's yeast, or an animal cell. More preferably, the host cell is an animal cell, preferably an insect cell or a mammalian cell, particularly a mouse or rat cell. Most preferably, the host cell is a human cell.

[0030] The term “radiation” is understood by those skilled in the art. Preferably, radiation is radiation that interacts with biological material, particularly cells, and preferably causes damage thereto. Preferably, radiation includes, preferably, particle beam irradiation, particularly helium ions (alpha beam irradiation), carbon ions, oxygen ions, and / or neutrons; or electron beam irradiation, particularly beta beam irradiation, and preferably so; or photon beam irradiation, particularly gamma beam irradiation, and preferably so. Preferably, radiation is the type of radiation used in radiotherapy, particularly in cancer radiotherapy.

[0031] The term “radiation burst,” as used herein, preferably refers to a short-term increase in radiation exposure that is foreseeable or predictable, where “short-term” refers to a time frame preferably up to 12 hours, preferably up to 6 hours, more preferably up to 2 hours, even more preferably up to 0.5 hours, and most preferably up to 1 minute. Corresponding radiation bursts are known in the art; for example, radiation bursts caused by solar flares typically last for several hours, while radiation bursts expected to be experienced by a radiation worker may last for several minutes or several hours, although, for example, a radiation therapy session may be completed within a few minutes or less than a minute. As a person skilled in the art will understand in consideration of the descriptions herein, the time frame (duration) of a radiation burst is preferably measured as beginning when the radiation intensity increases significantly above the background radiation and preferably ending when the radiation intensity returns to a value that does not significantly exceed the background radiation. Preferably, a radiation burst causes a radiation dose of 1 Gy to 100 Gy, preferably 2 Gy to 25 Gy, in the subject or a portion thereof. Accordingly, the radiation burst will cause the aforementioned radiation dose within a maximum of 12 hours, preferably within a maximum of 6 hours, more preferably within 2 hours, even more preferably within 0.5 hours, and most preferably within less than 1 minute.

[0032] Preferably, the radiation burst is radiotherapy, more preferably cancer radiotherapy. The term “radiotherapy” is understood by those skilled in the art and preferably relates to the use of ionizing radiation to kill or control an undesirable cell population within a subject, wherein the undesirable cell population preferably includes cancer cells. As referred herein, radiotherapy includes curative radiotherapy and adjuvant radiotherapy. Preferably, the radiation burst is administered to the subject as a radiotherapy session. In such a case, the radiation burst may be radiotherapy with a dose rate of up to 10 Gy / s, preferably up to 0.5 Gy / s, and more preferably up to 0.05 Gy / s. That is, radiotherapy is preferably radiotherapy with a conventional dose rate. However, radiotherapy with higher dose rates, e.g., above 10 Gy / s, preferably above 50 Gy / s, and more preferably above 100 Gy / s, may also be administered. The dose per treatment, i.e., the dose per session, is preferably 0.1 Gy to 10 Gy, preferably 1 Gy to 5 Gy. However, depending on the subject, the disease, particularly the type of cancer, the organ or organ system being treated, and other factors known to those skilled in the art, higher or lower doses may also be administered by radiotherapy. That is, preferably, radiotherapy is radiotherapy with a dose per treatment of 10 Gy to 100 Gy, preferably 20 Gy to 50 Gy. As those skilled in the art will understand, the doses expressed herein in Gy units are doses of absorbed radiant energy per kilogram. That is, the doses may be whole-body doses or organ or organ system doses. Preferably, the dose is a local dose at the site of radiotherapy. Those skilled in the art know how to adjust the dose of radiotherapy according to the relevant parameters, and appropriate guidelines for radiotherapy are available. Accordingly, SOD1 inhibitors may be used, in particular, for the treatment of subjects undergoing radiotherapy, especially for the protection of subjects from radiation burst damage in the treatment and / or prevention of cancer by radiotherapy.

[0033] As used herein, the term "cancer" refers to a disease in animals, including humans, characterized by the uncontrolled proliferation of a group of somatic cells ("cancer cells"). This uncontrolled proliferation may involve invasion and destruction of surrounding tissues, and possibly the spread of cancer cells to other parts of the body (metastasis). Preferably, the term cancer also includes cancer recurrence (relapse). That is, preferably, cancer is a solid tumor, metastasis, or a relapse thereof. Preferably, cancer is of stage 0 to stage III. Preferably, cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratomatous Teratoid tumors, basal cell carcinoma, cholangiocarcinoma, bladder cancer, brainstem glioma, breast cancer, Burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenesis, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastoma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and optic tract glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides The following are selected from a list consisting of nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade ovarian tumor, pancreatic cancer, papillomatosis, paranasal and nasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cervical cancer, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.More preferably, the cancer is a cancer of the brain, skin, lungs, liver, pancreas, intestines, esophagus, heart, kidneys, prostate, breast, head and neck, bone, bone marrow, genitals, and / or spinal cord pedicle, as described above and herein.

[0034] The terms “to treat” and “treatment” mean significant improvement of a disease or disorder or its associated symptoms as referred to herein; as used herein, the term includes prevention of exacerbation of the disease, disorder or its associated symptoms. As used herein, such treatment also includes complete recovery of health related to the disease or disorder as referred to herein. As used herein, it should be understood that treatment does not have to be effective in all subjects to be treated. However, the term would preferably require successful treatment of a statistically significant portion of subjects suffering from the disease or disorder as referred to herein. Whether a portion is statistically significant can be determined without further difficulty by those skilled in the art using various well-known statistical evaluation tools, such as confidence intervals, p-values, Student's t-tests, and Mann-Whitney tests. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment will be effective for at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of subjects in a given cohort or population. Preferably, the treatment includes inhibiting the growth of cancer cells, more preferably killing cancer cells. Preferably, the treatment of cancer is to reduce the tumor and / or cancer cell load in the subject. As will be understood by those skilled in the art, the effectiveness of the treatment of cancer, for example, depends on various factors, including, for example, the stage and type of cancer. Also preferably, the cancer treatment further includes at least one of chemotherapy, immunotherapy, surgery, and radiotherapy. Preferably, the treatment includes treating relapses. Also preferably, the treatment includes treating advanced-stage cancer.

[0035] The terms “prevention” and “prevention” mean maintaining health in a subject with respect to the disease or disorder referred to herein for a specific period of time. It will be understood that the period may depend on the dose and number of radiotherapy sessions administered, as well as the subject’s personal factors. It should be understood that prevention does not have to be effective in all subjects treated as specified herein. However, the term preferably requires that a statistically significant portion of a cohort or population of subjects is effectively prevented from developing the disease or disorder or its associated symptoms referred to herein. Preferably, in this context, a cohort or population of subjects is intended that would normally, i.e., without the use of preventive measures according to the invention, develop the disease or disorder as referred to herein. Whether the portion is statistically significant can be determined without further difficulty by those skilled in the art using various well-known statistical evaluation tools discussed elsewhere herein. In the context of cancer treatment, prevention refers, in particular, to preventing the development of cancer, preventing metastasis formation, and / or preventing recurrence, and preferably to preventing metastasis formation and / or preventing recurrence.

[0036] As will be understood by those skilled in the art, radiotherapy may be accompanied by or complemented by other treatment methods such as chemotherapy, surgery, and / or immunotherapy. The terms “chemotherapy” and “surgery” will be understood by those skilled in the art. Appropriate standard treatment protocols are available in the art. The term “immunotherapy,” as used herein, relates to the treatment and / or prevention of disease, preferably cancer, by modulating the immune response of a subject. Such modulation may involve inducing, enhancing, or suppressing the immune response, for example, by administering at least one immune checkpoint modulator and / or cytokine. Preferably, the cytokine is, in such cases, interferon, interleukin, or chemokine. Immunotherapy may also involve the administration of T cells, e.g., CAR T cells and / or recombinant T cell receptor T cells, and / or at least one T cell engager, i.e., molecules that tether T cells to target cells; corresponding T cell engagers, e.g., bispecific T cell engagers (BiTEs), such as bispecific antibodies, are known in the art. Furthermore, radiotherapy, particularly radiotherapy for cancer, may involve the administration of at least one radiosensitizer, i.e., a compound that increases the sensitivity of target cells, such as cancer cells, to ionizing radiation. Preferably, the radiosensitizer is a compound that increases radiation damage to DNA. That is, preferred radiosensitizers are selected from the list consisting of temozolomide, metronidazole, misonidazole, bromodeoxyuridine, motexafingadolinium, and efapoxyral, all of which are known in themselves to those skilled in the art as their use as radiosensitizers.

[0037] The term “injury due to a radiation burst,” which may also be called “radiation burst injury,” will be understood by those skilled in the art in light of the descriptions herein. Preferably, a radiation burst injury is at least one disease or injury or symptom caused by a dose of radiation exposure in a subject or host cell. Preferably, a radiation burst injury is either an acute or chronic radiation injury; those skilled in the art will know that the terms “acute” and “chronic” injuries are used in the context of radiation injury as relating to the timing between radiation exposure and injury, rather than the duration of radiation exposure. That is, preferably, an acute radiation injury is a reaction to radiation that occurs within minutes, hours, or up to seven days after exposure, while a chronic radiation injury is a reaction to radiation that occurs more than seven days after exposure. Typical radiation injuries, grouped by the organ system in which they most frequently occur, are summarized in Table 1 below.

[0038] [Table 1]

[0039] In other words, acute radiation injury is preferably selected from the list consisting of burns, ulceration, delayed fracture healing, decreased blood cell count, decreased white blood cell count, decreased platelet count, enteritis, nausea, vomiting, swelling, seizures, dysphagia, ulceration, inflammatory pneumonia, angioedema, hepatitis, nephritis, hemorrhage, and decreased fertility. Also preferably, chronic radiation injury is selected from the list consisting of fibrosis, osteoporosis, osteomalacia, bone mineral density, bone marrow failure, stenosis, fistula, sinus formation, cognitive impairment, vascular damage, heart attack, liver failure, kidney failure, and decreased fertility. In the case of radiotherapy for cancer, in addition to the above, radiation injury may preferably be cell death of non-cancerous cells, scarring, ulceration, wet desquamation, erythema, and / or dry desquamation, all at the treatment site. Preferably, radiation burst injury is not mutagenic injury, i.e., injury preferably not caused by the direct action of ionizing radiation on genetic material, particularly DNA.

[0040] The term “protection from radiation burst injury” will be understood by those skilled in the art in this specification, and especially in consideration of the disclosures herein and in the examples. Preferably, such protection means reducing the degree or frequency of at least one symptom of radiation burst injury by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%. More preferably, at least one type of radiation burst injury, particularly chronic radiation burst injury, is prevented by at least 75%, more preferably at least 90%. As will be understood by those skilled in the art, the degree of radiation burst injury is a measure of the intensity to which a given radiation burst injury occurs; that is, the degree of radiation burst injury can be measured in a single individual and compared to a reference of a control subject. However, the degree of radiation burst injury can also be determined by calculating, for example, the mean or a similar statistically significant parameter over a large number of subjects. As will also be understood, the frequency of radiation burst injury is a measure of how many of a group of subjects have a given radiation burst injury after a radiation burst, preferably to a certain degree. Therefore, the degree of radiation burst injury is, in advance, a measure of the severity of radiation burst injury, while the frequency of radiation burst injury is primarily a measure of how frequently radiation burst injury occurs in a given population. Where used herein, protection from radiation burst injury may mean reducing the degree of radiation burst injury, reducing the frequency of radiation burst injury, or both.

[0041] Preferably, the radiation burst is radiotherapy, and protecting the subject means protecting non-cancerous cells from the radiation therapy. In such cases, the SOD1 inhibitor is preferably administered locally, for example, near the irradiation site. However, the SOD1 inhibitor can also be administered systemically, especially when a specific body part is irradiated or when a large area or whole body is irradiated.

[0042] In consideration of the foregoing description herein, preferably, a radiation burst is radiotherapy, particularly a radiotherapy session; and also preferably, protecting the subject is protecting non-cancerous cells from radiotherapy. That is, preferably, the present invention relates to a copper-zinc superoxide dismutase inhibitor (SOD1 inhibitor) for use in protecting non-cancerous cells from radiotherapy for cancer in a subject. In such cases, protecting non-cancerous cells also preferably includes a reduction of at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50% of non-cancerous cell death after radiotherapy; and / or a reduction of at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50% of scarring. Also preferably, protecting non-cancerous cells includes avoiding necrosis and / or blister formation at the treatment site; and / or avoiding ulceration and / or moist desquamation at the treatment site. Even more preferably, protecting non-cancerous cells includes the development of erythema and / or dry desquamation at the treatment site as adverse effects of radiation.

[0043] According to the present invention, SOD1 inhibitors are intended for use in protecting subjects from radiation burst injury. That is, SOD1 inhibitors are preferably for medical use in subjects to prevent radiation burst injury; if the radiation burst is, for example, radiotherapy for cancer, the effect of radiotherapy is preferably the treatment of cancer, while the effect of SOD1 inhibitor administration is also preferably the protection of non-cancer cells. That is, by performing radiotherapy in the presence of at least one SOD1 inhibitor in non-cancer cells, cancer cells are killed or inhibited, while non-cancer cells are protected from the effects of radiation. That is, preferably, the use involves administering the SOD1 inhibitor within a time frame of 1 minute to 12 hours, preferably 1 minute to 6 hours, more preferably 1 minute to 3 hours, and even more preferably 1 minute to 2 hours, prior to the onset of the radiation burst. Also preferably, the use involves administering the SOD1 inhibitor within a time frame of 5 minutes to 60 minutes, preferably 5 minutes to 45 minutes, more preferably 5 minutes to 30 minutes, prior to the onset of the radiation burst. In consideration of the foregoing, those skilled in the art will understand that it is preferable for SOD1 to be inhibited over the entire duration of the radiation burst; that is, in the case of prolonged radiation bursts, repeated and / or continuous administration of SOD1 inhibitors may be considered. Preferably, the dose of the SOD1 inhibitor is adjusted so that SOD1 activity in a region, tissue, organ, or body is inhibited by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%, preferably over the entire duration of the radiation burst. However, higher doses of the SOD1 inhibitor may be considered, for example, to achieve further reduction of radiation burst injury. Appropriate doses of SOD1 inhibitors are generally known in the art and can be adjusted by those skilled in the art by measuring SOD1 activity and / or superoxide ion concentration.

[0044] Preferably, the SOD inhibitor is included in a pharmaceutical composition, which preferably further comprises a pharmaceutically acceptable carrier. The terms “pharmaceutical” and “pharmaceutical composition” are used essentially interchangeably herein and are, in principle, known to those skilled in the art. Where referred to herein, these terms relate to any composition of a substance comprising an activator identified as a pharmaceutically active compound and, optionally, one or more excipients. The pharmaceutically active compound may exist in liquid or dry form, for example, in lyophilized form. It will be understood that the form and properties of an acceptable pharmaceutical excipient, such as a carrier or diluent, are determined by the amount of the active ingredient to which it will be combined, the route of administration, and other well-known variability factors. The excipient must be acceptable in the sense that it is compatible with the other components of the formulation and is not harmful to its recipient. The excipients used may include solids, gels, or liquids. Exemplary solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Exemplary liquid carriers include phosphate-buffered salt solutions, physiological saline, Ringer's solution, dextrose solution, and Hanks' solution, syrups, oils, water, emulsions, and various types of wetting agents. Similarly, carriers or diluents may contain time-delaying materials well known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with wax. Suitable carriers include those described above and others well known in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. Excipients are selected so as not to affect the biological activity of the combination. However, excipients may also be selected to improve the uptake of the activator into cells, particularly non-cancer cells.

[0045] Preferably, SOD1 inhibitors and / or pharmaceuticals are administered systemically, preferably orally or parenterally, for example by intravenous administration, or locally, preferably intratumorally; in the case of cancer radiotherapy, local administration may be local to the peritumor and / or site of radiotherapy. However, administration may also be administered into afferent blood vessels, typically arteries, to the intended site of action, such as the peritumor region. However, depending on the nature of the formulation and the desired therapeutic application, SOD1 inhibitors and / or pharmaceuticals may also be administered by other routes.

[0046] The therapeutically effective dose refers to the amount of the active compound that protects the subject from radiation burst damage. The therapeutic efficacy and toxicity of a drug can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. The drug regimen will be determined by the attending physician and clinical factors, taking into account the parameters described herein above. As is well known in the medical field, the dosage for any one patient can depend on many factors, including the patient's size, age, the specific formulation of the drug to be administered, sex, time and route of administration, overall health status, and other drugs administered concurrently. The drugs referred to herein are preferably administered at least once, for example, as a bolus. However, the drugs may be administered multiple times, preferably at least twice, for example, permanently or periodically after a defined time frame. SOD1 inhibition can be monitored by periodic evaluation. Dosage recommendations may be provided in the prescriber's or user's instructions to anticipate dose adjustments depending on the recipient being considered. As will be understood by those skilled in the art, appropriate doses for the pharmaceutically active compounds described herein are known in the art for single-use of the compounds. That is, the dose may be such doses as are known in the art.

[0047] The pharmaceutical according to the present invention may contain further activators in addition to the activators described above. Preferably, the pharmaceutically active compound according to the present invention should be applied together with at least one further drug, that is, it may be formulated as a pharmaceutical together with this at least one further drug. More preferably, in the case of cancer treatment, the at least one further activator is a chemotherapeutic agent or an immunotherapy agent, such as a T cell or immune checkpoint modulator, or a radiosensitizer, all of which are preferably specified above herein. It should also be understood that the formulation of the pharmaceutical composition is preferably carried out under GMP standardized conditions, etc., to ensure the quality, pharmaceutically safe, and effectiveness of the pharmaceutical.

[0048] Advantageously, the research on which the present invention is based has found that administration of SOD1 inhibitors prior to radiation exposure can potently reduce the damage to non-cancerous cells typically caused by such radiation. In other words, the methods proposed herein enable a significant reduction in radiation damage, particularly to non-cancerous cells, during subject exposure to radiation, such as in radiotherapy. Furthermore, the means and methods of the present invention also enable high-dose radiotherapy with reduced or at least no increased radiation damage compared to conventional dose therapy. In particular, the present invention enables the mimicry of FLASH radiotherapy while using radiotherapy at conventional dose rates. Moreover, by combining the methods and compounds of the present invention with FLASH radiotherapy, radiation damage is further reduced compared to the effect caused by switching from conventional radiotherapy to FLASH radiotherapy; that is, the compounds and methods of the present invention have been found to provide further protection even in FLASH radiation application compared to FLASH alone.

[0049] The definitions made above shall apply mutatis mutandis to the following. Any further definitions and explanations made below shall also apply mutatis mutandis to all embodiments described herein.

[0050] The present invention also relates, preferably, to complex preparations as specified above herein, for medical use; and for use in the treatment and / or prevention of cancer. The present invention further relates to the use of SOD1 inhibitors for the manufacture of pharmaceuticals for protecting non-cancer cells from radiotherapy for cancer and / or reducing or avoiding the adverse effects of radiotherapy mediated by the action of ionizing radiation on non-cancer cells. The present invention further relates to a method for protecting non-cancer cells in radiotherapy of a subject, comprising the step of administering the subject with an SOD1 inhibitor prior to the radiotherapy. The method is preferably an in vivo method; preferably, the method further comprises administering the radiotherapy to the subject, preferably within a time frame as specified above herein. That is, the present invention also relates to a method for treating and / or preventing cancer in a subject by radiotherapy, comprising the steps of (a) administering at least one SOD1 inhibitor to the subject; (b) administering radiotherapy to the subject; and (c) thereby treating and / or preventing cancer in the subject.

[0051] The present invention also relates to a kit comprising an SOD1 inhibitor and a radiosensitizer for cancer cells, preferably contained in a common housing.

[0052] The term “kit,” as used herein, means an assembly of the compounds, means, or reagents described above, which may or may not be packaged together. The components of the kit may be contained in separate vials (i.e., as a kit of separated parts) or may be provided in a single vial, for example, as a pharmaceutical composition as specified herein. The housing of the kit preferably allows for the transfer of the compounds of the kit, in particular, general transfer; that is, the housing may be a transportable container containing all the specified components. Furthermore, it should be understood that the kits of the present invention may be used for carrying out the methods referred to herein. Preferably, all components are intended to be provided in a ready-to-use form for carrying out the methods referred to above. Furthermore, the kit preferably includes instructions for carrying out the methods. The instructions may be provided as a user manual in paper or electronic format. Preferably, the kit is adapted for use in the methods of the present invention, and more preferably to include all reagents required to carry out the methods. Preferably, the kit contains the compound as specified by the single dose, i.e., the compound in an amount corresponding to the single dose to be administered to the subject.

[0053] Preferably, the compounds in the kit are for separate administration or for combined administration. “Separate administration,” as used herein, refers to an administration in which at least two of the pharmaceutically active compounds of the present invention are administered via different routes and / or to different parts of the subject’s body. For example, one compound is administered intra-enterally (e.g., orally), while a second compound is administered parenterally (e.g., intravenously). Preferably, in such cases, the kit comprises at least two physically separated preparations for separate administration, each preparation containing at least one pharmaceutically active compound; for example, this option is preferred if, due to their chemical or physiological properties, the pharmaceutically active compounds in a combined preparation must be administered via different routes, e.g., parenterally and orally. Conversely, “combined administration” refers to an administration in which the pharmaceutically active compounds of the present invention are administered via the same route, e.g., orally, or preferably intravenously. That is, in such cases, the kit may include a preparation containing at least two, preferably all, pharmaceutically active compounds in a single preparation. In other words, the kit may preferably include the composite preparations as specified above herein.

[0054] Preferably, the compounds in the kit are for simultaneous or sequential administration. "Simultaneous administration," as used herein, refers to administration in which the pharmaceutically active compounds are administered simultaneously, i.e., preferably, the administration of the pharmaceutically active compounds is initiated within a time interval of less than 15 minutes, more preferably within a time interval of less than 5 minutes. Most preferably, the administration of the pharmaceutically active compounds is initiated simultaneously, for example, by swallowing tablets containing multiple pharmaceutically active compounds, or by swallowing a tablet containing one of the pharmaceutically active compounds and simultaneously injecting a second compound, or by applying an intravenous infusion of a solution containing one pharmaceutically active compound and injecting the second compound in different parts of the body. Conversely, "sequential administration," as used herein, refers to the administration of pharmaceutically active compounds such that an effective concentration is present in the relevant tissue for at least a portion of the duration of the radiation burst, but preferably not as simultaneous administration as specified above herein. Preferably, continuous administration is an administration in which the administration of the pharmaceutically active compounds, preferably all of the pharmaceutically active compounds, is initiated within a time interval of 12 hours, more preferably within a time interval of 4 hours, even more preferably within a time interval of 1 hour, and most preferably within a time interval of 5 minutes. As those skilled in the art will understand, continuous administration can be particularly intended when two pharmaceutically active compounds have significantly different pharmacokinetic properties.

[0055] The present invention also relates to the use of SOD1 inhibitors to protect non-cancer cells from ionizing radiation. Such use is preferably in vitro. Preferably, such use involves inducing mutations in the genome of the non-cancer cells.

[0056] In view of the above, the following embodiments are specifically intended: Embodiment 1: An intracellular copper-zinc superoxide dismutase inhibitor (SOD1 inhibitor) for use in protecting subjects from radiation burst injury (radiation burst injury).

[0057] Embodiment 2: An SOD1 inhibitor for use according to Embodiment 1, comprising a copper complexing agent.

[0058] Embodiment 3: An SOD1 inhibitor for use according to Embodiment 1 or 2, comprising tetrathiomolybdate ion, 4,5-dihalogeno-2-arylpyridazine-3(2H)-one, 2-methoxyestradiol or a derivative or prodrug thereof, and / or cyanide ion.

[0059] Embodiment 4: An SOD1 inhibitor for use according to any one of Embodiments 1 to 3, wherein the inhibitor is bis-cholinetetrathiomolybdate (ATN-224, CAS No. 649749-10-0), 4,5-dichloro-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1, CAS No. 41931-13-9), 4,5-dibromo-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1.28, CAS No. 1035450-90-8), 4,5-dichloro-2(2,4-dichlorophenyl)pyridazin-3(2H)-one (LCS-1.34, CAS No. 24725-65-3), or N,N'-bis(2-aminoethyl)-2-ethanediamine (trientine, CAS No. 112-24-3).

[0060] Embodiment 5: The SOD1 inhibitor for use according to any one of Embodiments 1 to 4, wherein the subject is a mammal.

[0061] Embodiment 6: The SOD1 inhibitor for use according to any one of Embodiments 1 to 5, wherein the subject is human.

[0062] Embodiment 7: An SOD1 inhibitor for use according to any one of Embodiments 1 to 6, wherein the SOD1 comprises the amino acid sequence shown in SEQ ID NO: 1 (Genbank registration number AAB05662.1) or a sequence that is at least 70% identical thereto.

[0063] Embodiment 8: An SOD1 inhibitor for use according to Embodiment 6, wherein the SOD1 comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence that is at least 90% identical thereto.

[0064] Embodiment 9: An SOD1 inhibitor for use according to any one of Embodiments 1 to 8, wherein the use comprises administering the SOD1 inhibitor within a time frame of 1 minute to 12 hours, preferably 1 minute to 6 hours, more preferably 1 minute to 3 hours, and even more preferably 1 minute to 2 hours, prior to the onset of the radiation burst.

[0065] Embodiment 10: An SOD1 inhibitor for use according to any one of Embodiments 1 to 9, wherein the use comprises administering the SOD1 inhibitor within a time frame of 5 to 60 minutes, preferably 5 to 45 minutes, more preferably 5 to 30 minutes, prior to the onset of the radiation burst.

[0066] Embodiment 11: An SOD1 inhibitor for use according to any one of Embodiments 1 to 10, wherein the radiation burst causes a radiation dose of 1 Gy to 100 Gy, preferably 2 Gy to 25 Gy.

[0067] Embodiment 12: An SOD1 inhibitor for use according to any one of Embodiments 1 to 11, wherein the radiation burst causes the radiation dose within a maximum of 12 hours, preferably within a maximum of 6 hours, more preferably within 2 hours, even more preferably within 0.5 hours, and most preferably within 1 minute.

[0068] Embodiment 13: An SOD1 inhibitor for use according to any one of Embodiments 1 to 12, wherein the dose is a whole-body dose or an organ dose.

[0069] Embodiment 14: An SOD1 inhibitor for use according to any one of Embodiments 1 to 13, wherein the radiation irradiation includes particle beam irradiation, particularly helium ions (alpha-ray irradiation), carbon ions, oxygen ions, and / or neutrons; electron beam irradiation, particularly beta-ray irradiation; or photon beam irradiation, particularly gamma-ray irradiation.

[0070] Embodiment 15: An SOD1 inhibitor for use according to any one of Embodiments 1 to 14, wherein the radiation burst injury is acute radiation injury or chronic radiation injury.

[0071] Embodiment 16: An SOD1 inhibitor for use according to any one of Embodiments 1 to 15, wherein the radiation burst injury is an acute radiation injury selected from the list consisting of burns, ulceration, delayed fracture healing, decreased blood cell count, decreased white blood cell count, decreased platelet count, enteritis, nausea, vomiting, swelling, seizures, dysphagia, ulceration, inflammatory pneumonia, angioedema, hepatitis, nephritis, bleeding, and decreased fertility.

[0072] Embodiment 17: An SOD1 inhibitor for use according to any one of Embodiments 1 to 16, wherein the radiation burst injury is a chronic radiation injury selected from the list consisting of fibrosis, osteoporosis, osteomalacia, bone mineral density, bone marrow failure, stenosis, fistula, sinus formation, cognitive impairment, vascular injury, heart attack, liver failure, kidney failure, and reduced fertility.

[0073] Embodiment 18: An SOD1 inhibitor for use according to any one of Embodiments 1 to 17, wherein the radiation burst injury is not a mutagenesis-related injury.

[0074] Embodiment 19: An SOD1 inhibitor for use according to any one of Embodiments 1 to 18, wherein protection from radiation burst injury reduces the severity of at least one symptom of at least one type of radiation burst injury by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%.

[0075] Embodiment 20: An SOD1 inhibitor for use according to any one of Embodiments 1 to 19, wherein the radiation burst is radiotherapy.

[0076] Embodiment 21: An SOD1 inhibitor for use according to any one of Embodiments 1 to 20, wherein the protection is to protect non-cancerous cells from radiotherapy for cancer.

[0077] Embodiment 22: An SOD1 inhibitor for use according to any one of Embodiments 1 to 22, wherein the use comprises local administration of the SOD1 inhibitor to the non-cancer cells.

[0078] Embodiment 23: An SOD1 inhibitor for use according to any one of Embodiments 1 to 22, wherein the radiation irradiation is radiotherapy with a dose rate of up to 10 Gy / s, preferably up to 0.5 Gy / s, and more preferably up to 0.05 Gy / s.

[0079] Embodiment 24: The SOD1 inhibitor for use according to any one of Embodiments 1 to 23, wherein the radiotherapy is radiotherapy with a dose rate greater than 10 Gy / s, preferably greater than 50 Gy / s, and more preferably greater than 100 Gy / s.

[0080] Embodiment 25: The SOD1 inhibitor for use according to any one of Embodiments 1 to 24, wherein the radiotherapy is radiotherapy with a dose per treatment of 0.1 Gy to 10 Gy, preferably 1 Gy to 5 Gy.

[0081] Embodiment 26: The SOD1 inhibitor for use according to any one of Embodiments 1 to 25, wherein the radiotherapy is radiotherapy with a dose per treatment of 10 Gy to 100 Gy, preferably 20 Gy to 50 Gy.

[0082] Embodiment 27: An SOD1 inhibitor for use according to any one of Embodiments 1 to 26, wherein the dose is a local dose at the site of radiotherapy.

[0083] Embodiment 28: An SOD1 inhibitor for use according to any one of Embodiments 1 to 27, wherein protecting non-cancer cells includes reducing post-radiotherapy non-cancer cell death by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%.

[0084] Embodiment 29: An SOD1 inhibitor for use according to any one of Embodiments 1 to 28, wherein protecting non-cancer cells includes a reduction of scarring by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%.

[0085] Embodiment 30: An SOD1 inhibitor for use according to any one of Embodiments 1 to 29, comprising protecting non-cancer cells and avoiding necrosis and / or blister formation at the treatment site.

[0086] Embodiment 31: An SOD1 inhibitor for use according to any one of Embodiments 1 to 30, comprising protecting non-cancerous cells and avoiding ulcer formation and / or moist desquamation at the treatment site.

[0087] Embodiment 32: An SOD1 inhibitor for use according to any one of Embodiments 1 to 31, wherein protection of non-cancer cells includes, at most, erythema and / or dry desquamation at the treatment site as an adverse effect of radiation.

[0088] Embodiment 33: An SOD1 inhibitor for use according to any one of Embodiments 1 to 32, wherein the cancer is a solid tumor.

[0089] Embodiment 34: The SOD1 inhibitor for use according to Embodiment 33, wherein the cancer is stage 0 to stage III.

[0090] Embodiment 35: An SOD1 inhibitor for use according to any one of Embodiments 1 to 34, wherein the cancer is a cancer of the brain, skin, lung, liver, pancreas, intestine, esophagus, heart, kidney, prostate, breast, head and neck, bone, bone marrow, genitals, and / or spinal pedicle.

[0091] Embodiment 36: An SOD1 inhibitor for use in protecting non-cancerous cells from radiotherapy for cancer in a subject.

[0092] Embodiment 37: An SOD1 inhibitor for use in radiotherapy for cancer, comprising protecting non-cancerous cells from radiotherapy.

[0093] Embodiment 38: An SOD1 inhibitor for use in radiotherapy for cancer, comprising reducing or avoiding the adverse effects of radiotherapy mediated by the action of ionizing radiation on non-cancer cells.

[0094] Embodiment 39: An SOD inhibitor for any one of Embodiments 36 to 38, further having the features of any one of Embodiments 1 to 35.

[0095] Embodiment 40: Use of an SOD1 inhibitor for the manufacture of a pharmaceutical product for protecting non-cancerous cells from radiotherapy for cancer, and / or for reducing or avoiding the adverse effects of radiotherapy mediated by the action of ionizing radiation on non-cancerous cells.

[0096] Embodiment 41: A method for protecting non-cancer cells in a subject during radiotherapy, comprising the step of administering an SOD1 inhibitor to the subject before radiotherapy.

[0097] Embodiment 42: (a) A step of administering at least one SOD1 inhibitor to a subject, (b) the step of administering radiotherapy to the subject; and (c) A step of treating and / or preventing cancer in the subject thereby. A method for treating and / or preventing cancer in a subject by radiotherapy, including [specific example of radiotherapy].

[0098] Embodiment 43: A kit comprising an SOD1 inhibitor and a radiosensitizer for cancer cells.

[0099] Embodiment 44: The kit according to Embodiment 43, wherein the radiosensitizer is not the SOD1 inhibitor.

[0100] Embodiment 45: The kit according to Embodiment 43 or 44, wherein the radiosensitizer is selected from the list consisting of temozolomide, metronidazole, misonidazole, bromodeoxyuridine, motexafingadolinium, and efapoxyral.

[0101] Embodiment 46: The kit according to any one of Embodiments 43 to 46, wherein the SOD1 inhibitor is an SOD1 inhibitor as specified in any one of Embodiments 1 to 4.

[0102] Embodiment 47: Use of an SOD1 inhibitor to protect non-cancer cells from ionizing radiation.

[0103] Embodiment 48: The use according to Embodiment 48, wherein the use is an in vitro use.

[0104] Embodiment 49: The use according to Embodiment 47 or 48, wherein the use includes inducing mutations in the genome of the non-cancer cells.

[0105] Embodiment 50: A compound comprising a tetrathiomolybdate ion, 4,5-dihalogeno-2-arylpyridazine-3(2H)-one, 2-methoxyestradiol or a derivative or prodrug thereof, and / or a cyanide ion.

[0106] Embodiment 51: A composition comprising a compound selected from the list consisting of bis-cholinetetrathiomolybdate (ATN-224, CAS No. 649749-10-0), 4,5-dichloro-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1, CAS No. 41931-13-9), 4,5-dibromo-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1.28, CAS No. 1035450-90-8), 4,5-dichloro-2(2,4-dichlorophenyl)pyridazin-3(2H)-one (LCS-1.34, CAS No. 24725-65-3), and N,N'-bis(2-aminoethyl)-2ethanediamine (trientine, CAS No. 112-24-3).

[0107] Embodiment 52: The compound according to Embodiment 50 or the composition according to Embodiment 51, wherein the compound is bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0).

[0108] Embodiment 53: A compound or composition according to any one of Embodiments 50 to 52 for use in protecting a subject from radiation burst injury (radiation burst injury).

[0109] Embodiment 54: A compound or composition for use according to Embodiment 53, further having one of the features of any one of Embodiments 5 to 35.

[0110] Embodiment 55: Use of the compound described in Embodiment 50 or the composition described in Embodiment 51 for the manufacture of a pharmaceutical product for protecting non-cancerous cells from radiotherapy for cancer and / or for reducing or avoiding the adverse effects of radiotherapy mediated by the action of ionizing radiation on non-cancerous cells.

[0111] Embodiment 56: A method for protecting non-cancer cells in a subject's radiotherapy, comprising the step of administering to the subject the compound described in Embodiment 50 or the composition described in Embodiment 51 before radiotherapy.

[0112] Embodiment 57: (a) A step of administering to a subject at least one compound described in Embodiment 50 and / or at least one composition described in Embodiment 51, (b) the step of administering radiotherapy to the subject; and (c) A step of treating and / or preventing cancer in the subject thereby. A method for treating and / or preventing cancer in a subject by radiotherapy, including [specific example of radiotherapy].

[0113] Embodiment 58: (i) a compound described in Embodiment 50 or a composition described in Embodiment 51, and (ii) a radiosensitizer for cancer cells.

[0114] Embodiment 59: The kit according to Embodiment 58, wherein the radiosensitizer is selected from the list consisting of temozolomide, metronidazole, misonidazole, bromodeoxyuridine, motexafingadolinium, and efapoxyral.

[0115] Embodiment 60: Use of the compound described in Embodiment 50 or the composition described in Embodiment 51 to protect non-cancer cells from ionizing radiation.

[0116] Embodiment 61: The use described in Embodiment 60, which is for in vitro use.

[0117] Embodiment 62: The use according to Embodiment 60 or 61, comprising inducing mutations in the genome of the non-cancer cells.

[0118] All references cited herein are incorporated herein by reference with respect to their entirety and to the disclosures specifically referred to herein. [Examples]

[0119] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention in any way.

[0120] Example 1 1.1 Cell culture Human non-small lung cells (H460) were cultured in RPMI1640 medium (Thermo Fischer Scientific) supplemented with 10% fetal bovine serum (Thermo Fischer Scientific) and 1% Pen / Strep (Thermo Fischer Scientific), and maintained at 37°C in a humidified 5% CO2 atmosphere.

[0121] 1.2 Cloning Assay To study the effects of ATN224 after radiation treatment using various dose rates, a cloning assay experimental setup was used. The day before treatment, 400,000 H460 cells were seeded in a 35 mm Petri dish (Greiner) and incubated overnight. Cells were treated with 60 μM ATN-224 for 30 minutes prior to irradiation and incubated under the same conditions as cell culture. After incubation, cells were irradiated with 10 Gy using a MultiRad225 X-ray source (Faxitron Bioptics) at a conventional dose rate of 2.15 Gy / s and a high dose rate (HDR) of approximately 10 Gy / s. After irradiation, cells were detached, collected, and plated in double repeats at a density of 400,000 cells per flask. Two non-irradiated controls were prepared similarly (with and without ATN-224 treatment) and plated in quadruple repeats at a density of 1200 cells per flask. All samples were plated in T175 flasks. The cloning assay was stopped 10 days after irradiation. All samples were fixed in 100% ethanol, stained with crystal violet, scanned, and counted using the in-house ImageJ macro tool.

[0122] 1.3 Dose Measurement To achieve HDR, the petri dish was placed very close to the radiation source. By placing the RF at the bottom of the petri dish, dose measurements were performed using EBT XD (Ashland) radiochromic film (RF) only for irradiation using HDR. Previous research made it possible to determine the dose delivered inside the dish for the experimental setup used.

[0123] 1.4 SOD1 activity SOD1 activity was measured using a superoxide dismutase assay (order number CS0009) manufactured by Sigma-Aldrich, in accordance with the manufacturer's instructions.

[0124] 1.4 Results The results of the SOD1 activity measurement are shown in Table 2 below.

[0125] [Table 2]

[0126] The results of the cloning assay are shown in Figure 1. Using unirradiated cells (survival rate = 1) as a positive control, conventional irradiation with a dose of 10 Gy killed more than 99.95% of the cells, while HDR at essentially the same dose killed 99–99.9% of the cells. Cell pretreatment with ATN-224 or sodium cyanide resulted in significantly increased viability, which was similar to that of HDR, particularly with ATN-224.

[0127] literature - Che et al. (2016), Drug Discovery Today 21(1), 143 - Donate et al. (2008), Br J Cancer 98:776 - Evans et al. (1985), Int J Rad Oncol Biol Phys 11(6):1163 - Jiang et al. (2018), Cancer Lett 438:154 - Mashiba et al. (1991), Life Sciences 49:1419 - Milas et al. (1984), Int J Rad Oncol Biol Phys 10(12):2335 - Misra et al. (1979), JBC 254(22):11623 - Trapp et al. (2009), Melanoma Res 19(6):350 - U.S. Patent No. 10,722,526 B2 - Vonk et al, (2010), JBC 285(37):2891 - Vozenin et al. (2022), Nat Rev Clin Oncol19:791

Claims

1. An intracellular copper-zinc superoxide dismutase inhibitor (SOD1 inhibitor) for use in protecting subjects from radiation burst injury (radiation burst injury).

2. An SOD1 inhibitor for use according to claim 1, comprising tetrathiomolybdate ion, 4,5-dihalogeno-2-arylpyridazine-3(2H)-one, 2-methoxyestradiol or a derivative or prodrug thereof, and / or cyanide ion.

3. An SOD1 inhibitor for use according to claim 1 or 2, which is bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0), 4,5-dichloro-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1, CAS number 41931-13-9), 4,5-dibromo-2-(m-tolyl)-pyridazin-3(2H)-one (LCS-1.28, CAS number 1035450-90-8), 4,5-dichloro-2(2,4-dichlorophenyl)pyridazin-3(2H)-one (LCS-1.34, CAS number 24725-65-3), or N,N'-bis(2-aminoethyl)-2ethanediamine (trientine, CAS number 112-24-3).

4. An SOD1 inhibitor for use according to any one of claims 1 to 3, comprising a tetrathiomolybdate ion.

5. A SOD1 inhibitor for use according to any one of claims 1 to 4, wherein the SOD1 inhibitor is bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0).

6. The SOD1 inhibitor for use according to any one of claims 1 to 5, wherein the subject is a human.

7. The SOD1 inhibitor for use according to any one of claims 1 to 6, wherein the use comprises administering the SOD1 inhibitor within a time frame of 1 minute to 12 hours, preferably 1 minute to 6 hours, more preferably 1 minute to 3 hours, and even more preferably 1 minute to 2 hours, prior to the onset of the radiation burst.

8. The SOD1 inhibitor for use according to any one of claims 1 to 7, wherein the use comprises administering the SOD1 inhibitor within a time frame of 1 minute to 2 hours prior to the onset of the radiation burst.

9. An SOD1 inhibitor for use according to any one of claims 1 to 8, wherein the radiation burst causes a radiation dose of 1 Gy to 100 Gy, preferably 2 Gy to 25 Gy, in the subject.

10. An SOD1 inhibitor for use according to any one of claims 1 to 9, wherein the radiation burst causes a radiation dose of 2 Gy to 25 Gy in the subject.

11. An SOD1 inhibitor for use according to any one of claims 1 to 10, wherein the radiation burst injury is an acute radiation injury selected from the list consisting of burns, ulcer formation, delayed fracture healing, decreased blood cell count, decreased white blood cell count, decreased platelet count, enteritis, nausea, vomiting, swelling, seizures, dysphagia, ulcer formation, inflammatory pneumonia, angioedema, hepatitis, nephritis, bleeding, and decreased fertility.

12. An SOD1 inhibitor for use according to any one of claims 1 to 11, wherein the radiation burst injury is a chronic radiation injury selected from the list consisting of fibrosis, osteoporosis, osteomalacia, bone mineral density, bone marrow failure, stenosis, fistula, sinus formation, cognitive impairment, vascular injury, heart attack, liver failure, kidney failure, and reduced fertility.

13. An SOD1 inhibitor for use according to any one of claims 1 to 12, wherein protection from radiation burst injury reduces the severity of at least one symptom of at least one type of radiation burst injury by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%.

14. An SOD1 inhibitor for use according to any one of claims 1 to 13, wherein the radiation burst is radiotherapy, and the protection is to protect non-cancerous cells from cancer radiotherapy.

15. The SOD1 inhibitor for use according to claim 14, wherein the radiotherapy is radiotherapy with a dose rate of up to 10 Gy / s, preferably up to 0.5 Gy / s, and more preferably up to 0.05 Gy / s.

16. An SOD1 inhibitor for use according to any one of claims 1 to 15, wherein protecting non-cancer cells includes reducing post-radiotherapy non-cancer cell death by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%.

17. An SOD1 inhibitor for use according to any one of claims 1 to 16, wherein the cancer is cancer of the brain, skin, lung, liver, pancreas, intestine, esophagus, heart, kidney, prostate, breast, head and neck, bone, bone marrow, genitals, and / or spinal pedicles.

18. A kit comprising an SOD1 inhibitor and a radiosensitizer for cancer cells, wherein the radiosensitizer is selected from a list consisting of temozolomide, metronidazole, misonidazole, bromodeoxyuridine, motexafingadolinium, and efapoxiral.

19. The kit according to claim 18, wherein the SOD1 inhibitor comprises a tetrathiomolybdate ion, a 4,5-dihalogeno-2-arylpyridazine-3(2H)-one, a 2-methoxyestradiol or a derivative or prodrug thereof, and / or a cyanide ion.

20. The kit according to claim 18 or 19, wherein the SOD1 inhibitor comprises a tetrathiomolybdate ion.

21. The kit according to any one of claims 18 to 20, wherein the SOD1 inhibitor is bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0).

22. In vitro use of SOD1 inhibitors to protect non-cancer cells from ionizing radiation.

23. The in vitro use according to claim 22, wherein the SOD1 inhibitor comprises a tetrathiomolybdate ion, a 4,5-dihalogeno-2-arylpyridazine-3(2H)-one, a 2-methoxyestradiol or a derivative or prodrug thereof, and / or a cyanide ion.

24. The in vitro use according to claim 22 or 23, wherein the SOD1 inhibitor comprises a tetrathiomolybdate ion.

25. The in vitro use according to any one of claims 22 to 24, wherein the SOD1 inhibitor is bis-cholinetetrathiomolybdate (ATN-224, CAS number 649749-10-0).

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