Pterostilbene and silybin for preventing, ameliorating or reducing radiation-induced disease

Abstract

The present invention relates to compositions comprising pterostilbene or a pharmaceutically acceptable salt thereof in combination with silybin or a pharmaceutically acceptable salt thereof, and uses thereof for treating, preventing, ameliorating or reducing ionizing radiation-induced diseases in a subject. The compositions and uses can further comprise at least one or more radiation mitigant compounds, preferably NAD+ boosters and / or FSL-1.

Description

Technical Field

[0001] This invention relates to the field of medicine. Specifically, this invention relates to the field of compositions and methods for preventing, improving, or reducing radiation-induced diseases. Background Technology

[0002] Today, ionizing radiation is used for various medical purposes. X-rays and gamma rays primarily damage cells through direct ionization of DNA and other cellular targets, as well as indirectly through the effects of reactive oxygen species (ROS). The difference between gamma rays and X-rays lies in their generation. Gamma rays originate from the settling of excited atomic nuclei of radioactive isotopes after radioactive decay, while X-rays are produced when electrons bombard targets or rearrange electrons within atoms. Both X-rays and gamma rays are high-energy (high-frequency) electromagnetic radiation. They are energy packets (photons) without charge or mass. X-rays and gamma rays share similar properties and health effects.

[0003] Electromagnetic radiation, including X-rays and gamma rays, damages cells directly by ionizing DNA and other molecules, and indirectly by generating reactive oxygen species. The response to radiation exposure depends on cell type and radiation dose, inherent tissue sensitivity and repair, and modulating intracellular factors, including cell cycle state, O2 stress, and levels of thiols and other antioxidants. However, radiation protection and mitigation strategies are scarce and inefficient. Millions of cancer patients receive radiation therapy annually as part of their treatment, and recent radiological or nuclear events pose a threat, thus necessitating radiation protection or mitigation strategies.

[0004] For decades, the development of protective agents against harmful radiation exposures has been a subject of in-depth research. Currently, there are no effective and safe radiation protectants. To date, no drugs have been developed for treating high levels of ionizing radiation exposure, such as ionizing radiation exposure leading to acute hematopoietic and gastrointestinal radiation syndrome (ARS).

[0005] Radiation protectants are compounds that reduce direct damage caused by radiation. Radiation modifiers are compounds that minimize toxicity even after radiation has been released. Generally, an ideal radiation protectant or modifier should be stable, easily administered, free of associated toxicity, and should protect normal tissues that are considered sensitive, such that acute or late-stage toxicity in these tissues is either dose-limiting or causes a significant decline in quality of life (e.g., mucositis, pneumonia, myelopathy, xerostomia, proctitis, fibrosis, or leukoencephalopathy).

[0006] Many phytochemicals (compounds produced by plants that typically help them thrive or deter competitors, predators, or pathogens) are antioxidants and therefore have potential radiation protection. However, their radiation-protective or radiation-mitigating properties are usually effective only for a short period, typically limited to a maximum of one month after radiation exposure. Therefore, there is a need for new compounds and compositions capable of treating, reducing, preventing, or mitigating the harmful effects of radiation on subjects over a sustained period.

[0007] This invention provides compositions for long-term protection and mitigation of diseases caused by radiation exposure, and their uses. Attached Figure Description

[0008] Figure 1 A) Radiation dose response in Swiss albino mice subjected to whole-body gamma irradiation for 30-day survival; B) 60-day survival in mice treated with gamma rays (LD50 / 30) and different doses of pterostilbene (PT). Amifostine was used as a control for approved radiation protection agents; C) 60-day survival in mice treated with gamma rays (LD50 / 30) and different doses of resveratrol (Resv). The compounds were administered orally. Twenty mice were used in each group in all cases.

[0009] Figure 2 The combination of pterostilbene (PT) with other polyphenols was used to prevent gamma radiation-induced lethality (LD50 / 30) at irradiation days A) 6, B) 30, and C) 60. Doses for each phytochemical were selected from the maximum published non-toxic dose. Compounds were administered orally. Twenty mice were used in each group in all cases.

[0010] Figure 3 A) Effects of the combination of pterostilbene (PT) and silymarin (SIL) on long-term survival in mice subjected to whole-body gamma irradiation (LD50 / 30). B) Radiation protection induced by the combination of PT and SIL: a comparison between administration of polyphenols before or after gamma irradiation. Compounds were administered intraperitoneally. Twenty mice were used in each group in all cases.

[0011] Figure 4 A) Effects of pterostilbene (PT) and silymarin (SIL) on survival and body weight in γ-irradiated (LD50 / 30) mice. B) Effects of PT and SIL administration on systemic γ-irradiation-induced neuromotor function changes in mice. The compounds were administered intraperitoneally. Twenty mice were used in each group in all cases.

[0012] Figure 5Protective effects of pterostilbene (PT) and silymarin (SIL) on the expression of different antioxidant defense-related enzymes in isolated cells against γ-radiation-induced oxidative damage (RT-PCR). GCL, gamma-glutamylcysteine ​​ligase; GPX, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; EIC, intestinal epithelial cells. Fold change in expression rate compared to control (untreated) cells (n=5 for each molecule, cell type, and treatment).

[0013] Figure 6 Protective effects of pterostilbene (PT) and silymarin (SIL) against γ-radiation-induced oxidative damage of different molecular markers. A) 8-OHdG, B) 8-isoprostane and C) protein carbonyl (n=5 for each molecule, cell type and treatment).

[0014] Figure 7 A) γ-glutamylcysteine ​​ligase, B) the protective effects of pterostilbene (PT) and silymarin (SIL) against γ-radiation-induced oxidative damage in glutathione-related antioxidant defenses (n=5 for each molecule, cell type, and treatment).

[0015] Figure 8 Protective effects of pterostilbene (PT) and silymarin (SIL) against γ-radiation-induced oxidative damage by different antioxidant defense-related enzyme activities: A) catalase, B) glutathione peroxidase, and C) and D) superoxide dismutase 1 and 2 (n=5 for each molecule, cell type and treatment).

[0016] Figure 9 Effects of nicotinamide nucleoside (NR) and fibroblast-stimulating lipopeptide (FSL-1) on γ-irradiation (LD50 / 30)-induced mortality. NR was administered daily for 30 days as a single oral dose, starting immediately after irradiation (60 minutes). FSL-1 was administered intraperitoneally only once, 24 hours after irradiation. Twenty mice were used in each group in all cases.

[0017] Figure 10 The levels of A) pterostilbene (PT), B) silymarin (SIL) and C) NAD+ in the circulating blood of irradiated mice (6 mice per group in all cases).

[0018] Figure 11 Potential radiation modifiers (nicotinamide nucleoside, NR; and fibroblast-stimulating lipopeptide-1, FSL-1) were added to the radiation protection combination of pterostilbene (PT) and silymarin (SIL). A) Administration regimen, B) Survival versus time post-irradiation (in days). Twenty mice were used in each group in all cases.

[0019] Figure 12 The causes of death (LD50 / 30) in γ-irradiated mice and the effects of combined administration of radiation protectants and radiation modifiers (PT, SIL, NR, and FSL-1) were studied. In all cases, twenty mice were studied in each group.

[0020] Figure 13 Mice were treated with radiation protection / mitigation combination PT+SIL+NR+FSL-1 (PSNF) and X-rays in combination with human A549 (lung adenocarcinoma) xenografts growing in groups A) and MDA-MB-231 (triple-negative breast cancer) in groups B. Five mice were used in each group in all cases.

[0021] Figure 14 Effects of radiation protectants and radiation modifiers on cellular NAD+ content, DNA damage, and hematopoietic recovery in γ-irradiated mice. A) Effects of specific non-competitive inhibitors of PT, SIL, NR, and nicotinamide phosphoribosyltransferase (NAMPT) FK866 on NAD+ content in cells isolated from γ-irradiated mice (per group, cell type, and treatment, n=5). FK866 was administered in vivo (15 mg / kg, twice daily via intraperitoneal injection for 6 days, starting 3 days prior to irradiation, see [reference]). Figure 11 a) It was also present in cell cultures (100 nM). Cells were isolated 72 hours after irradiation. Statistical analysis was performed using Student's t-test (P < 0.01; * compare all groups with controls; + compare mice treated with PT+SIL+NR+γ-rays or mice treated with PT+SIL+NR+FK866+γ-rays with mice treated with γ-rays only). B) Effect of FK866 on cellular NAMPT activity (P < 0.01; * compare mice treated with FK866 with controls, n = 5). Mice were treated with the inhibitor for 6 days as described in (a) above. C) Effects of PT, SIL, NR, and FK866 on DNA damage in cells isolated from γ-irradiated mice (per group, cell type, and treatment, n = 5). Statistical analysis was performed using Student's t-test (P < 0.01; * compare all groups with γ-irradiated controls; + compare γ-irradiated groups with FK866 with their respective controls). Mice were treated and cells were isolated as described in (a) above. D) Effects of PT, SIL, NR, and FSL1 on hematopoietic recovery in γ-irradiated mice. Hematopoietic colony-forming cells (CFU-GM, granulocytes, macrophages) and CFU-GEMM, granulocytes, erythrocytes, macrophages, and megakaryocytes, were quantified from the femur of control and treated mice (n=5 each). Figure 11Mice were treated as shown in figure a, and bone marrow cells were isolated 7 days after irradiation. Statistical analysis was performed using Student's t-test (P<0.01; * compare all groups with control; + compare different treatments + γ irradiation with γ irradiation only).

[0022] Figure 15 Effects of treatments with pterostilbene and silymarin on Nrf2, NF-κB, PARP1, and PGC1α-dependent mechanisms. Figure 11 Intestinal epithelial cells were isolated from mice according to protocol a and then immediately irradiated. A) Western blot analysis of p65NF-κB, Nrf2, PARP1, and Sirt1 in nuclear extracts, Sirt3 in mitochondrial extracts, and phosphorylated PGC-1α in total extracts. Densitometry (Western blot) represents the mean ± standard deviation for each molecule and 5 to 6 different mice under experimental conditions (P < 0.01; * compares PT-treated, SIL-treated, or PT+SIL-treated mice with saline (PS)-treated mice; + compares PT+SIL-treated mice with PT-treated mice; # compares PT+SIL-treated mice with SIL-treated mice). B) Potential molecular interactions and relationships between different signal transduction mechanisms. Ub, ubiquitin; IκB, nuclear factor of the κ light polypeptide gene enhancer in B cell inhibitors; Keap1, Kelch-like ECH-associated protein 1; RE, reactive element; ARE, antioxidant reactive element; MAF, myofascial fibrosarcoma oncogene homolog; Ac, acetylation; IMF, inflammation mediator. C) Gene silencing of specific signal transduction-related molecules. This figure shows representative experiments with n=5 to 6 different experiments and experimental conditions. PS, physiological saline. D) Isolated IECs were cultured for 24 hours in the presence or absence of specific siRNA, followed by irradiation with gamma rays (at the same dose used in in vivo experiments). Cell death in isolated and cultured IEC cells was analyzed 12 hours after gamma irradiation, as described in the Methods section. PT (20 µM) and SIL (15 µM) were present in cultures containing IECs isolated from PT+SIL-treated mice. Thus, the effects induced in vivo were retained under in vitro conditions. Data are mean ± standard deviation of 4 to 5 different experiments for each molecule and experimental condition (p<0.01; * compares treatment with specific siRNA under PS or PT+SIL with control treated with solvent; p<0.01; + compares data under PT+SIL with equivalent data under PS). Summary of the Invention

[0023] On one hand, the present invention relates to a composition comprising a) pterostilbene or a salt thereof, and b) silymarin or a salt thereof. Preferably, the composition further comprises at least one or more radiation-mitigating compounds, which are preferably selected from a) NAD+ enhancers and / or b) fibroblast-stimulating lipopeptide-1, or any salt thereof.

[0024] Preferably, the composition further comprises at least two or more radiation mitigating compounds, wherein the at least two or more radiation mitigating compounds are NAD+ enhancers and fibroblast-stimulating lipopeptide-1, or any salt thereof.

[0025] Preferably, pterostilbene is pterostilbene disodium phosphate, and / or silymarin is silymarin-C-2',3-dihydrosuccinate disodium salt. Preferably, the NAD+ enhancer is nicotinamide nucleoside.

[0026] Preferably, the composition is a pharmaceutical composition or a cosmetic composition.

[0027] On the other hand, the present invention relates to pterostilbene or a pharmaceutically acceptable salt thereof for the prevention, improvement or reduction of ionizing radiation-induced diseases in a subject, in combination with silymarin or a pharmaceutically acceptable salt thereof, wherein the application is performed before, after or during radiation; and wherein the combination is understood herein to mean applying a) pterostilbene or a pharmaceutically acceptable salt thereof and b) silymarin or a pharmaceutically acceptable salt thereof to the subject simultaneously or sequentially in any order.

[0028] Preferably, pterostilbene or a pharmaceutically acceptable salt thereof and silymarin or a pharmaceutically acceptable salt thereof are administered as a single pharmaceutical composition or as separate pharmaceutical compositions. Preferably, the administration of pterostilbene or a pharmaceutically acceptable salt thereof and silymarin or a pharmaceutically acceptable salt thereof is topical, oral, or parenteral.

[0029] Preferably, pterostilbene or a pharmaceutically acceptable salt thereof and silymarin or a pharmaceutically acceptable salt thereof are applied several times, simultaneously or sequentially, before and / or after radiation, or both. Preferably, the dosage of this combination is sufficient to provide radiation protection.

[0030] Preferably, the use further includes application in combination with at least one or more radiation-mitigating compounds, wherein the combination is understood herein to mean simultaneous or sequential application to the subject of a) pterostilbene or a pharmaceutically acceptable salt thereof, b) silymarin or a pharmaceutically acceptable salt thereof, and c) at least one or more radiation-mitigating compounds or any pharmaceutically acceptable salt thereof.

[0031] Preferably, at least one or more radiation mitigating compounds are NAD+ enhancers and / or fibroblast-stimulating lipopeptides, or any pharmaceutically acceptable salt thereof.

[0032] Preferably, the use further includes administration in combination with at least two or more radiation-mitigating compounds, wherein the at least two or more radiation-mitigating compounds are NAD+ enhancers and fibroblast-stimulating lipopeptides, or any pharmaceutically acceptable salts thereof. Preferably, the NAD+ enhancer is nicotinamide nucleoside.

[0033] Preferably, the ionizing radiation-induced diseases are associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage, more preferably with acute radiation syndrome and / or chronic radiation syndrome.

[0034] Preferably, the ionizing radiation is gamma rays or X-rays.

[0035] Preferably, pterostilbene is pterostilbene disodium phosphate, and / or silymarin is silymarin-C-2',3-dihydrosuccinate disodium salt.

[0036] On the other hand, the present invention relates to the non-therapeutic cosmetic use of pterostilbene or its salts in combination with silymarin or its salts, wherein the combination is understood herein to mean the simultaneous or sequential application of a) pterostilbene or its salts and b) silymarin or its salts to the subject in any order. Preferably, the use is to protect against, improve, and / or reduce the adverse effects of sunlight on human skin and lips, such as age spots, sunburn, sunspots, lines, fine lines, wrinkles, crow's feet, spider lines, stretch marks, dark circles, pigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, acne breakouts, blemishes, fragile skin, dryness, patches, roughness, cracking, sagging, thinning, and enlarged pores.

[0037] On the other hand, the present invention relates to pterostilbene or a salt thereof for increasing intracellular levels of sirtuin in patients in need, in combination with silybin or a salt thereof, wherein the increase is an increase compared to the intracellular levels of sirtuin in the same patient prior to treatment with the combination of pterostilbene or a salt thereof and silybin or a salt thereof. Preferably, the patients in need are those suffering from neurodegenerative, cardiovascular, pulmonary, metabolic, or age-related conditions. Preferably, the condition is selected from the list consisting of Alzheimer's disease or other age-related dementia or cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal and bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

[0038] On the other hand, the present invention relates to a multi-component kit comprising at least two containers containing at least pterostilbene or a salt thereof and silybin or a salt thereof, and optionally at least one or more radiation-mitigating compounds. Preferably, the optionally at least one or more radiation-mitigating compounds are selected from a) NAD+ enhancers and / or b) fibroblast-stimulating lipopeptides, or any pharmaceutically acceptable salt thereof. Preferably, the NAD+ enhancer is nicotinamide nucleoside. Preferably, pterostilbene is pterostilbene disodium phosphate, and / or silybin is silybin-C-2',3-dihydrosuccinate disodium salt. Detailed Implementation

[0039] General definition

[0040] It should be noted that, as used herein, the singular forms “a” and “the” include plural references unless the context clearly indicates otherwise. Furthermore, unless otherwise stated, the term “at least” preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Such equivalents are intended to be covered in this invention.

[0041] The term “about” is intended to include a deviation of plus or minus five percent when referring to a given quantity or amount.

[0042] As used herein, the connecting term “and / or” between multiple referenced elements is understood to encompass both individual and combined options. For example, when two elements are connected by “and / or”, the first option means that the first element applies in the absence of the second element. The second option means that the second element applies in the absence of the first element. The third option means that the first and second elements apply together. Any of these options is understood to fall within this meaning and therefore satisfies the requirement of the term “and / or” as used herein. The simultaneous application of more than one option is also understood to fall within this meaning and therefore satisfies the requirement of the term “and / or”.

[0043] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the specified integer or step, or a group of integers or steps, but not to exclude any other integer or step, or a group of integers or steps. When used herein, the term “comprise” may be replaced by the terms “containing” or “including,” or sometimes by the term “having.” Any of the foregoing terms (comprising, containing, including, having), whenever used in the context of aspects or embodiments of the invention, may be replaced by the term “consisting of,” albeit less preferred.

[0044] When used herein, "consisting of" excludes any element, step, or component not specified in the elements of the claims. When used herein, "consisting substantially of" does not exclude materials or steps that do not substantially affect the essential and novel features of the claims.

[0045] The term “disease” as used in this article is generally synonymous and is used interchangeably with the terms “pathology,” “symptom,” and “condition” (as in medical condition), because all of these reflect an abnormal condition of the body or a part thereof that impairs normal function and is usually manifested as obvious signs and symptoms.

[0046] The term "therapeutic effective amount" as used herein is defined as the amount of a compound sufficient to achieve such treatment of the condition when applied to a subject. The "therapeutic effective amount" can vary depending on the compound, the condition being treated, the subject's age and relative health status, the route and form of administration, the judgment of a person skilled in the art, and other factors.

[0047] As used herein, the term "treatment" refers to medical care given to a patient in response to a disease or injury. This term includes curing the disease, as well as improving, alleviating, or reducing the symptoms of said disease. Therefore, as used herein, the terms "therapeutic treatment" or "treatment" refer to restoring the body from a pathological state or disease to its normal, healthy state. As used herein, "therapeutic treatment" or "treatment" also refers to alleviating, improving, or reducing the harmful effects of radiation on a subject, or in some way reducing symptoms associated with such effects. As used herein, the term "preventive treatment" refers to preventing a pathological state.

[0048] "Prevention" refers to preventing the occurrence of disease. "Improvement" refers to the improvement of a patient's condition, or efforts to correct the condition or at least make it more acceptable. "Reduction" or "mitigation" refers to reducing the severity, intensity, or suffering of the disease.

[0049] As used herein, the term "object" refers to a mammalian object. Preferably, it is selected from humans, companion animals, non-domesticated livestock, or zoo animals. For example, the object may be selected from humans, dogs, cats, cows, pigs, sheep, horses, bears, etc. In a preferred embodiment, the mammalian object is a human object.

[0050] "Ionizing radiation" refers to radiation composed of particles, X-rays, or gamma rays, which possess sufficient energy to cause ionization in the medium they pass through. "Gamma rays" or "gamma radiation" refers to the penetrating form of electromagnetic radiation produced by the radioactive decay of atomic nuclei. It consists of the shortest wavelength electromagnetic waves and therefore has the highest photon energy. "X-rays" or X-ray radiation refers to X-ray radiation, a penetrating form of high-energy electromagnetic radiation. Most X-rays have wavelengths ranging from 10 picometers to 10 nanometers, corresponding to frequencies ranging from 30 petahertz to 30 ehrtz, and energies ranging from 124 electron volts to 124 kiloelectron volts.

[0051] "Pterostilbene" or PT refers to 3,5-dimethoxy-4'-hydroxystilbene or its salts, solvates, isomers (enantiomers, diastereomers), hydrates, or any known derivatives, synthetic variants, and acceptable formulations (e.g., pharmaceuticals, cosmetics). Pterostilbene (PT) is a natural antioxidant and a natural analogue of resveratrol, but its antifungal effects are almost 60 to 100 times stronger than resveratrol, and it possesses similar anticancer properties. It is primarily found in blueberries and red grapes, but quantitative studies have shown that only 1 to 2 parts trans-pterostilbene are present per 10 parts trans-resveratrol. Pterostilbene corresponds to the following formula:

[0052]

[0053] Silybinin, or SIL, also known as silybin or silymarin (both derived from milk thistle), is a flavonoid compound obtained from a genus of thistle species in the Asteraceae family. Silybinin includes any salts, solvates, isomers (enantiomers and diastereomers), hydrates, or any known derivatives, synthetic variants, and acceptable formulations (e.g., pharmaceuticals, cosmetics). These plants are native to the Mediterranean region of Europe, North Africa, and the Middle East. Silybinin (SIL) is the main active ingredient of silymarin, a standardized extract of milk thistle seeds containing a mixture of flavonoid lignans, including silybinin, isosilybinin, silymarin, and silymarinine. Silybinin itself is a mixture of two diastereomers, silybinin A and silybinin B, in approximately equimolar ratios. SIL, also known as 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxane-6-yl]-2,3-dihydrochromene-4-one, has the following formula:

[0054]

[0055] Nicotinamide adenine dinucleotide enhancer, NAD+ precursor, or NAD+ enhancer refers to any compound explicitly disclosed in literature such as Rajman et al., Cell Metab. 2018 March 06; 27(3): 529–547. doi:10.1016 / j.cmet.2018.02.011, such as an NAD precursor, an activator of NAD synthesis, or an inhibitor of NAD+ degradation. Nicotinamide includes any salt, solvate, isomer (enantiomer, diastereomer), hydrate, or any known derivative, synthetic variant, and acceptable formulation (e.g., pharmaceutical, cosmetic).

[0056] "Fibroblast-stimulating lipopeptide" or FSL-1 refers to a Toll-like receptor 2 / 6 agonist or its salts, solvates, isomers (enantiomers, diastereomers), hydrates, or any known derivatives, synthetic variants, and acceptable formulations (e.g., pharmaceuticals, cosmetics), and corresponds to the following formula:

[0057]

[0058] Nicotinamide nucleoside is a pyridine nucleoside similar to vitamin B3, acting as a precursor to nicotinamide adenine dinucleotide or NAD+. NR has the following formula:

[0059]

[0060] As used in this article, the term "multipart kit" refers to a combination formulation in which the active ingredients are physically separated for use in combination therapy by simultaneous or sequential administration to a patient. Detailed Implementation

[0062] It is known that applying various natural compounds to a target before irradiation provides radiation protection. For example... Figure 2 As shown in Figure A, compounds such as pterostilbene (PT), gallic acid, and EGCG resulted in approximately 100% survival of Swiss albino mice 6 days after radiation exposure. However, some compounds failed to protect the mice for longer periods. For example, after 30 days (… Figure 2 B), the survival percentage of luteolin decreased by about 40%, while after 60 days ( Figure 2 (C), with a survival rate of 0%. Therefore, compounds that can provide a high level of protection over a long period of time are needed.

[0063] Although these compounds can be combined and applied together, the resulting radiation protection effect cannot be predicted. In some cases, the radiation protection properties of a compound do not change when it is combined with another compound. For example, as... Figure 2 As shown in Figure C, the survival rate of mice treated with PT at 60 days post-irradiation was 40%. Delphinidin provided approximately 12% survival. However, when PT was used in combination with delphinidin, the resulting survival rate remained around 40%, which is approximately the same as the survival rate provided by PT alone. In this case, the effect of delphinidin did not additively contribute to the effect of PT.

[0064] In other cases, surprisingly, the radiation-protective effect of one compound is negatively affected by the co-application of another compound. For example... Figure 2 As shown in Figure C, PT provides a 40% survival rate after 60 days of radiation protection, while gallic acid provides approximately 5% protection. However, when the two compounds are used in combination, the overall survival rate is approximately 35%, resulting in a lower radiation protection effect compared to using PT alone.

[0065] In other cases, the radiation-protective effects of the two compounds are additive when they are combined, resulting in protection that is the sum of their individual effects. For example, Figure 2 C shows that PT provides approximately 40% protection, while genistein provides approximately 7%. Therefore, when the two compounds are combined, the resulting protective effect is approximately 45%, meaning that both compounds are working, and the overall protection is roughly the sum of the effects of the two compounds.

[0066] More interestingly, the authors of this invention have surprisingly discovered that the combination of two compounds can not only lead to no change, or loss of protection, or additive protection, but also, in rare cases, an enhanced radiation protection performance rather than a simple additive effect. That is, a specific combination of specific compounds provides enhanced protection that cannot be explained by the sum of their individual effects, implying a synergistic effect. This is as follows: Figure 2 As shown in B and 2C. In Figure 2 In C, silymarin (SIL) provides approximately 10% survival, while PT provides approximately 40% survival, but the combination of the two provides a survival rate greater than 70%. This clearly demonstrates that the combination of SIL and PT not only produces an additional effect on their radiation protection performance but also creates a synergistic effect, thereby enhancing radiation protection.

[0067] These findings suggest that, in order to obtain enhanced radiation protection, attention should be paid to the combination of compounds, as some combinations can enhance radiation protection while others are detrimental. In the latter case, it would be preferable to apply the compound alone.

[0068] Therefore, to obtain enhanced radiation protection, compounds with different radiation protection properties cannot be arbitrarily combined, as such combinations do not always lead to overall enhanced radiation protection. Depending on the compounds used together, the resulting radiation protection may be worse, either the same compounds or a combination of two compounds. However, some combinations of compounds provide synergistic effects and enhanced protection, which cannot be derived from the simple sum of their radiation protection properties.

[0069] Another aspect discovered by the inventors is that a specific combination of PT and SIL provides protection for an extended period. This is surprising because, as mentioned above, not all compounds provide radiation protection for more than 30 days; for example, see [link to relevant documentation]. Figure 2 B (30 days) of EGCG, quercetin, naringin or phlorizin compounds, whose radioprotective effects decreased to 0% after 60 days. Figure 2 C). However, as Figure 3 As shown, the combination of PT and SIL provided approximately 25% survival at day 360 (i.e., one year later) (5 out of 20 mice survived). Therefore, the combination of PT and SIL not only produces a synergistically enhanced protective effect, but this effect is also durable. Furthermore, as... Figure 11As shown in B, when PT and SIL are combined with other radiation-mitigating compounds such as nicotinamide nucleoside (NR) or fibroblast-stimulating lipopeptide-1 (FSL-1), the survival rate after one year is further increased to as high as 50 to 60%. Notably, when the four compounds (PT, SIL, NR and FSL-1) are combined, they provide almost complete protection (90%) to the animals after 360 days.

[0070] In summary, this invention provides two key findings: First, the combined application of PT and SIL, two natural compounds with radiation-protective properties, provides enhanced and durable synergistic radiation protection, which is not derived from a simple additive effect of their respective radiation-protective properties. Second, the combination of these two compounds with radiation-mitigating compounds such as NR and / or FSL-1 further enhances radiation protection, achieving a long-term survival rate of up to 90% even after one year.

[0071] To investigate the synergistic effect of SIL and PT in detail, the inventors further studied the potential signal transduction pathways that may be involved in the protective effect caused by the PT+SIL combination. For example... Figure 15 As shown in figure a, both PT and SIL increased nuclear Nrf2 and decreased nuclear NF-κB levels compared to the control. These effects generally suggest a synergistic effect in promoting antioxidant defense while downregulating NF-κB-dependent pro-inflammatory responses. pPGC-1α was also increased by PT ( Figure 15 a). Interestingly, although SIL alone did not affect Sirt1 levels, the combination of the two polyphenols increased Sirt1 levels more than PT alone. Figure 15 a). These results lead to the conclusion that the synergistic effect of PTE and SIL is based on the combined effect of these two compounds in reducing oxidative stress and DNA damage in cells, and pave the way for the use of compositions containing these two compounds in cosmetic and medical fields.

[0072] Pterostilbene is used to prevent, improve, or reduce diseases induced by ionizing radiation.

[0073] A first aspect of the invention relates to a stilbene compound (preferably resveratrol or pterostilbene) or a pharmaceutically acceptable salt or derivative thereof for treating, preventing, improving, or reducing ionizing radiation-induced diseases in a subject, in combination with silybin or a pharmaceutically acceptable salt or derivative thereof. Preferably, the stilbene compound used is pterostilbene or a pharmaceutically acceptable salt thereof. Therefore, in a first aspect, the invention provides pterostilbene or a pharmaceutically acceptable salt thereof for treating, preventing, improving, or reducing ionizing radiation-induced diseases in a subject, in combination with silybin or a pharmaceutically acceptable salt thereof, wherein the application is performed before, after, or during radiation; and wherein the combination is understood herein to mean, in any order, simultaneous or sequential application of a) pterostilbene or a pharmaceutically acceptable salt thereof, and b) silybin or a pharmaceutically acceptable salt thereof to the subject. In a specific embodiment, ionizing radiation-induced diseases are associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage. In a preferred embodiment, the ionizing radiation-induced diseases are acute radiation syndrome and / or chronic radiation syndrome. In another embodiment, the disease is selected from the group consisting of skin diseases, acute radiation syndrome, cancer, and cardiovascular diseases.

[0074] According to a first aspect of the invention, pterostilbene and silymarin are used in combination. PT and SIL can be administered as a single composition or as separate compositions.

[0075] In a preferred embodiment, pterostilbene is the disodium salt of pterostilbene phosphate. In another preferred embodiment, silybin is the disodium salt of silybin-C-2',3-dihydrosuccinate.

[0076] As described above, PT and SIL, or any pharmaceutically acceptable salt or derivative thereof, are administered in combination to prevent, improve, or reduce ionizing radiation-induced diseases in a subject. In a preferred embodiment, the subject is a mammal. More preferably, the subject is a human.

[0077] Application schedule and route

[0078] Application can be simultaneous (at the same time) or sequential. As used herein, “sequential application” means applying SIL or a composition containing PT shortly before or shortly after application of PT or a composition containing PT. “Shortly before or shortly after” as used herein should be understood to mean within an interval of 1 to 24 hours, preferably 1 to 12 hours, preferably 1 to 3 hours, preferably within 2 hours, more preferably within 1 hour or less, for example, within 45 minutes, 30 minutes, 15 minutes or less. In one embodiment, PT is applied first, followed by SIL. In another embodiment, SIL is applied first, followed by PT. In a preferred embodiment, PT and SIL are applied simultaneously. In one embodiment, PT and SIL are applied simultaneously as part of a single composition or as part of two different compositions.

[0079] In one embodiment, the pharmaceutically acceptable compounds and compositions are formulated to be compatible with their intended route of administration. Methods of administration are known to those skilled in the art. This includes, for example, injection via parenteral routes, such as intravascular, intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, intravenous, epidural, or other routes, as well as oral, topical, nasal, ocular, rectal, or local administration. Sustained-release administration via methods such as cumulative injection or perforated implants is also particularly contemplated. A preferred route of administration is parenteral administration, which is understood herein as administration within one or more blood vessels and generally includes intravenous or intra-arterial administration. Currently, the most preferred routes of administration are parenteral, oral, or topical administration.

[0080] The application may be performed once or more. In a preferred embodiment, the compound is applied several times. In a specific embodiment, the application may include at least one, two, three, four, five, six, seven, or more doses.

[0081] Application can be made before, during, or after radiation. For example, it can be made at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days before radiation. In another embodiment, it is made after radiation. In one embodiment, it is made 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after radiation, or more than 10 days, such as 20 or 30 days, or more than one month, such as 2, 3, 5, 6, 8, or 9 months after radiation. In one specific embodiment, it is made on the same day as, before, or after radiation. In one embodiment, the application consists of several doses administered over several days before, on, and / or after radiation. In a preferred embodiment, pterostilbene and silymarin or any pharmaceutically acceptable salt thereof are administered simultaneously or sequentially several times before and / or after radiation, or both.

[0082] Dosage

[0083] The optimal dosage will be selected based on the route of administration, treatment regimen, and / or administration schedule, taking into account available toxicity and efficacy data. In a preferred embodiment, PT and SIL, according to the use of the first aspect of the invention, are administered in doses capable of providing radiation protection. As mentioned above, "radiation protection" is understood as the ability to reduce direct damage caused by radiation. Therefore, in this invention, there are no particular limitations on the dosage of PT and SIL.

[0084] Furthermore, it will be apparent to those skilled in the art that the doses disclosed herein for use in mouse models can be converted to equivalent doses for use in humans. For this purpose, those skilled in the art can refer to well-known general practice guidelines for dose conversion between animals and humans, see, for example, Nair AB, Jacob S. A simple practice guide fordose conversion between animals and human. J Basic Clin Pharma 2016;7:27-31. In short, the therapeutic dose in mouse models can be converted to a therapeutic dose in humans by dividing by approximately 12.3. That is, if a dose of 100 mg / kg has a therapeutic effect in mice, the equivalent dose in humans is 8.13 mg / kg.

[0085] In one embodiment, PT or a pharmaceutically acceptable salt thereof is administered at a maximum dose of 500 mg / kg. PT may be administered at doses from 300 mg / kg to 30 mg / kg. Preferably, PT is administered at doses from 200 mg / kg to 40 mg / kg or from 150 mg / kg to 40 mg / kg. More preferably, PT is administered at a dose of 100 mg / kg. In one embodiment, PT or a pharmaceutically acceptable salt thereof is administered at a maximum dose of 40.65 mg / kg. PT may be administered to humans at doses from 24.39 mg / kg to 2.43 mg / kg. Preferably, PT is administered to humans at doses from 16.2 mg / kg to 3.25 mg / kg or from 12.19 mg / kg to 3.25 mg / kg. More preferably, PT is administered to humans at a dose of 8.13 mg / kg.

[0086] In another embodiment, SL or a pharmaceutically acceptable salt thereof is administered at a dose of up to 500 mg / kg. SL may be administered at a dose of 200 mg / kg to 35 mg / kg. Preferably, SL is administered at a dose of 150 mg / kg to 40 mg / kg or 100 mg / kg to 50 mg / kg. More preferably, PT is administered at a dose of 70 mg / kg or a human equivalent dose. In another embodiment, SL or a pharmaceutically acceptable salt thereof is administered to a human at a dose of up to 40.65 mg / kg. SL may be administered to a human at a dose of 16.26 mg / kg to 2.84 mg / kg. Preferably, SL is administered to a human at a dose of 12.19 mg / kg to 3.25 mg / kg or 8.13 mg / kg to 4.06 mg / kg. More preferably, PT is administered to a human at a dose of 5.69 mg / kg or a human equivalent dose.

[0087] Application of other compounds

[0088] In embodiments of the first aspect, any of the uses described in the above embodiments further includes administration in combination with at least one other radiation-mitigating compound, wherein the uses described herein are understood herein to mean simultaneous or sequential administration to the subject of a) pterostilbene or a pharmaceutically acceptable salt thereof, b) silymarin or a pharmaceutically acceptable salt thereof, and c) one or more radiation-mitigating compounds. In a preferred embodiment, at least one or more radiation-mitigating compounds refer to nicotinamide adenine dinucleotide enhancer (hereinafter referred to as NAD+ enhancer) and / or fibroblast-stimulating lipopeptide-1 (hereinafter referred to as FSL-1), or any pharmaceutically acceptable salt thereof. Note that in the context of this invention, although FSL-1 is preferred, FSL-1 is a type of fibroblast-stimulating lipopeptide and can therefore be replaced by any other fibroblast-stimulating lipopeptide. Therefore, the term "FSL-1" used in any implementation of radiation of this invention can be replaced by any other suitable fibroblast stimulant.

[0089] As defined above, a “radiation modifier” is a compound that minimizes toxicity even after radiation has been released. Therefore, in embodiments of the first aspect, the combined application of PT and SIL also includes the application of one or more radiation modifier compounds. The combination of PT, SIL, and one or more radiation modifier compounds is not limited; that is, PT may be applied together with SIL and one or more radiation modifiers in a single composition, or they may be applied individually in different compositions. All possible combinations of three (or four or more) compounds are included herein.

[0090] The above-described administration routes and schedules also apply to cases where PT and SIL are administered together with one or more radiation mitigating compounds.

[0091] In a preferred embodiment, the use of PT in combination with SIL, its salts, or derivatives thereof in the prevention, improvement, or reduction of ionizing radiation-induced diseases in a subject also includes the application of an NAD+ enhancer. Therefore, in this embodiment, at least three compounds are applied together, and these at least three compounds may be contained in the same or different compositions and may be applied sequentially or simultaneously.

[0092] In another preferred embodiment, the use of PT in combination with SIL, its salts, or derivatives thereof in the prevention, improvement, or reduction of ionizing radiation-induced diseases in a subject also includes the administration of FSL-1. Thus, in this embodiment, at least three compounds are administered together, and these at least three compounds may be contained in the same or different compositions and may be administered sequentially or simultaneously.

[0093] In a preferred embodiment, the use of PT in combination with SIL, its salts, or derivatives thereof in the prevention, improvement, or reduction of ionizing radiation-induced diseases in subjects also includes the application of an NAD+ enhancer and FSL-1. Therefore, in this embodiment, the four compounds may be contained in the same or different compositions and may be applied sequentially or simultaneously.

[0094] In a more preferred embodiment, the NAD+ enhancer is nicotinamide nucleoside or 1-(β-D-rifuranosyl)nicotinamide (hereinafter referred to as NR), a pharmaceutically acceptable salt or derivative thereof.

[0095] In a more preferred embodiment, the use of PT in combination with SIL, its salts, or derivatives thereof in preventing, improving, or reducing ionizing radiation-induced diseases in a subject also includes the administration of NR and FSL-1. Thus, in this embodiment, the four compounds are administered together in one or more doses, the four compounds being contained in the same or different compositions and administered sequentially or simultaneously before, during, or after radiation.

[0096] In a preferred embodiment, PT and SIL are applied once daily for several days before and / or several days after radiation. More preferably, PT and SIL are applied daily for 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 days (preferably 5 days) before radiation. In another embodiment, PT and SIL are applied daily for up to 5, 4, 3, 2, and 1 days before and after radiation. Preferably, PT and SIL are applied daily for 5 days before radiation and 2 days after radiation.

[0097] In another embodiment, the NAD+ enhancer is applied once daily on the same day as the radiation and for the following month. In yet another embodiment, the NAD+ enhancer is applied at least 60 minutes after the radiation and further applied for the next 30 days following the radiation.

[0098] In another embodiment, FSL-1 is administered only once, preferably after radiation, more preferably at least one day after radiation.

[0099] In one embodiment, the NAD+ enhancer or a pharmaceutically acceptable salt thereof is administered at a maximum dose of 500 mg / kg. In one embodiment, the NAD+ enhancer or a pharmaceutically acceptable salt thereof is administered at a dose of 400 mg / kg to 100 mg / kg. Preferably, the NAD+ enhancer is administered at a dose of 300 mg / kg to 150 mg / kg or 200 mg / kg to 150 mg / kg. More preferably, the NAD+ enhancer is administered at a dose of 185 mg / kg. In one embodiment, the NAD+ enhancer or a pharmaceutically acceptable salt thereof is administered to a human at a maximum dose of 40.65 mg / kg. In one embodiment, the NAD+ enhancer or a pharmaceutically acceptable salt thereof is administered to a human at a dose of 32.52 mg / kg to 8.13 mg / kg. Preferably, the NAD+ enhancer is administered to a human at a dose of 24.39 mg / kg to 12.19 mg / kg or 16.26 mg / kg to 12.19 mg / kg. More preferably, the NAD+ enhancer is administered to humans at a dose of 15.04 mg / kg.

[0100] In another embodiment, the NAD+ enhancer is nicotinamide nucleoside or any pharmaceutically acceptable salt thereof, and it is administered at an amount from 400 mg / kg to 100 mg / kg. Preferably, nicotinamide nucleoside is administered at an amount from 300 mg / kg to 150 mg / kg or from 200 mg / kg to 150 mg / kg. More preferably, nicotinamide nucleoside is administered at an amount of 185 mg / kg. In another embodiment, the NAD+ enhancer is nicotinamide nucleoside or any pharmaceutically acceptable salt thereof, and it is administered to humans at an amount from 32.52 mg / kg to 8.13 mg / kg. Preferably, nicotinamide nucleoside is administered to humans at an amount from 24.39 mg / kg to 12.19 mg / kg or from 16.26 mg / kg to 12.19 mg / kg. More preferably, nicotinamide nucleoside is administered to humans at an amount of 15.04 mg / kg.

[0101] In one embodiment, FSL-1 or a pharmaceutically acceptable salt thereof is administered at a maximum dose of 1 mg / kg. In another embodiment, the FSL-1 compound or a pharmaceutically acceptable salt thereof is administered at a dose of 1 mg / kg to 0.1 mg / kg. Preferably, the FSL-1 compound is administered at a dose of 0.75 mg / kg to 0.1 mg / kg or 0.5 mg / kg to 0.1 mg / kg. More preferably, the FSL-1 compound is administered at a dose of 0.25 mg / kg. In one embodiment, FSL-1 or a pharmaceutically acceptable salt thereof is administered at a maximum dose of 0.08 mg / kg. In another embodiment, the FSL-1 compound or a pharmaceutically acceptable salt thereof is administered at a dose of 0.08 mg / kg to 0.008 mg / kg. Preferably, the FSL-1 compound is administered at a dose of 0.06 mg / kg to 0.008 mg / kg or 0.04 mg / kg to 0.081 mg / kg. More preferably, the FSL-1 compound is applied at a dose of 0.020 mg / kg.

[0102] In one implementation, the ionizing radiation is gamma (γ) rays or X-rays.

[0103] Compositions containing PT and SIL

[0104] In a second aspect, the present invention relates to a composition comprising a) a stilbene compound (preferably resveratrol or pterostilbene) or a salt or derivative thereof, and b) silymarin or a salt or derivative thereof. Preferably, the stilbene compound is pterostilbene or a salt thereof. Therefore, the second aspect of the invention provides a composition comprising a) pterostilbene or a salt thereof and b) silymarin or a salt thereof. Preferably, the composition is a pharmaceutical composition or a cosmetic composition.

[0105] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. They may also include pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, such as acetate, propionate, malonate, benzoate, etc. It is also preferred that the formulation contains a pharmaceutically acceptable excipient that acts as a stabilizer, particularly if the vaccine composition is to contain peptides, proteins, or other similar molecules, then the stabilizer is a stabilizer for those peptides, proteins, or other similar molecules. Examples of suitable carriers include, but are not limited to, pharmaceutical-grade dextran, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, etc. Other suitable carriers include starch, cellulose, sodium or calcium phosphate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycol (PEG), and combinations thereof. A comprehensive discussion of pharmaceutically acceptable excipients, additives, and carriers can be found in Remington Pharmaceutical Sciences (2012, 22nd edition).

[0106] Different choices regarding drug formulations, structural modifications, and delivery systems can help facilitate oral administration of PTE and SIL, and are included herein. Some examples include prodrugs (carboxylates, sulfonates, sulfates, phosphates, acetals, carbamates, or carbonates), nanoemulsions, polyionic / polymer shells encapsulating nanoparticles, liposomes, or exosomes. In particular, any pharmaceutically acceptable formulation of PTE and SIL is included herein.

[0107] One or more compositions according to the second aspect comprise PT and SIL in amounts defined in the first aspect of the invention, and are administered according to the administration route and schedule defined in the first aspect of the invention. Further details and preferred embodiments regarding the dosage, administration route, and schedule of said compositions are also provided according to the first aspect of the invention.

[0108] The composition comprises PT and SIL or any salts or derivatives thereof, and may also comprise one or more radiation moderating compounds as defined in the first aspect of the invention. As described above, in one embodiment, the radiation moderating compound may be an NAD+ enhancer and / or FSL-1. In a preferred embodiment, the composition comprising PT and SIL further comprises at least two radiation moderating compounds, preferably NR and FSL-1.

[0109] Preferably, the one or more compositions are in a form suitable for parenteral, oral, or topical application. In a preferred embodiment, the composition is an aqueous composition, more preferably a stable aqueous composition. As used herein, "stable composition" can mean a formulation in which the active ingredient substantially retains its physical and / or chemical stability and / or biological activity after storage.

[0110] The present invention also relates to the compositions for use as defined in the first aspect of the invention.

[0111] Multipart kit containing PT and SIL

[0112] In a third aspect, the present invention relates to a multi-component kit comprising at least two containers containing at least a) a stilbene compound (preferably resveratrol or pterostilbene) and silymarin or any salt or derivative thereof, and optionally at least one or more radiation mitigating compounds as defined above. Preferably, the stilbene compound contained in the multi-component kit is pterostilbene.

[0113] Typically, two or more ingredients are provided to formulate a composition, and these two or more ingredients are provided in two or more separate containers. For example, PT and SIL may be combined in one container, while the radiation modifier is in another. In one embodiment, each compound is contained in a separate container. In a further embodiment, the radiation modifier compound is present in a container similar to the radiation modifier compound.

[0114] Preferably, the composition is used for the purposes defined in the foregoing aspects of the invention, preferably for the therapeutic treatment of diseases induced by ionizing radiation.

[0115] Methods to prevent, improve, or reduce diseases induced by ionizing radiation

[0116] In a fourth aspect, the present invention relates to a method for preventing, treating (including improving or reducing) ionizing radiation-induced diseases in a subject, comprising administering to the subject a combination of a stilbene compound (preferably resveratrol or pterostilbene) or a pharmaceutically acceptable salt or derivative thereof with silymarin or any pharmaceutically acceptable salt or derivative thereof. Preferably, the stilbene compound is pterostilbene. Therefore, in a fourth aspect, the present invention provides a method for preventing, treating (including improving or reducing) ionizing radiation-induced diseases in a subject, comprising administering a combination of pterostilbene and silymarin, or any pharmaceutically acceptable salt thereof, wherein the administration is performed before, after, or during radiation; and wherein the combination is understood herein to mean simultaneously or sequentially administering to the subject a) pterostilbene or a pharmaceutically acceptable salt thereof, and b) silymarin or any pharmaceutically acceptable salt thereof, and optionally c) at least one or more radiation-mitigating compounds, as defined throughout this specification. Similar to those described above in the first and second aspects of the invention, at least one or more radiation-mitigating compounds may be NAD+ enhancers and / or FSL-1.

[0117] This fourth aspect relates to the first and second aspects described above. Therefore, compounds and pharmaceutical compositions, dosages, routes of administration, and schedules are provided and applicable herein according to the foregoing aspects of the invention.

[0118] In a preferred embodiment, the use is for the treatment or prevention of diseases induced by ionizing radiation, for the treatment or prevention of neurodegenerative, cardiovascular, pulmonary, metabolic, or age-related conditions, and / or for the protection or repair of the adverse effects of sunlight on human skin or lips. The term "repair of the adverse effects of sunlight on human skin or lips" is used herein to refer to preventing, reversing, improving, reducing, and / or minimizing skin defects, blemishes, or unpleasant aesthetic conditions, including but not limited to: age spots, sunburn, sunspots, lines, fine lines, wrinkles, crow's feet, spider lines, stretch marks, dark circles, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, acne breakouts, blemishes, fragile skin, dryness, patches, roughness, cracking, sagging, thinning, enlarged pores, milia formation, acne formation, rosacea, psoriasis, and eczema. The term "skin" includes facial or body skin.

[0119] Preferably, the ionizing radiation-induced diseases are associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage. More preferably, the ionizing radiation-induced diseases are selected from a list consisting of acute radiation syndromes and / or chronic radiation syndromes.

[0120] The combination of pterostilbene and silymarin is used as a cosmetic composition for non-therapeutic purposes.

[0121] In a sixth aspect, the invention also relates to the non-therapeutic cosmetic use of a stilbene compound (preferably resveratrol or pterostilbene) in combination with silybin, or any salt or derivative thereof. Furthermore, the sixth aspect also relates to the non-therapeutic cosmetic use of the composition according to the second aspect. Preferably, the stilbene compound used is pterostilbene. Therefore, the sixth aspect of the invention provides pterostilbene or a salt thereof in combination with silybin or a salt thereof for non-therapeutic cosmetic use or as a cosmetic composition, wherein the combination is understood herein to mean the simultaneous or sequential application of a) pterostilbene or a salt thereof and b) silybin or a salt thereof to the subject in any order.

[0122] As used herein, the term "non-therapeutic cosmetic use" refers to methods for improving a person's appearance, i.e., beautifying one's appearance. This does not involve treatments performed through surgery or therapy in cosmetic applications. Cosmetic care includes the topical application of PTE and SIL to the skin area to be treated. Preferably, the cosmetic use of the composition is for non-therapeutic purposes of cleansing, beautifying, increasing attractiveness, altering appearance, or maintaining or promoting the good condition of skin or hair. These purposes may also include skin whitening, minimizing wrinkles on the face and body, and protecting against sun and sunlight. In one embodiment, the non-therapeutic cosmetic use is for preventing, reducing, and / or improving the natural processes of skin aging.

[0123] Preferably, non-therapeutic uses are for the protection, improvement, and / or reduction of the adverse effects of sunlight on human skin and lips, including but not limited to age spots, sunburn, sunspots, lines, fine lines, wrinkles, crow's feet, spider lines, stretch marks, dark circles, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, acne breakouts, blemishes, fragile skin, dryness, patches, roughness, cracking, sagging, thinning, and enlarged pores. The term "skin" includes facial or body skin.

[0124] This sixth aspect relates to the first and second aspects described above. Therefore, the compounds and compositions, dosages, routes of administration, and schedules are compatible with cosmetic applications and, as provided by the foregoing aspects of the invention, also apply herein.

[0125] The combination of pterostilbene and silymarin is used to increase the level of intracellular deacetylase.

[0126] based on Figures 5 to 8 According to data reported in the Chinese study, PT+SIL has been shown to exert a protective effect against induced oxidative damage. Furthermore, as... Figure 15As shown, this mechanism was found to be directly related to a synergistic increase in deacetylase (Sirt) levels, particularly Sirt1, induced by PTE+SIL. Deacetylases are a family of signaling proteins involved in metabolic regulation, controlling many important functions and participating in various pathologies, such as metabolic, cardiovascular, and pulmonary diseases, neurodegenerative and age-related conditions, and cancer. Deacetylases have been shown to be regulators of inflammation and autophagy. Growing evidence suggests that compounds activating or increasing deacetylases show promise as therapeutic agents for metabolic disorders, cancer, and age-related diseases, strongly supporting the use of PTE combined with SIL for the treatment or prevention of these diseases. In particular, some studies have shown the role of deacetylases in neuroprotection, especially in neurodegenerative diseases such as Alzheimer's disease or other dementias or age-related cognitive impairments, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal and bulbar muscular atrophy), amyotrophic lateral sclerosis, and other neuron-related diseases (see Kim D et al. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amytrophic lateral sclerosis. EMBO J. 2007;26(13):3169-3179; Donmez G et al. SIRT1 protects against α-synuclein aggregation by activating molecular chaperones. J Neurosci. 2012;32(1):124-132; Outeiro TF et al. Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease). Science. 2007;317(5837):516-519; Pallos J et al. Inhibition of specificHDACs and sirtuins suppresses pathogenesis in a Drosophila model of Huntington's disease. Hum Mol Genet. 2008;17(23):3767-377; Luthi-Carter R et al.SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis. Proc Natl Acad Sci US A. 2010;107(17):7927-7932; Shindler KS et al. SIRT1 modulates aggregation and toxicity through deacetylation of the androgen receptor incell models of SBMA. J Neurosci. 2011;31(48):17425-17436). .

[0127] In view of this, in a seventh aspect, the present invention provides a stilbene compound (preferably resveratrol or pterostilbene) combined with silymarin, or any salt or derivative thereof, or a composition according to the second aspect, for increasing the intracellular level of deacetylase in patients in need of this, wherein the increase is an increase compared to the intracellular level of deacetylase in the same patient before treatment with the combination of the stilbene compound (preferably resveratrol or pterostilbene) and silymarin, or any salt thereof, or before treatment with the composition according to the second aspect. Preferably, the stilbene compound is pterostilbene. Preferably, the deacetylase whose intracellular level is increased is Sirt1, Sirt2, or Sirt3, most preferably Sirt1. Compared with untreated patients, the increase in intracellular deacetylase levels is preferably statistically significant and can be measured by any suitable technique designed to measure the amount of protein, such as, but not limited to, the Bradford assay, colorimetric protein assay BCA, antibody-based assays such as Western blot or ELISA, Lowry protein assay, fluorescence-based assays, protein mass spectrometry-based assays, nanoparticle and nanopore-based methods, etc.

[0128] "A statistically significant increase in intracellular levels of deacetylase" herein means that the analyst determines that the increase in the expression or activation level of the protein (in this case, deacetylase) cannot be explained by mere chance. Statistical hypothesis testing is a method by which those skilled in the art make such a determination. This test provides a p-value, which is the probability that the hypothetical result is indeed caused solely by chance and that an outcome as extreme as the result in the data is observed. A p-value of 0.1 or less (preferably 0.05, 0.01, 0.001 or less) is considered statistically significant herein. For example, an increase in intracellular levels of deacetylase is statistically significant when a statistical test is performed to compare intracellular levels of deacetylase to baseline intracellular levels of deacetylase in reference or untreated cells, and when the p-value obtained from said statistical test is 0.1 or less, preferably 0.05, 0.01, 0.001 or less.

[0129] Preferably, the patient in need suffers from a disease or condition characterized by decreased intracellular levels of deacetylases, preferably decreased intracellular levels of Sirt1. Preferably, the patient in need suffers from a neurodegenerative, cardiovascular, pulmonary, metabolic, or age-related condition characterized by decreased intracellular levels of deacetylases, preferably Sirt1, compared to intracellular levels of deacetylases in healthy individuals. Preferably, the condition is selected from the list consisting of Alzheimer's disease or other age-related dementia or cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal and bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

[0130] Preferably, pterostilbene or its salts are combined with silymarin or its salts for the treatment, prevention, improvement, or reduction of neurodegenerative diseases, wherein the combination is understood herein to mean the simultaneous or sequential application of a) pterostilbene or its salts and b) silymarin or its salts to the subject in any order. Preferably, the disease is characterized by a decrease in intracellular deacetylase, preferably Sirt1, levels compared to healthy individuals. In one embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease or other age-related dementia or cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal and bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes. In one embodiment, the cells from which the intracellular levels of deacetylase (preferably Sirt1) increase after treatment are derived from neural tissue. Preferably, the cells are neurons or glial cells.

[0131] Furthermore, it is well known that deacetylases are potential therapeutic targets for treating metabolic diseases such as diabetes. Multiple studies have shown that overexpression of Sirt1 in pancreatic cells can be used to control hyperglycemia in diabetic patients (see Nerurkar et al. Respected Sir(2): magic target for diabetes. Cellscience. 2008;4(4):82-96; Moynihan et al. Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice. Cell Metab. 2005;2(2):105-117; and Bordone et al. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS Biol. 2006;4(2):e31). In view of this and based on the findings presented herein, stilbene compounds (preferably resveratrol or pterostilbene) in combination with silymarin or in combination according to the second aspect can also be used to treat, prevent, improve, or reduce diabetes. Preferably, the stilbene compound is pterostilbene. Therefore, in a preferred embodiment, the present invention also provides pterostilbene or a salt thereof in combination with silymarin or a salt thereof for treating, preventing, improving or reducing diabetes, wherein the combination is understood herein to mean applying a) pterostilbene or a salt thereof and b) silymarin or a salt thereof to the subject simultaneously or sequentially in any order.

[0132] This seventh aspect relates to the first and second aspects described above. Therefore, compounds and pharmaceutical compositions, dosages, routes of administration, and schedules are provided and applicable herein according to the foregoing aspects of the invention.

[0133] Similar to that defined in the first aspect, and also included in the embodiments of the fifth, sixth, and seventh aspects, the combination of PT and SIL may be administered in separate compositions or in separate compositions. In the embodiments of the fifth, sixth, and seventh aspects, pterostilbene is pterostilbene disodium phosphate. In other preferred embodiments of the aspects, silybin is silybin-C-2',3-dihydrosuccinate disodium salt. It is noteworthy that the use described in any of the foregoing aspects also includes the combined administration of PTE and SIL with at least one additional radiation-mitigating compound, wherein the use described herein is understood to mean the simultaneous or sequential administration of a) pterostilbene or a salt thereof, b) silybin or a salt thereof, and c) one or more radiation-mitigating compounds to the subject. In preferred embodiments, at least one or more radiation-mitigating compounds refer to nicotinamide adenine dinucleotide enhancer (hereinafter referred to as NAD+ enhancer) and / or fibroblast-stimulating lipopeptide-1 (hereinafter referred to as FSL-1) or any salt thereof.

[0134] Similar to that defined in the first aspect, and also included in the embodiments of the fifth, sixth, and seventh aspects, PT is applied to the object with SIL, or any salt or derivative thereof. In a preferred embodiment, the object is a mammal. Preferably, the mammal is a human or livestock such as a dog, cat, or horse. More preferably, the mammal is a human.

[0135] The present invention also provides a method for increasing intracellular levels of deacetylases in a subject to prevent, treat (including improve or reduce) a disease in the subject characterized by low deacetylase levels and reduced deacetylase activation, as described in the foregoing aspects. In one embodiment, the method comprises administering a combination of pterostilbene and silymarin, or any salt thereof, wherein the combination is understood herein to mean simultaneously or sequentially administering to the subject a) pterostilbene or a salt thereof, b) silymarin or a salt thereof, and optionally c) providing at least one or more radiation-mitigating compounds, as defined throughout this specification. Similar to the above, at least one or more radiation-mitigating compounds may be NAD+ enhancers and / or FSL-1. Importantly, the dosage and route of administration defined in the first aspect also apply to uses according to the fifth, sixth, and seventh aspects.

[0136] It is worth noting that this invention also relates to the following terms or embodiments:

[0137] 1. Pterostilbene or a pharmaceutically acceptable salt thereof for the prevention, improvement or reduction of ionizing radiation-induced diseases in a subject, in combination with silybin or a pharmaceutically acceptable salt thereof, wherein the administration is performed before, after or during radiation; and wherein the combination is hereby understood to mean, in any order, simultaneous or sequential administration of a) pterostilbene or a pharmaceutically acceptable salt thereof and b) silybin or a pharmaceutically acceptable salt thereof to the subject.

[0138] 2. Pterostilbene for use as described in Clause 1, wherein pterostilbene and silymarin or a pharmaceutically acceptable salt thereof are administered as a single pharmaceutical composition or as separate pharmaceutical compositions.

[0139] 3. Pterostilbene for use according to any one of Clauses 1 to 2, wherein the application of pterostilbene and silymarin or a pharmaceutically acceptable salt thereof is topical, oral or parenteral.

[0140] 4. Pterostilbene for use according to any one of clauses 1 to 3, wherein pterostilbene and silymarin, or a pharmaceutically acceptable salt thereof, are applied several times simultaneously or sequentially before and / or after irradiation, or both.

[0141] 5. Pterostilbene for use according to any one of Clauses 1 to 4, wherein the combined dosage is sufficient to provide radiation protection.

[0142] 6. Pterostilbene for use according to any one of clauses 1 to 5, wherein pterostilbene or a pharmaceutically acceptable salt thereof is administered at a dose of 300 mg / kg to 30 mg / kg, preferably 100 mg / kg, and silymarin or a pharmaceutically acceptable salt thereof is administered at a dose of 200 mg / kg to 35 mg / kg, preferably 70 mg / kg, or equivalent human doses.

[0143] 7. Pterostilbene for use according to any one of Clauses 1 to 6, wherein the use further comprises administration in combination with at least one or more radiation-mitigating compounds, wherein the combination is understood herein to mean simultaneous or sequential administration to the subject of a) pterostilbene or a pharmaceutically acceptable salt thereof, b) silymarin or a pharmaceutically acceptable salt thereof, and c) at least one or more radiation-mitigating compounds or any pharmaceutically acceptable salt thereof.

[0144] 8. Pterostilbene for use as described in Clause 7, wherein at least one or more radiation-mitigating compounds are NAD+ enhancers and / or fibroblast-stimulating lipopeptides, or any pharmaceutically acceptable salt thereof.

[0145] 9. Pterostilbene for use as described in Clause 7, wherein the use further comprises administration in combination with at least two or more radiation-mitigating compounds, wherein the at least two or more radiation-mitigating compounds are NAD+ enhancers and fibroblast-stimulating lipopeptides, or any pharmaceutically acceptable salt thereof.

[0146] 10. Pterostilbene for use according to any one of Clauses 7 to 9, wherein the NAD+ enhancer or any pharmaceutically acceptable salt thereof is administered at an amount of 400 mg / kg to 100 mg / kg, preferably 185 mg / kg, and / or wherein the fibroblast-stimulating lipopeptide or any pharmaceutically acceptable salt thereof is administered at an amount of 1 mg / kg to 0.1 mg / kg, preferably 0.25 mg / kg, or equivalent human doses.

[0147] 11. Pterostilbene for use according to any one of clauses 8 to 10, wherein the NAD+ enhancer is nicotinamide nucleoside.

[0148] 12. Pterostilbene for use according to any one of Clauses 1 to 11, wherein the ionizing radiation-induced disease is associated with DNA damage, radiation-induced cytotoxicity, genotoxicity and / or oxidative cell damage.

[0149] 13. Pterostilbene for use according to any one of Clauses 1 to 12, wherein the ionizing radiation-induced disease is acute radiation syndrome and / or chronic radiation syndrome.

[0150] 14. Pterostilbene for use according to any one of clauses 1 to 13, wherein the ionizing radiation is gamma rays or X-rays.

[0151] 15. Pterostilbene for use according to any one of Clauses 1 to 14, wherein pterostilbene is pterostilbene disodium phosphate, and / or wherein silybin is silybin-C-2',3-dihydrosuccinate disodium salt.

[0152] 16. A pharmaceutical composition comprising a) pterostilbene or a pharmaceutically acceptable salt thereof, and b) silymarin or a pharmaceutically acceptable salt thereof.

[0153] 17. The pharmaceutical composition according to Clause 16, wherein pterostilbene or a pharmaceutically acceptable salt thereof is present in the composition at an amount of 300 mg / kg to 30 mg / kg, preferably 100 mg / kg, and wherein silymarin or a pharmaceutically acceptable salt thereof is present in the composition at an amount of 200 mg / kg to 35 mg / kg silymarin, preferably 70 mg / kg, or equivalent human doses.

[0154] 18. The pharmaceutical composition according to any one of clauses 16 and 17, wherein the pharmaceutical composition further comprises at least one or more radiation-mitigating compounds, preferably selected from:

[0155] a. NAD+ enhancer, and / or

[0156] b. Fibroblast-stimulating lipopeptide-1,

[0157] Or any pharmaceutically acceptable salt thereof.

[0158] 19. The pharmaceutical composition according to Clause 18, wherein the pharmaceutical composition further comprises at least two or more radiation-mitigating compounds, wherein the at least two or more radiation-mitigating compounds are NAD+ enhancers and fibroblast-stimulating lipopeptide-1, or any pharmaceutically acceptable salt thereof.

[0159] 20. A pharmaceutical composition according to any one of Clauses 18 to 19, wherein the NAD+ enhancer or a pharmaceutically acceptable salt thereof is present in the composition at an amount of 400 mg / kg to 100 mg / kg, preferably 185 mg / kg, and / or wherein fibroblast-stimulating lipopeptide-1 or a pharmaceutically acceptable salt thereof is present in the composition at an amount of 1 mg / kg to 0.1 mg / kg, preferably 0.25 mg / kg, or equivalent to a human dose.

[0160] 21. The pharmaceutical composition according to any one of Clauses 18 to 20, wherein the NAD+ enhancer is nicotinamide nucleoside.

[0161] 22. A pharmaceutical composition according to any one of Clauses 16 to 21, wherein the composition further comprises a pharmaceutically acceptable carrier, additive, and / or excipient.

[0162] 23. The pharmaceutical composition according to any one of Clauses 16 to 22, wherein pterostilbene is pterostilbene disodium phosphate, and / or wherein silybin is silybin-C-2',3-dihydrosuccinate disodium salt.

[0163] 24. A multipart kit comprising at least two containers containing at least pterostilbene and silymarin, or any pharmaceutically acceptable salt thereof, and optionally at least one or more radiation mitigating compounds.

[0164] 25. The multipart kit according to Clause 24, wherein pterostilbene or a pharmaceutically acceptable salt thereof is present in an amount of 300 mg / kg to 30 mg / kg, preferably 100 mg / kg, and silymarin or a pharmaceutically acceptable salt thereof is present in an amount of 200 mg / kg to 35 mg / kg silymarin, preferably 70 mg / kg, or equivalent human doses.

[0165] 26. The multipart kit according to clauses 24 and 25, wherein optional at least one or more radiation mitigating agent compounds are selected from the group consisting of:

[0166] a. NAD+ enhancer, and / or

[0167] b. Fibroblast-stimulating lipopeptides,

[0168] Or any pharmaceutically acceptable salt thereof.

[0169] 27. The multipart kit according to Clause 26, wherein an NAD+ enhancer or a pharmaceutically acceptable salt thereof is present in an amount of 400 mg / kg to 100 mg / kg, preferably 185 mg / kg, and / or wherein a fibroblast-stimulating lipopeptide or a pharmaceutically acceptable salt thereof is present in an amount of 1 mg / kg to 0.1 mg / kg, preferably 0.25 mg / kg, or equivalent human doses.

[0170] 28. A multipart kit according to any one of Clauses 26 and 27, wherein the NAD+ enhancer is nicotinamide nucleoside.

[0171] 29. A multipart kit according to any one of Clauses 24 to 27, wherein pterostilbene is pterostilbene disodium phosphate, and / or wherein silybin is silybin-C-2',3-dihydrosuccinate disodium salt.

[0172] 30. A method for preventing, improving or reducing ionizing radiation-induced diseases in a subject, comprising: administering a combination of pterostilbene and silymarin, or a pharmaceutically acceptable salt thereof, wherein the administration is performed before, after or during radiation; and wherein the combination is understood herein to mean administering simultaneously or sequentially to the subject a) pterostilbene or a pharmaceutically acceptable salt thereof, and b) silymarin or a pharmaceutically acceptable salt thereof.

[0173] 31. The method according to clause 30 further comprises applying at least one radiation moderating compound, wherein said at least one or more radiation moderating compounds are preferably selected from:

[0174] a. NAD+ enhancer, and / or

[0175] b. Fibroblast-stimulating lipopeptides,

[0176] Or any pharmaceutically acceptable salt thereof.

[0177] 32. The method according to Clause 31, wherein the NAD+ enhancer is nicotinamide nucleoside.

[0178] It should be understood that the specific embodiments described herein are shown by way of illustration and not intended to limit the invention. The main features of the invention may be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to determine many equivalents of the specific processes described herein using only conventional experimentation. These equivalents are considered to be within the scope of the invention and are covered by the claims.

[0179] Example

[0180] Example 1: General Materials and Methods

[0181] This document discloses the general materials and methods used in this invention. Specific procedures are further described in each of Examples 2 through 4.

[0182] Mice and treatment procedures

[0183] Swiss albino mice (male, 9-10 weeks old, 30-32 g) were obtained from Charles River Laboratories (Wilmington, Massachusetts). This strain is one of the most commonly used strains in radiation research (Williams 2010). Mice were fed a complete diet (standard diet, SD) before irradiation. This was Panlab (Barcelona, ​​Spain) A03, with 3200 kcal / kg and the following nutritional composition: crude protein, 23.5%; crude fat, 4.3%; crude fiber, 3.7%; and carbohydrates, 51.1%; providing the same vitamins and minerals as the NIH-7 open formula used for rats and mice. This diet conforms to the dietary intake recommended by the National Institute of Nutrition for adult mice. These animals were allowed free access to food and housed in individual metabolic cages, with body weight and food intake monitored daily. The animal room was maintained at 22°C with a 12-hour light / 12-hour dark cycle. Free access to drinking water was also permitted. The care of irradiated mice followed the Robert Wood Johnson Medical School (Piscataway, NJ) recommendations for whole-body irradiation of mice (Strategy 15), which included: a) administering antibiotics in drinking water (134 mg ampicillin / kg / day, 40 mg Baytril / kg / day, and 220 / 42 mg sulfamethoxazole / trimethoprim / kg / day); b) ensuring easy access to drinking water; c) providing Napa nectar (Systems Engineering Lab Group Inc., Napa, CA) at the bottom of the cages for the first 14 days and changing it daily; d) providing softened food in small petri dishes on the cage floor; and e) maintaining a completely sterile environment (cages, food, and water) to avoid potential immunosuppression-related infections. Each cage also included a separate wheel for animal exercise. All experiments began at 10:00 AM to minimize potential circadian rhythm variations. The procedures involving the animals complied with international laws and policies (EEC Directive 86 / 609, OJ L 358. 1, December 12, 1987; and NIH Guidelines for the Care and Use of Laboratory Animals, NIH Publ. No. 85-23, 1985). Mice were exposed to gamma radiation in the BIOBEAM 8000 (Gamma-Service Medical GmbH, Leipzig, Germany). 137(Cs) (Total Irradiation, TBI). Single-dose radiation therapy with a total dose range of approximately 5.0 to 10.0 Gy, administered at a rate of approximately 5.0 Gy / min. The irradiation distance from the source is limited to 12 cm. Radiation dosimetry is controlled using a Bruker (Bielelica, Massachusetts) alanine dosimeter with a dose variation of + / - 0.09 Gy. For oral administration, PTER and SIL (Merck Chemicals GmbH, Darmstadt, Germany) are dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethyl cellulose. For intraperitoneal administration, PTER salt (disodium pterostilbene phosphate, SYNCOM, Groningen, Netherlands) and SIL salt (disodium silymarin-C-2',3-dihydrosuccinate, Rottapharm / Madaus, Cologne, Germany) are dissolved in sterile vaccine-grade water. All other polyphenolic phytochemicals measured (Merck Chemicals GmbH; and Cayman Chemical Co., Ann Arbor, Michigan) represent major flavonoids and non-flavonoids (Estrela 2017), namely gallic acid (a phenolic acid), caffeic acid (a hydroxycinnamic acid), curcumin (a curcuminoid compound), epigallocatechin gallate (a flavanol), genistein (an isoflavone), quercetin (a flavonol), luteolin (a flavonoid), naringenin (a flavanol), delphinidin (an anthocyanin), and phlorizin (a chalcone), dissolved prior to oral administration as with PTER and SIL (Wani 2016). NR (nicotinamide nucleoside, 3-aminocarbonyl-1-bD-rifuranosyl-pyridinium, ElysiumHealth, Inc., NY, New York) was dissolved in vaccine-grade water and the solution was neutralized to pH 7.0 before oral administration (185 mg / kg × day, Obrador 2020). FSL-1 (InvivoGen, San Diego, CA) was resuspended in sterile vaccine-grade water and administered intraperitoneally (0.25 mg / kg × day, 50 to 60 mL, depending on mouse weight, as recommended by Kurkjian 2017). Pharmaceutical-grade amifostine (Ethyol) was purchased from Cumberland Pharmaceuticals (Nashville, Tennessee) in 500 mg sterile lyophilized powder vials, reconstituted with physiological saline before use.

[0184] Pathology

[0185] Mice that were dead, dying, or scheduled for euthanasia were excluded from the study. All mice underwent necropsy, and approximately 30 tissues or organs, along with all gross lesions, were collected for microscopic examination. After fixation in 10% neutral buffered formalin, all tissues were routinely processed and stained with hematoxylin and eosin for histopathological evaluation.

[0186] Fourteen days after irradiation, mice were euthanized, and the femur was dissected. Bone marrow cells were flushed from the dissected femur using a 26-gauge needle with pre-cooled phosphate-buffered saline. Single-cell suspensions were prepared by gently agitating the flushed cells multiple times, and the number of viable bone marrow nucleated cells (BMNCs) was counted using a hematology counter (Neubauer, Marienfeld, Germany) and expressed as ×10⁻⁶. 6 / femur.

[0187] Rotating bar test

[0188] The test functions included assessing the subjects' balance, grip strength, and motor coordination, particularly in testing the effects of experimental drugs. For this test (Rota Rod, Harvard Apparatus, Holliston, MA), each animal underwent three trials, and the longest duration (in seconds) it could remain on a rotating axis (3.5 cm in diameter; rotation speed: 15 rpm) without falling was measured. Each mouse could attempt up to three times within any limit of 1200 seconds, and the longest time was recorded. Changes over time were monitored weekly for different groups.

[0189] Pterocaryonium level

[0190] As previously mentioned (Ferrer 2005), the TSQ Vantage uses an SLC-10Avp controller system equipped with a Shimadzu LC-10ADvp pump and a SIL-10ADvp autosampler. TM Liquid chromatography and mass spectrometry (LC-MS / MS) were performed using a triple quadrupole mass spectrometer (ThermoFisher Scientific).

[0191] Silybum level

[0192] The measurement is based on the method described by Song et al. (2019). In short, in Hypurity... TMSamples were analyzed using HPLC (Ultimate 3000, Thermo Fisher Scientific) and MS / MS (Waters TQ detector triple quadrupole) systems with an injection volume of 5 μL on a C18 column (50 × 4.6 mm). The mobile phase consisted of water (A) and acetonitrile (B). The gradient elution system was optimized as follows: 0 to 3.0 min 20% to 100% B, 3.0 to 5.0 min 100% to 100% B, 5.0 to 5.5 min 100% to 20% B, and 5.5 to 7.5 min 20% B. The flow rate was 0.5 mL / min. MS / MS data acquisition was performed in negative electrospray ionization mode. Multiple reaction monitoring (MRM) was used to monitor SIL, with the transition from precursor ion to product ion at m / z 481.3 / 125.0. The fragmentation voltage was 150 V and the collision energy was 15 eV. Tissue sample preparation was performed as described for PTER level determination (above).

[0193] Blood NAD + level

[0194] Following the method described by Yoshino and Imai (Yoshino et al. 2013), NAD in whole blood was measured by HPLC. + Levels were established. In this procedure, an HPLC system (Shimadzu, Kyoto, Japan) equipped with a Supelco LC-18-T column (15cm × 4.6cm; Sigma Aldrich) and a Hypercarb column (15cm × 4.6cm; Thermo Fisher Scientific, Waltham, MA) was used. Whole blood samples (0.5ml) were collected in sodium citrate tubes at 4°C. Then, 0.1ml aliquots of whole blood were transferred to cryovials containing 0.9ml of 0.5M ice-cold perchloric acid to lyse all blood cells. Samples were centrifuged at 10,000g for 10 minutes (4°C). The supernatant was stored at -80°C until analysis.

[0195] Assessment of treatment-induced in vivo toxicity

[0196] This includes blood cell counting and chemical analysis based on NIH standard methods.

[0197] Chromosomal aberrations and double-strand DNA breaks

[0198] Following the technique described by M'kacher et al. (2015), where telomere and centromere staining facilitates the easy detection of dicentric, centromere loops, and acentric chromosomes in lymphocytes by using optimized hybridization conditions and interphase chromosome fusion capture. A major advantage of this method is that the damage can be observed within hours of blood collection. Blood was collected 8 hours after irradiation as recommended in the procedure (M'kacher 2015), allowing time for DNA repair and the appearance of dicentric, centromere loops, and acentric chromosomes.

[0199] Peripheral blood lymphocytes were isolated as described below. Precocious chromosome aggregation (PCC) was performed using CHO cells (ATCC) as previously described (Pantelias 1983). Based on morphological characteristics, the aggregated human chromosomes were distinguished from the CHO chromosomes (M'kacher 2015). This process takes approximately 4 hours.

[0200] Telomeres and centromeres of PCC fusions were stained using Q-FISH technology, employing Cy-3-labeled peptide nucleic acid (PNA) probes specific to telomeric TTAGGG and fluorescein isothiocyanate-labeled PNA probes specific to centromere sequences (M'kacher 2015) (both from ThermoFisher Scientific, Waltham, MA). The stained fusions were automatically recorded using Metasystems' Autocapt software (Altrusheim, Germany) and a high-resolution CCD camera (C91002-23B, Hamamatsu Photonics, Helsing, Germany).

[0201] In the same slide, two different manual procedures were performed as described by M'kacher et al. (2015). Briefly, firstly, loops and excessive acentric chromosomes were scored using trans-4',6-diamidindo-2-phenylindole. Secondly, telomere and centromere staining was used to score dicentric chromosomes, centromere loops, and acentric chromosomes with intermediate deletions from telomere-free staining, acentric chromosomes with only one telomere (representing terminal deletions), and acentric chromosomes with two telomeres (derived from the fusion of two acentric chromosomes with dicentric or acentric loop formation), and the resulting double-stranded DNA breaks (DSBs) were calculated (M'kacher 2015).

[0202] Micronucleus formation

[0203] For this purpose, femoral bone marrow was collected from euthanized mice, smears were prepared, and fixed with methanol. After drying, double staining was performed using May Grunwald and Giemsa staining solution (Schmid 1975). According to this method, polychromatic erythrocytes (PCEs) stained red-blue, orthochromatic erythrocytes (NCEs) stained orange, and nuclear material appeared deep purple. Micronucleated polychromatic (MnPCE) cells and micronucleated orthochromatic (MnNCE) cells were counted from 2000 cells under oil immersion.

[0204] Isolation and incubation of lymphocytes, hepatocytes and intestinal epithelial cells

[0205] Hepatocytes were isolated according to a previously reported method (Berry 1969). Blood mononuclear cells were obtained from heparinized blood by Accuspin-Histopaque (Sigma-Aldrich, St. Louis, Missouri) gradient centrifugation. The mononuclear cells were washed twice and centrifuged at 20 × 10⁻⁶. 6 Cells / ml were resuspended in Krebs-Henseleit bicarbonate medium (pH 7.4) containing 0.3% bovine serum albumin. Lymphocyte subsets were further positively selected by monodisperse immunomagnetic Dynabead (Dynal) at 4°C, as described elsewhere (Thornton 2003). Hepatocyte and lymphocyte volumes were obtained as previously described (Estrela 1992).

[0206] The isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate medium (KHBM, pH 7.4) at 37°C in flasks (approximately 10 mg dry weight / ml) containing 1.3 mM CaCl2, 5% defatted bovine serum albumin, and glucose (5 mM). The gas atmosphere was O2 / CO2 (19:1). The isolated lymphocytes (10... 6 Cells (per flask) were incubated at 37°C in Krebs-Ringer medium containing 5% defatted bovine serum albumin and glucose (5 mM) and glutamine (2 mM). After incubation, cells were disrupted by adding 0.2 ml of 25% perchloric acid. Proteins were removed by centrifugation, and the supernatant was neutralized with 20 ml of 40% KOH solution and Tris-(hydroxymethyl)aminomethane / KOH (0.5 to 2.0 M) solution for metabolite measurement.

[0207] Primary intestinal epithelial cells (IECs) were isolated using a protocol adapted from Graves et al. (2014). The longitudinal muscle layer was removed and the cells were immersed in ice-cold, Mg-free tubing containing 100 U penicillin, 100 μg / ml streptomycin, 25 μg / ml gentamicin, and 0.5 mM dithiothreitol (DTT). 2+ and Ca2+ Colon tissue was prepared by washing with Hank's balanced salt solution (HBSS). The tissue was cut into small pieces, suspended in 50 ml of HBSS washing solution, agitated, and allowed to settle for 2 minutes. The supernatant was removed, and the settled contents were washed three times. The precipitated contents were then chopped and suspended in HBSS. The resulting suspension was filtered through a 1000 μm filter. 2 The tissue was filtered through a sieve. The remaining tissue was digested in a buffer solution containing 75 U / ml type XI collagenase (Sigma-Aldrich), 25 μg / ml dispersant neutral proteinase II (Thermo Fisher Scientific, Waltham, MA), 0.5 mM DTT, and 2% v / v fetal bovine serum (FBS) (Thermo Fisher Scientific) in Duchenne Modified Eagle Medium (DMEM) (Sigma-Aldrich). The digestion buffer containing the tissue was then placed in a 37°C incubator and shaken at 200 rpm for 2 hours. The resulting digestion mixture was then filtered again through a 1000 μm sieve. 2 The filter was cleaned, and the tissue debris at the top of the filter was washed with 25 ml of complete growth medium (DMEM, 8.5 g / L sodium pyruvate, 2% v / v FBS, 0.25 U / ml insulin, 100 U penicillin, 100 μg / ml streptomycin, 25 μg / ml gentamicin, 5 μg / ml transferrin, and 10 ng / ml EGF containing 2% w / v D-sorbitol). The remaining tissue debris in the filter was discarded, and the effluent containing proliferative crypt structures was centrifuged at 200 g for 5 min at 4 °C. The remaining precipitate containing isolated intestinal crypts was resuspended in DMEM. The crypts were resuspended in complete growth medium, spread at a density of approximately 2000 crypts / ml / well in 12-well type I collagen-coated culture dishes (Thermo Fisher Scientific), and incubated at 37 °C, O2 / CO2 (19:1).

[0208] Glucose and glutamine utilization were measured as previously described (Newsholme 1987).

[0209] Cell culture

[0210] The separated hepatocytes were divided into 10 6 Cells / mL were cultured in William complete medium. The isolated C2D cells were... + Lymphocytes at 2×10 6 Cells / mL were cultured in PB-MAX medium (GIBCO, Thermo Fisher Scientific). IEC (10 6Cells (10 cells / mL) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 300 mg / mL L-glutamine. All cells were cultured in a humid atmosphere at 37°C, 95% air and 5% CO2.

[0211] DNA oxidation

[0212] Tissue samples were homogenized in 10 mM Tris (pH 7.0) + 1 mM EDTA + 150 mM NaCl. They were then incubated with RNase A at 37 °C for 30 min, followed by incubation with proteinase K at 55 °C overnight. Chloroform / isoamyl alcohol extraction was then performed. 100 μg of isolated DNA samples were digested into nucleosides using nuclease P1 and alkaline phosphatase (Crain 1990). Analysis of the modified DNA base 8-hydroxy-2'-deoxyguanosine (8OHdG) was performed by UPLC-MS / MS.

[0213] Lipid peroxidation

[0214] For isoprostane assays, tissue samples were homogenized in 0.1 M phosphate buffer (pH 7.4) + 1 mM EDTA + 0.005% butylated hydroxytoluene. Isoprostane was measured using the 8-isoprostane EIA kit (Cayman Chemical Co.) following the manufacturer's protocol.

[0215] Protein carbonylation

[0216] Protein carbonylation is an irreversible oxidative damage, and measurements were performed using a protein carbonyl fluorescence assay kit (Cayman Chemical Co.) following the manufacturer’s protocol.

[0217] Enzyme activity and metabolic level

[0218] The isolated cells were homogenized in 0.1 M phosphate-buffered saline (pH 7.2) at 4 °C. The activities of γ-glutamylcysteine ​​ligase (GCL), glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase 1 and 2 (SOD1 and SOD2) were measured as previously described in detail (Benlloch 2016). Protein concentrations were determined using the Pierce BCA protein assay (Thermo Fisher Scientific). Glutathione (γ-L-glutamyl-L-cysteine, GSH) was determined by LC / MS as previously reported (Obrador 2014). Cell treatment was performed according to published methods, in which rapid N-ethylmaleimide derivatization was used to prevent GSH auto-oxidation (Asensi 1994). NAD and NADH in the isolated cells were quantified using a assay kit from MyBiosource (San Diego, CA).

[0219] RT-PCR and mRNA detection

[0220] Total RNA was isolated using the Invitrogen (San Diego, CA) TRIzol kit, following the manufacturer's instructions. cDNA was obtained using random hexamer primers and the MultiScribe reverse transcriptase kit as recommended by the manufacturer (Taq-ManRT Reagents; Applied Biosystems, Foster City, CA). The PCR master mixture and AmpliTaq Gold DNA polymerase (Applied Biosystems) were added to the previously reported primers (Benlloch 2016). Real-time quantification of mRNA was performed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as previously reported (Benlloch 2016).

[0221] Comet determination

[0222] This assay is a single-cell gel electrophoresis assay used for a simple assessment of cellular DNA damage. We used the Abcam (Cambridge, UK) assay kit (ab238544). Follow the manufacturer's instructions step by step.

[0223] Nicotinamide phosphoribosyltransferase activity assay

[0224] The formation of 14C-nicotinamide mononucleotide (NMN) from 14C-nicotinamide (NAM, GE Healthcare Life Sciences, Björkgatan, Uppsala, Sweden) can be measured by precipitating 14C-NMN in acetone onto filter paper and scintillation counting. Cell dispersion was performed using a trypsin assay (in a Mg-free environment supplemented with 0.2% trypsin, 0.5 mM EDTA and 5 mM glucose). 2+ -and Ca 2+ The procedure was performed in PBS (37°C for 3 minutes). Cell pellets were obtained by centrifugation (800 g × 5 minutes, 4°C), and 100 µl of phosphate-buffered saline [0.01 M Na₂HPO₄ / NaH₂PO₄ buffer, pH 7.4, containing protease inhibitors (Sigma-Aldrich)] was added to each cell sample. Samples were further processed as previously described, with NAMPT activity determined based on 30 µg of protein (Elliott GC et al., 1980; Schuster S et al., 2014). Radioactivity (disintegration / min) was measured using a Perkin-Elmer Tri-Carb® liquid scintillation counter. Background disintegration / min of the scintillation solution was subtracted from each measurement.

[0225] Colony-forming cells in bone marrow

[0226] Mice were euthanized under aseptic conditions, and femurs were isolated on day 7 post-irradiation. Bone marrow cells were flushed from the isolated femurs (see above) with phosphate-buffered saline (4°C) supplemented with 5% fetal bovine serum. Single-cell suspensions were passed through a 100 μm nylon mesh filter to remove debris and clumps. Cells were washed twice with phosphate-buffered saline (4°C) and counted using a hemocytometer. Cells were cultured at 5 × 10⁶ cells per 35 mm cell culture dish according to the manufacturer's protocol. 4 Cells at a concentration of [number] were seeded in Methodcult GF M3434 (Stemcell Technologies, Vancouver, Canada) growth medium and incubated for 14 days at 37°C, 5% CO2, and 95% humidity. On day 14, colonies were observed under polarized light using an optical microscope (AxioCam MRc5, Zeiss, Upper Köhgen, Germany) and scanned in 60mm grid scoring dishes (Stemcell Technologies). CFU-GM and CFU-GEMM hematopoietic colony-forming cells were quantified.

[0227] Preparation of cell, nucleus and mitochondrial extracts

[0228] Cell extracts were prepared using Sigma Aldrich's Whole Cell Extraction Kit. The NE-PER™ Nuclear and Cytoplasmic Extraction Kit (ThermoFisherScientific) allows for stepwise isolation of cytoplasmic and nuclear extracts from mammalian cells or tissues. Mitochondrial extracts were prepared according to ThermoFisher Scientific's standardized cell grading and organelle isolation procedures.

[0229] Gene silencing

[0230] Long-term gene silencing was achieved using the PSilencer 3.1-H1 linear vector from Ambion Inc. (Austin, Texas). To target mouse Nfkbie, the siRNA OligoDuplex (Locus ID 18037) from Origen (Rockville, Maryland) was used. The siRNAs targeting mouse Nfe2l2 (RefSeq NM_010902.3), Ppargc1 (RefSeq NM_008904.2), Sirt1 (RefSeq NM_019812.2), Sirt3 (RefSeq MN_022433.2), Parp1 (RefSeq NM_007415.2), SOD2 (RefSeq NM_013671.3), and GPX1 (RefSeq NM_008160.6) were derived from Invitrogen.

[0231] In all cases, the silencing procedure was performed following the manufacturer's technical recommendations. Control experiments were conducted using equal amounts of the corresponding sense oligonucleotide and a randomized oligonucleotide with the same base composition and random sequence. Silencing was confirmed by Western blotting. siRNA was delivered using the Invitrogen Lipofectamine RNAiMAX transfection reagent and the manufacturer's protocol.

[0232] Cell death analysis

[0233] Apoptotic and necrotic cell death were distinguished using fluorescence microscopy. For this purpose, isolated MN was incubated for 3 minutes with Hoescht 33342 (10 mM; staining all cell nuclei) and propidium iodide (10 mM; staining the nuclei of cells with ruptured plasma membranes), and analyzed using a Diaphot 300 fluorescence microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 360 nm. The nuclei of live cells, necrotic cells, and apoptotic cells were observed as blue round nuclei, pink round nuclei, and fragmented blue or pink nuclei, respectively. Approximately 1000 cells were counted each time.

[0234] Protein blot

[0235] As previously described, Western blot analysis was performed (Benlloch M et al., 2016). Proteins were transferred to nitrocellulose membranes and Western blotted with specific anti-human monoclonal antibodies (OriGene, Rockville, MD; and Abcam). The blots were developed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL system; GE HealthCare LifeSciences). Protein bands were quantified using laser densitometer.

[0236] Xenografts for Tumors

[0237] Human A549 and MDA-MB-231 cells were obtained from ATCC (Manassas, Virginia). Cells were grown in DMEM (pH 7.4) (Invitrogen, San Diego, California) supplemented with 10% heat-inactivated FCS (Biochrom KG, Berlin, Germany), 100 IU / mL penicillin, and 100 μg / mL streptomycin. Cells were plated (20,000 cells / cm²). 2 Cells were cultured at 37°C in a humid atmosphere with 5% CO2. Cells were harvested by incubating them for 5 minutes with 0.05% (w / v) trypsin (Sigma-Aldrich, St. Louis, Missouri) containing 0.3 mM EDTA in PBS (pH 7.4) containing 0.3 mM EDTA, followed by inactivation of the trypsin with 10% FCS. Cells were allowed to adhere for 12 hours before any further treatment. Cell number and viability were determined using a BioRad (Hercules, CA) TC20 automated cell counter. Male nude (nu / nu) mice (9–10 weeks old, Charles River Laboratories, Wilmington, MA) were allowed free access to a standard diet (Letica, Rochester Hills, Michigan) and maintained in a 12-hour light / 12-hour dark cycle at 22°C. The procedure complied with international laws and policies (EEC Directive 86 / 609, OJ L 358. 1, December 12, 1987; and NIH Guidelines for the Care and Use of Laboratory Animals, NIH Publ. No. 85-23, 1985). Tumor cells were harvested from culture flasks by exposure to 0.02% EDTA (37°C for 5 minutes), washed twice in DMEM, resuspended in the same medium, and subcutaneously injected into the back of the animals (5 × 10⁻⁶ cells). 6 (cells / mouse). Starting from the second day of treatment, local tumor growth was measured every two days using calipers.

[0238] statistics

[0239] Data are expressed as mean ± standard deviation of different experimental runs. Statistical analysis was performed using Student's t-test, and P < 0.05 was considered significant.

[0240] Sample size was determined using G*Power 3.1.9.2 software (ANOVA test, effect size = 0.21, α = 0.05, β = 0.80, and specified groups). Survival data were analyzed using Kaplan-Meier curves and LogRank (Mantel-Cox) tests.

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[0281] Example 2: PT and radiation protection effects

[0282] First, the effects of gamma radiation and mouse survival were investigated. The study examined mice that received whole-body gamma radiation (…). Figure 1 A). Mice were given whole-body gamma radiation (a single dose); 137Cs, 0.5 to 0.6 Gy / min; Biobeam 8000, GSM GmbH. 1 Gy = 1 J of energy absorbed per kilogram of material. LD50 / 30 is approximately 7.2 Gy (per radiation dose, n=20).

[0283] Next, the 30-day survival rate of mice treated with gamma rays (LD50 / 30) and pterostilbene (PT) was analyzed. Figure 1 B). For this purpose, Swiss albino mice (Charles River, n=20 / group) were irradiated with amifostine (2-(3-aminopropylamino)ethylthioalkylphosphonic acid) (dissolved in physiological saline; a single oral dose 30 minutes before irradiation; the recommended human dose of 200 mg / m²). 2 According to FDA guidelines, this was indicated for use in mice. Alternatively, pterostilbene (dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethyl cellulose; orally administered once daily for 5 days before and 2 days after irradiation). Mice received whole-body gamma radiation (a single dose of 7.2 Gy). 137 Cs, 0.5 to 0.6 Gy / min, equipment = Biobeam 8000, GSM GmbH. 1 Gy = 1 J of energy absorbed per kilogram of substance. Data were analyzed using the LogRank (Mantel-Cox) test (*Compare all groups with the solvent-treated control). Figure 1 The results in B indicated that a dose of 100 mg / kg of Pterocarya stenoptera had the best effect on improving survival rate (60 days).

[0284] The survival rate of mice treated with gamma rays (LD50 / 30) and resveratrol (Resv) was also analyzed, and the results were as follows: Figure 1 As shown in C. Swiss albino mice (n=20 / group) were treated with resveratrol (dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethyl cellulose; orally administered once daily for 5 days before and 2 days after irradiation). Mice were also treated with whole-body gamma radiation (a single dose of 7.2 Gy). 137 Cs, 0.5 to 0.6 Gy / min, Mortazavi SMJ et al. Dose-Response 11:281–292, 2013; Equipment = Biobeam 8000, GSM GmbH). 1 Gy = 1 J of energy absorbed per kilogram of substance. The results showed that resveratrol had a lower radiation protection effect than pterostilbene.

[0285] The plasma and extravascular levels of pterostilbene following oral administration to mice were investigated. Mice were treated with 200 mg pterostilbene (Pter) / kg body weight. Groups of 4 to 5 animals were sacrificed at each time point. Pter was dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethyl cellulose. Undetectable = nd * P < 0.1. Data obtained from 10 to 1440 minutes were compared with data obtained from 5 minutes. The results are shown in Table 1 below:

[0286]

[0287] These results indicate that in all the tissues studied, pterostilbene levels peaked 60 minutes after administration.

[0288] In conclusion, the above results confirm the potential of PTER as a radiation shield.

[0289] Example 3: Combination of PT and SIL

[0290] To clarify whether pterostilbene can be combined with other polyphenols to achieve better radiation protection, Swiss albino mice (n=20 / group) were treated with pterostilbene and / or other polyphenols. For oral administration, PT was dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethyl cellulose. All other polyphenolic phytochemicals tested, representing major flavonoids and non-flavonoids, namely gallic acid (a phenolic acid), caffeic acid (a hydroxycinnamic acid), curcumin (a curcuminoid compound), epigallocatechin gallate (a flavanol), genistein (an isoflavone), quercetin (a flavonol), luteolin (a flavonoid), naringenin (a flavanol), delphinidin (an anthocyanin), and phlorizin (a chalcone), were dissolved as before oral administration, as with PT. The dosage of each phytochemical was selected from the maximum published non-toxic dose. The data were analyzed using the LogRank (Mantel-Cox) test (*Compare all groups with the solvent-treated control, +Compare all groups with the PT-treated group only). Figure 2 ).

[0291] The results showed that PT combined with different polyphenols produced different effects:

[0292] a) In some cases, the survival rate obtained by combination is lower than that provided by PT alone. For example, this can be seen when PT is used in combination with gallic acid 60 days after irradiation.

[0293] (b) In some cases, the survival rate obtained by combining the two compounds is similar to that provided by applying PT alone, indicating that adding the other compound has little effect on the overall survival rate. This can be seen, for example, when PT is used in combination with delphinidin 60 days after irradiation.

[0294] c) In some other cases, the survival rate obtained is approximately equal to the sum of the survival rates produced by applying the two compounds alone. This can be seen, for example, when PT is used in combination with genistein 60 days after irradiation.

[0295] d) Finally, and most importantly, the combination of the two compounds resulted in a synergistic effect, producing an abnormally high survival rate that cannot be derived from the sum of the effects of the two compounds applied individually. This last case occurred only in the combination of silymarin (SIL) and PT, and both occurred at 30 and 60 days post-irradiation.

[0296] In summary, these results demonstrate that the combination of SIL and PT provides synergistic radiation protection, achieving high survival percentages at 30 and 60 days post-gamma radiation.

[0297] Based on these results, the improved survival rate and enhanced radiation protection over a longer period were investigated. Swiss albino mice (n=20 / group) received intraperitoneal treatment with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (5 days before and 2 days after gamma irradiation). For intraperitoneal administration, the PT salt (disodium pterostilbene phosphate, SYNCOM, Groningen, Netherlands) and the SIL salt (disodium silymarin-C-2',3-dihydrosuccinate, Rottapharm / Madaus, Cologne, Germany) were dissolved in sterile vaccine-grade water. Figure 3 A shows the Kaplan-Meier curve and LogRank (Mantel-Cox) test (*comparing the PT+SIL treatment group with the solvent-treated control). Intraperitoneal administration was chosen to facilitate routine administration to a large number of mice. Figure 3 As shown in Figure A, the PT+SIL combination provided long-term protection, with a survival rate of approximately 25% (5 / 20) one year after irradiation. These results confirm the synergistic enhancement effect of the two compounds when administered in combination and demonstrate that their combined radiation protection can last for a long time.

[0298] also, Figure 3B shows a comparison between PT and SIL administration before and after γ-irradiation: Swiss albino mice (n=20 / group) were treated intraperitoneally with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (LD50 / 30: light gray bars for 5 days before and 2 days after γ-irradiation; or dark gray bars for 7 days after γ-irradiation). Data were analyzed using the LogRank (Mantel-Cox) test (*comparing the post-irradiation treatment group with the pre-irradiation treatment group). These results indicate that survival rates are higher when polyphenols are administered before irradiation.

[0299] Next, the effects of pterostilbene (PT) and silymarin (SIL) on the survival rate and body weight of γ-irradiated (LD50 / 30) mice were investigated: Swiss albino mice (n=20 / group) received intraperitoneal treatment with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (5 days before and 2 days after γ-irradiation). Statistical analysis was performed using Student's t-test (*P<0.01, comparing PT+SIL with the solvent-treated control). Results are as follows. Figure 4 As shown in Figure A, it can be seen that polyphenols reduce radiation-induced weight loss.

[0300] Effects of combined administration of pterostilbene (PT) and silymarin (SIL) on radiation-induced neuromotor function changes in mice: Swiss albino mice (n=20 / group) received intraperitoneal treatment with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (5 days before and 2 days after γ-irradiation). To investigate neuromotor function, a standardized rotating bar test was performed as follows: the test assessed the subjects' balance, grasping ability, and motor coordination, particularly in terms of the effects of the experimental drugs. For this test (rotating bar, Harvard Apparatus, Holliston, MA), each animal underwent three trials, and the longest duration (in seconds) of not falling from the rotating shaft (3.5 cm in diameter; rotation speed: 15 rpm) was measured. Each mouse could attempt up to three times within any limit of 1200 seconds, and the longest time was recorded. Changes over time were monitored weekly in different groups. Figure 4 As shown in Figure B, the results indicate that neuromotor function was not significantly altered by the application of radiation and / or PT and SIL.

[0301] Next, the expression of different antioxidant defense-related enzymes in the isolated cells was investigated by RT-PCR. Statistical analysis was performed using Student's t-test (P < 0.01; * compares mice treated with PT+SIL+γ-rays with mice treated with γ-rays only; + compares mice treated with PT+SILs with controls treated with solvent). These results are as follows: Figure 5As shown, the conclusion is that radiation reduced the expression of antioxidant defense-related molecules in all studies, while PT and SIL offset the radiation-induced effects.

[0302] Furthermore, the protective effects of PT and SIL against gamma radiation-induced oxidative damage were investigated by evaluating different molecular markers. For this purpose, PT, SIL, and gamma rays were administered at previously specified doses (n=5 for each molecule, cell type, and treatment), and statistical analysis was performed using Student's t-test (P<0.01; * compare all groups with controls; + compare mice treated with PT+SIL+γ rays with mice treated with gamma rays only). Figure 6 As shown, PT and SIL offset the increase in different oxidative stress-related biomarkers induced by radiation.

[0303] To provide a complete analysis of the radiation protection effect of the combined administration of PT and SIL, GCL- and GSH-mediated antioxidant defenses were also measured. Therefore, PT, SIL, and gamma rays were administered at previously specified doses (n=5 for each molecule, cell type, and treatment), and statistical analysis was performed using Student's t-test (P<0.01; * compare all groups with controls; + compare mice treated with PT+SIL+γ rays with mice treated with gamma rays only). Figure 7 As shown, PT and SIL offset the reduction in different glutathione-related biomarkers induced by radiation.

[0304] exist Figure 8 In this study, catalase and glutathione peroxidase were measured when PT, SIL, and γ-rays were administered at previously specified doses (n=5 per molecule, cell type, and treatment). Figure 8 A and Figure 8 B) and superoxide dismutase levels ( Figure 8 C and Figure 8 D). Statistical analysis was performed using Student's t-test (P<0.01; * compare all groups with control; + compare mice treated with PT+SIL+γ-rays with mice treated with γ-rays only). The results showed that PT and SIL counteracted the radiation-induced decrease in the activity of different antioxidant enzymes.

[0305] Finally, the effects of PT and SIL on γ-radiation-induced chromosomal aberrations and micronucleus formation in circulating CD2+ lymphocytes were investigated. Telomeres and centromeres of precocious chromosome condensates were stained using Q-FISH. Using telomere and centromere staining, dicentric chromosomes, centromere loops, and acentric chromosomes with intermediate deletions (derived from telomere-free staining), acentric chromosomes with only one telomere (representing terminal deletion), and acentric chromosomes with two telomeres (derived from fusions of two acentric chromosomes accompanied by dicentric or acentric loop formation) were scored, and the resulting double-strand DNA breaks (DSBs) were calculated. In bone marrow, double staining was performed using May Grunwald and Giemsa staining agents. According to this method, polychromatic erythrocytes (PCEs) stained red-blue, orthochromatic erythrocytes (NCEs) stained orange, and nuclear material appeared deep purple. Micronucleated polychromatic (MnPCE) cells and micronucleated orthochromatic (MnNCE) cells were counted. Statistical analysis was performed using Student's t-test (P<0.01; * compare all groups with control; + compare mice treated with PT+SIL+γ-rays with mice treated with γ-rays only). The results are shown in Table 2 below:

[0306]

[0307] As can be seen from Table 2, PT and SIL reduced all radiation-induced cytogenetic alterations, namely chromosomal changes and micronucleus formation.

[0308] Example 4: Combination of PT and SIL with radiation mitigating agents

[0309] The next step was to investigate whether the combination of radiation protectants (molecules that can reduce direct damage from radiation) and radiation modifiers (compounds that can minimize toxicity even after radiation has been released) could extend survival. To investigate this, PT and SIL were combined with the NAD+ enhancer nicotinamide nucleoside (NR) and the Toll-like receptor 2 / 6 agonist FSL-1 lipopeptide (FSL-1).

[0310] First, the effects of the two molecules individually and in combination were investigated to confirm their radiation-mitigating properties. For this purpose, NR (nicotinamide nucleoside, 3-aminocarbonyl-1-bD-furanibrosy-pyridinium, Elysium Health, Inc., NY, New York) was dissolved in vaccine-grade water, and the solution was pH-neutralized (pH 7.0) before oral administration (185 mg / kg × day). FSL-1 (fibroblast-stimulating lipopeptide 1, InvivoGen, San Diego, CA) was resuspended in sterile vaccine-grade water and administered intraperitoneally (0.25 mg / kg × day, administration volume 50 to 60 mL, depending on mouse weight). NR was administered daily for 30 days as a single daily dose, starting immediately after irradiation. FSL-1 was administered only once every 24 hours after irradiation. A total of 20 mice were used. Results are as follows: Figure 9 As shown, it was observed that the 60-day survival rate after γ-irradiation provided by applying each of these molecules alone was 0% for NR, 5% for FSL-1, and 10% for the combination of NR+FSL1.

[0311] Next, Swiss albino mice (n=6 / group) were treated with PT, SIL, or NR according to the previously planned dosage, and statistical analysis was performed using Student's t-test (P<0.01; * compare 120 minutes vs. 30 minutes; + compare 2 days vs. 3 days; # compare 3 days or 15 days vs. 0 days). The results are as follows: Figure 10 As shown in the figure, PT and SIL levels were high 30 minutes after administration, while NR-induced NAD+ levels were similar at both time points (30 minutes and 120 minutes).

[0312] Next, the four compounds PT, SIL, NR and FSL-1 were administered to n=20 mice / group at the above dosage. Figure 11 A shows the application schedule that was followed. Figure 11 B shows the Kaplan-Meier curves and LogRank (Mantel-Cox) test (* compares all groups with the solvent-treated control group; + compares the PT+SIL+NR+FSL1 treatment group with the PT+SIL+NR- or PT+SIL+FSL1 treatment group). Surprisingly, a synergistic effect was also found when NR and FLS1 were used in combination with PT and SIL, as mouse survival increased to up to one year. Importantly, the combination of the four components provided protection and survival in up to 90% of the treated mice, indicating that the combination of the four components enabled long-term survival of irradiated mice.

[0313] The study investigated the causes of death in γ-irradiated mice (LD50 / 30) and the effects of combining four compounds, such as... Figure 12As shown, Swiss albino mice (n=20 / group) were treated with PT, SIL, NR, and FSL-1 according to the previously planned regimen and at the above doses. The results showed that the complete combination (PT+SIL+NR+FSL-1) indeed avoided all deaths associated with a) gastrointestinal and hematopoietic ARS and b) kidney disease. The complete combination also minimized deaths associated with myeloid leukemia and infection / inflammation.

[0314] Next, the toxicity induced by gamma radiation and the prevention caused by the combination of PT, SIL, NR, and FSL-1 (PSNF) were investigated. The results are shown in Table 3 below:

[0315]

[0316] As can be seen from Table 3, treatment with the complete combination (PT+SII+NR+FSL-1) helps to improve radiation-induced toxicity.

[0317] Finally, to clarify whether treatment with the four combinations of compounds (PT+SIL+NR+FSL-1; PSNF) interfered with the therapeutic effect of anticancer radiotherapy (X-ray) in tumor xenografts, female nu / nu nude mice (6 to 8 weeks old) (n=5 mice / group) were subcutaneously inoculated with 5 × 10⁻⁶ compounds. 6 The tumor contains either A549 (lung adenocarcinoma) or MDA-MB-231 (triple-negative breast cancer) cells. Tumor volume is calculated based on two dimensions measured using calipers, according to the formula V = 0.5a × b. 2 The values ​​are expressed in cubic millimeters, where a and b are the major and minor diameters of the tumor, respectively. The tumor was irradiated with X-rays (10 Gy) using a Philips 6 keV SL75 linear accelerator. A single irradiation was administered at a rate of 5.0 Gy / min. PSNF administration followed the same protocol as described above, with irradiation performed on day 21. Statistical analysis was performed using Student's t-test (P < 0.01; * compares all groups with the control group; + compares the gamma-ray + PSNF treatment group with the gamma-ray treatment group only).

[0318] The results are as follows Figure 13 As shown, the combination of the four compounds did not weaken the efficacy of X-ray anticancer therapy, demonstrating the feasibility of using these compounds in patients receiving the radiotherapy.

[0319] In summary, these results support the use of compositions containing at least PT and SIL, and optionally NR and FSL1, to protect against and mitigate radiation-induced damage. Notably, these compounds provide radiation protection for an extended period (one year or more), such as... Figure 3 A and Figure 11 As shown in B.

[0320] Example 5: Pterostilbene, silymarin, and nicotinamide nucleoside reduce cellular DNA damage, while FSL1 lipopeptide promotes the recovery of the hematopoietic system.

[0321] like Figure 14 As shown in Figure a, NR treatment increased NAD+ levels in different cell types and also prevented γ-radiation-induced NAD+ reduction. Administration of PT+SIL alone did not affect NAD+ levels in any of the studied cells, nor did it affect the increase induced by NR (results not shown). Administration of FK866 (a specific, non-competitive inhibitor of nicotinamide phosphoribosyltransferase (NAMPT)) reduced NAD+ levels in mice treated with PT+SIL+NR or PT+SIL+NR+γ-rays. Figure 14 a). FK866-induced NAD+ depletion is associated with a sharp decline in NAMPT activity ( Figure 14 (b) (PT, SIL, NR, FSL1, and / or γ radiation do not affect the rate of FK866-induced inhibition of NAMPT activity, not shown). Importantly, as Figure 14 As shown in c, based on DNA damage comet assays, PT+SIL or NR are beneficial for DNA recovery, and their combination is the most effective. Figure 14 c). It is speculated that the role of polyphenols is related to antioxidant mechanisms, while NR-induced NAD+ increase directly promotes DNA repair activity. Figure 14 As shown in d, FSL-1 increases hematopoietic colony-forming cells CFU-GM (colony-forming units - granulocytes, macrophages) and CFU-GEMM (colony-forming units - granulocytes, erythrocytes, macrophages, megakaryocytes). This confirms the post-irradiation hematopoietic recovery indicated by the hematological data in Table 3 above.

[0322] Interactions of Nrf2, NF-κB, PGC-1α and PARP1 in radiation protection mechanisms

[0323] Isolated intestinal epithelial cells were used to investigate potential signaling pathways involved in the protective effect induced by the PT+SIL combination. Figures 5 to 8 According to data reported in the Chinese study, PT+SIL exerts a protective effect against γ-radiation-induced oxidative damage. It is speculated that the observed protective effect is due to the induction of Nrf2 (nuclear factor erythrocyte-associated factor 2)-dependent antioxidant defense. Figures 5 to 8Both polyphenols have been reported to possess anti-inflammatory properties, thus suggesting involvement in NF-κB (nuclear factor-activated B cell κ light chain enhancer)-dependent signaling. NR-induced NAD+ increase also favors PARP1 (poly[ADP-ribose] polymerase 1)-dependent DNA repair, deacetylase activity, and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1α) activation. Furthermore, PT can induce PGC-1α and Nrf2 activation and Sirt1 expression; while SIL can affect Sirt3 expression and PARP1 activity. Figure 15 As shown in figure a, both PT and SIL increased nuclear Nrf2 and decreased nuclear NF-κB levels compared to the control. In the Nrf2 setting, the effect of PT was more significant than that of SIL. These effects generally suggest a synergistic effect in promoting antioxidant defense while downregulating NF-κB-dependent pro-inflammatory responses. pPGC-1α was also increased by PT ( Figure 15 a). Sirt1 levels increased with PT. Interestingly, although SIL alone did not affect Sirt1 levels, the combination of the two polyphenols increased Sirt1 levels more than PT alone. Figure 15 a). Both PT and SIL increase Sirt3 levels, although SIL has a stronger effect on Sirt3 ( Figure 15 a). Both PT and SIL increased PARP1 ( Figure 15 a). Figure 15 b outlines the possible molecular interactions between these signaling cascades and the steps by which PT and SIL may exert their effects. To further investigate which specific molecules may be decisive for radiation protection, isolated IECs (from control or PT+SIL-treated mice, such as...) were used. Figure 11 (as shown in a) and gene silencing-based methods. For example... Figure 15 As shown in c, siRNAs targeting the expression of mouse Nfκbie, Nfe2l2, Ppargc1, Sirt1, Sirt3, Parp1, SOD2, or GPX1 genes promote an increase in free NF-κB and a downregulation of all other molecular targets. The isolated IEC is used at concentrations that, in the absence or presence, ensure the retention of in vivo induced radiation protection (see [reference]). Figure 15 (Explanation) Cultured for 24 hours in the presence of PT+SIL and specific siRNA. For example... Figure 15 As shown in d, PT and SIL significantly reduced γ-irradiation-induced cell death observed in control (solvent-treated) cells. Cell death analysis based on the effects of specific RNAs on γ-ray-exposed cells indicated that Nrf2, pPGC-1α, SOD2, and Sirt3 were primarily responsible for ( Figure 15 d) Radiation protection effect caused by polyphenol treatment.

[0324] in conclusion

[0325] Here, the effects of systemically administered PT on gamma radiation were investigated, and it was found that combining PT and SIL improved survival. Furthermore, the combination of potential radioprotective and radiation-mitigating agents as a strategy to reduce radiation-induced damage and improve survival remains an unexplored area. This work demonstrates the efficacy of this combination in achieving long-term survival in mice exposed to lethal (LD50 / 30) gamma radiation. This novel strategy, demonstrating prolonged survival, can serve as a paradigm for further progress. Therefore, considering the large number of molecules being tested as radioprotective and / or radiation-mitigating agents, this paradigm may help to open up new avenues in this field. PT and SIL upregulate antioxidant defense in different cell types and reduce gamma radiation-induced oxidative damage and cytogenetic alterations (chromosomal aberrations and micronucleus formation). Figure 15 As shown, the protection induced by PT and SIL may involve different interconnected signaling mechanisms. Oxidative stress and inflammation are direct pathophysiological mechanisms activated by cellular exposure to harmful ionizing radiation. PT and SIL increase nuclear Nrf2 and antioxidant responses in different cell types. Figure 15 ), and they reduce NF-κB ( Figure 15 ), and may reduce radiation-related inflammatory responses. Furthermore, PT increases Sirt1 levels, thereby promoting the deacetylation of PGC-1α ( Figure 15 PGC-1α is an inducer of Sirt3 expression, such as... Figure 15 As shown, SIL treatment was also associated with increased Sirt3 levels. Mitochondrial SOD2 (deacetylated by Sirt3) and GSH peroxidase (dependent on GSH input from the cytoplasm) help reduce mitochondrial-derived ROS and prevent apoptosis activation. Importantly, as Figure 15 As shown in c and 15d, the gene silencing-based approach identified the pathway linking Nrf2, Sirt3, and SOD2 as key to the radioprotective effect induced by the combined PT and SIL treatments. Furthermore, NR generates NAD+ to support Sirt1 and Sirt3 activity and PARP1-dependent DNA repair (…). Figure 15 PARP1 activity is also induced by two polyphenols ( Figure 15 NR is the pyridine nucleoside form of vitamin B3 and a precursor to NAD+. It has superior pharmacokinetic characteristics compared to other forms of B3 (nicotinic acid and nicotinamide), thus representing the optimal NAD+ enhancer.

[0326] This work demonstrates that two radiation protectants, pterostilbene and silymarin, together with two radiation modifiers, nicotinamide nucleoside and fibroblast-stimulating lipoprotein 1, can provide long-term protection of normal tissues against lethal doses of radiation. The established safety profiles of these four molecules clearly require further investigation to apply this combination to humans. An ideal radiation protectant or modifier should be stable, offer ease of administration, have no associated systemic toxicity, and protect normal tissues from radiation-induced damage. The proposed combination meets these criteria while exhibiting high efficacy.

Claims

1. A pharmaceutical composition comprising: a) pterostilbene or its salt, b) silymarin or its salt, and c) nicotinamide nucleoside; or a) Pterostilbene or its salt, b) silymarin or its salt, and c) fibroblast-stimulating lipopeptide-1; or a) Pterostilbene or its salt, b) Silymarin or its salt, and c) Nicotinamide nucleoside and fibroblast-stimulating lipopeptide-1.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises nicotinamide nucleoside and fibroblast-stimulating lipopeptide-1, or any salt thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pterostilbene is pterostilbene disodium phosphate, and / or wherein the silybin is silybin-C-2',3-dihydrosuccinate disodium salt.

4. Use of pterostilbene or a pharmaceutically acceptable salt thereof in combination with silymarin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, improvement or reduction of ionizing radiation-induced disease in a subject, wherein the administration is performed before, after or during radiation; and wherein the combination herein is understood to mean, in any order, simultaneous or sequential administration of a) pterostilbene or a pharmaceutically acceptable salt thereof and b) silymarin or a pharmaceutically acceptable salt thereof to the subject.

5. The use according to claim 4, wherein pterostilbene or a pharmaceutically acceptable salt thereof and silymarin or a pharmaceutically acceptable salt thereof are administered as a single pharmaceutical composition or separate pharmaceutical compositions.

6. The use according to claim 4 or 5, wherein the application of pterostilbene or a pharmaceutically acceptable salt thereof and silymarin or a pharmaceutically acceptable salt thereof is topical, oral or parenteral.

7. The use according to claim 4 or 5, wherein pterostilbene or a pharmaceutically acceptable salt thereof and silymarin or a pharmaceutically acceptable salt thereof are applied several times, simultaneously or sequentially, before and / or after irradiation or both.

8. The use according to claim 4 or 5, wherein the use further comprises administration in combination with at least one of nicotinamide nucleoside and fibroblast-stimulating lipopeptide-1, wherein the combination is understood herein to be administration to the subject simultaneously or sequentially of a) pterostilbene or a pharmaceutically acceptable salt thereof, b) silymarin or a pharmaceutically acceptable salt thereof, and c) at least one of nicotinamide nucleoside and fibroblast-stimulating lipopeptide-1 or any pharmaceutically acceptable salt thereof.

9. The use according to claim 4 or 5, wherein the ionizing radiation-induced disease is associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage.

10. The use according to claim 4 or 5, wherein the ionizing radiation-induced disease is acute radiation syndrome and / or chronic radiation syndrome.

11. The use according to claim 4 or 5, wherein the ionizing radiation is gamma rays or X-rays.

12. The use according to claim 4 or 5, wherein the pterostilbene is pterostilbene disodium phosphate, and / or wherein the silybin is silybin-C-2',3-dihydrosuccinate disodium salt.

13. The use according to claim 4 or 5, wherein the drug is used to increase the intracellular level of deacetylase in a patient in need, wherein the increase is an increase compared to the intracellular level of deacetylase in the same patient prior to treatment with a combination of pterostilbene or a salt thereof and silymarin or a salt thereof.

14. A multi-part kit comprising the pharmaceutical composition according to claim 1.

15. The multipart kit according to claim 14, wherein the pterostilbene is pterostilbene disodium phosphate, and / or wherein the silybin is silybin-C-2',3-dihydrosuccinate disodium salt.

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