Pterostilbene and silibinin prevent, ameliorate, or reduce radiation-induced diseases

Abstract

The present invention relates to compositions comprising a combination of pterostilbene or a pharma- ceutically acceptable salt thereof and silibinin or a pharma- ceutically acceptable salt thereof, and their use in treating, preventing, ameliorating, or alleviating diseases induced by ionizing radiation in a subject. The compositions and the uses include at least one or more radiation mitigating compounds, preferably NAD + It may further include a booster and / or FSL-1.

Description

[Technical field]

[0001] The present invention relates to the field of medicine. In particular, the present invention relates to the field of compositions and methods for preventing, ameliorating, or reducing radiation-induced illness. [Background technology]

[0002] Currently, ionizing radiation is used for various medical purposes. X-rays and gamma rays mainly damage cells by direct ionization of DNA and other cellular targets and indirect effects via reactive oxygen species (ROS). The difference between gamma rays and X-rays is how they are produced. Gamma rays come from the settling process of excited nuclei of radionuclides after undergoing radioactive decay, whereas X-rays are produced when electrons collide with a target or when electrons rearrange within atoms. Both X-rays and gamma rays are types of high-energy (high frequency) electromagnetic radiation. They are packets of energy (photons) with no charge or mass. Both X-rays and gamma rays have the same properties and health effects.

[0003] Electromagnetic radiation, including X-rays and gamma rays, damages cells directly by ionization of DNA and other molecules and indirectly by the generation of reactive oxygen species. Responses to radiation exposure vary depending on cell type and radiation dose, intrinsic tissue sensitivity and tissue repair, and regulation of intracellular factors including cell cycle state, O2 tension, and levels of thiols and other antioxidants. However, radioprotection and radiation mitigation strategies are lacking and inefficient. Millions of cancer patients undergo radiotherapy during the course of their treatment every year, and in recent years, some radiological or nuclear events have posed a threat to people, so radioprotection or radiation mitigation strategies are needed.

[0004] The development of protective agents against exposure to harmful radiation has been the subject of intense research for decades. Effective and safe radioprotectors do not exist. To date, no drugs have been developed to treat exposure to high levels of ionizing radiation, such as those that cause hematopoietic and gastrointestinal acute radiation syndrome (ARS).

[0005] Radioprotectors are compounds that can reduce the direct damage caused by radiation. Radiomitigators are compounds that can minimize toxicity even after radiation has been administered. In general, an ideal radioprotector or radiomitigator should be stable, easily administered, have no associated toxicity, and protect normal tissues that are deemed sensitive such that acute or delayed toxicity is dose-limiting or contributes to a significant decrease in quality of life (e.g., causing mucositis, pneumonitis, myelopathy, xerostomia, proctitis, fibrosis, or leukoencephalopathy).

[0006] A wide range of phytochemicals (compounds produced by plants that generally aid in plant growth or thwart competitors, predators, or pathogens) are antioxidants and therefore potentially radioprotective. However, their radioprotective or radiomitigating properties are usually effective for a short period of time, usually limited to a maximum of one month after radiation occurs. Thus, new compounds and compositions that can treat, reduce, prevent, or ameliorate the harmful effects of radiation in a subject over a sustained period of time are desirable. Summary of the Invention

[0007] The present invention provides compositions and uses thereof for the long-term protection and mitigation of diseases caused by radiation exposure. [Brief description of the drawings]

[0008] [Figure 1]A) Radiation dose-response for a 30-day survival study in Swiss albino mice subjected to total body gamma irradiation. B) 60-day survival of mice treated with gamma radiation (LD50 / 30) and various doses of pterostilbene (PT). Amifostine was used as a control approved radioprotector. C) 60-day survival of mice treated with gamma radiation (LD50 / 30) and various doses of resveratrol (Resv). Compounds were administered orally. In all cases, 20 mice per group were used. [Diagram 2] Figure 1 shows the combination of pterostilbene (PT) and other polyphenols preventing lethality induced by gamma radiation (LD50 / 30) after A) 6 days, B) 30 days, and C) 60 days of irradiation. The doses for each phytochemical were selected from the published maximum non-toxic doses. Compounds were administered orally. In all cases, 20 mice per group were used. [Diagram 3] A) Effect of the combination of pterostilbene (PT) and silibinin (SIL) on the long-term survival of mice subjected to total body gamma irradiation (LD50 / 30). B) Radioprotection elicited by the combination of PT and SIL: comparison between administration of polyphenols before or after gamma irradiation. Compounds were administered IP. In all cases, 20 mice per group were used. [Figure 4] A) Effect of pterostilbene (PT) and silibinin (SIL) on survival and body weight of γ-irradiated (LD50 / 30) mice. B) Effect of administration of PT and SIL on whole body γ-radiation-induced changes in neuromotor function in mice. Compounds were administered via IP. In all cases, 20 mice per group were used. [Diagram 5]Protective effects of pterostilbene (PT) and silibinin (SIL) against γ-radiation-induced oxidative damage expression (RT-PCR) of various antioxidant defense-related enzyme activities in isolated cells. GCL, γ-glutamyl-cysteine ​​ligase; GPX, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; EIC, intestinal epithelial cells. Fold change relative to expression rate in control (untreated) cells (n=5 per molecule, cell type, and treatment). [Figure 6] Figure 1 shows the protective effect of pterostilbene (PT) and silibinin (SIL) against gamma radiation-induced oxidative damage in various molecular markers: A) 8-OHdG, B) 8-isoprostanes, and C) protein carbonyls (n=5 per molecule, cell type, and treatment). [Figure 7] FIG. 1 shows the protective effect of pterostilbene (PT) and silibinin (SIL) against γ-radiation-induced oxidative damage in glutathione-related antioxidant defenses in A) γ-glutamyl-cysteine ​​ligase, B) glutathione (n=5 per molecule, cell type, and treatment). [Figure 8] FIG. 1 shows the protective effect of pterostilbene (PT) and silibinin (SIL) against gamma radiation-induced oxidative damage in various antioxidant defense-related enzyme activities: A) catalase, B) glutathione peroxidase, and C) and D) superoxide dismutase 1 and superoxide dismutase 2 (n=5 per molecule, cell type, and treatment). [Figure 9] Figure 1 shows the effect of nicotinamide riboside (NR) and fibroblast stimulating lipopeptide (FSL-1) on gamma-irradiation (LD50 / 30)-induced mortality. NR was administered daily for 30 days in a single dose (orally) starting immediately (60 min) after irradiation. FSL-1 was administered IP only once, 24 h after irradiation. In all cases, 20 mice per group were used. [Figure 10]FIG. 1 shows A) pterostilbene (PT), B) silibinin (SIL), and C) NAD+ levels in circulating blood of treated and irradiated mice (6 mice per group in all cases). [Figure 11] Addition of potential radiomitigating agents (nicotinamide riboside (NR) and fibroblast stimulating lipopeptide-1 (FSL-1)) to the radioprotective combination of pterostilbene (PT) and silibinin (SIL). A) Dosing scheme, B) Survival versus time after irradiation in days. In all cases 20 mice per group were used. [Figure 12] Figure 1 shows the cause of death (LD50 / 30) in γ-irradiated mice and the effect of combined administration of radioprotectors and radiomitigators (PT, SIL, NR, and FSL-1). In all cases, 20 mice per group were studied. [Figure 13] Combined administration of the radioprotective / radiomititive combination PT+SIL+NR+FSL-1 (PSNF) and X-rays to A) growing human A549 (lung adenocarcinoma) and B) MDA-MB-231 (triple-negative breast cancer) xenografts. In all cases, 5 mice per group were used. [Figure 14]Figure 1. Effect of radioprotectors and radiomitigators on cellular NAD+ content, DNA damage, and hematopoietic recovery in γ-irradiated mice. A) Effect of FK866, a specific noncompetitive inhibitor of PT, SIL, NR, and nicotinamide phosphoribosyltransferase (NAMPT), on NAD+ content in cells isolated from γ-irradiated mice (n=5 per group, cell type, and treatment). FK866 was administered in vivo (15 mg / kg, intraperitoneal injection twice daily for 6 days starting 3 days before irradiation, see Figure 11a). FK866 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; *comparing all groups to control; +PT+SIL+NR+γ-ray or PT+SIL+NR+FK866+γ-ray to mice treated with γ-ray alone). B) Effect of FK866 on cellular NAMPT activity (P<0.01; *comparing FK866-treated mice to control, n=5). Mice were treated with inhibitors for 6 days as in (a) above. C) Effect of PT, SIL, NR, and FK866 on DNA damage in cells isolated from γ-irradiated mice (n=5 per group, cell type, and treatment). Statistical analysis was performed using Student's t-test (P<0.01; *comparing all groups to γ-irradiated control; +γ-irradiated and FK866 present groups to their respective controls). Mice were treated and cells isolated as in (a) above. D) Effect of PT, SIL, NR, and FSL-1 on hematopoietic recovery in γ-irradiated mice. Hematopoietic colony forming cells CFU-GM (colony forming units-granulocyte, macrophage) and CFU-GEMM (colony forming units-granulocyte, erythrocyte, macrophage, megakaryocyte) were quantified in femurs excised from control and treated mice (n=5 per group and treatment). Mice were treated as in Fig. 11a and bone marrow cells were isolated 7 days after irradiation.Statistical analysis was performed using Student's t-test (P<0.01; *comparing all groups to control; +comparing different treatments + gamma irradiation to gamma irradiation alone). [Figure 15]Figure 11 shows the effect of treatment with pterostilbene and silibinin on Nrf2-, NF-κB-, PARP1-, and PGC1α-dependent mechanisms. Immediately prior to irradiation, intestinal epithelial cells were isolated from mice undergoing the protocol shown in Figure 11a. A) Western blots to detect p65NF-κB, Nrf2, PARP1, and Sirt1 in nuclear extracts, Sirt3 in mitochondrial extracts, and phosphorylated PGC-1α in total extracts. Densitometric analysis (Western blots) represents the mean ± SD for 5 or 6 different mice per molecule and experimental condition (P<0.01; *Comparing mice treated with PT, SIL, or PT+SIL to mice treated with saline, PS, +Comparing mice treated with PT+SIL to mice treated with PT, #Comparing mice treated with PT+SIL to mice treated with SIL). B) Potential molecular interactions and interrelationships between different signaling mechanisms. Ub, ubiquitin; IkB, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor; Keap1, Kelch-like ECH-associated protein 1; RE, response element; ARE, antioxidant response element; MAF, musculoaponeurotic fibrosarcoma oncogene homolog; Ac, acetylation; IMF, inflammatory mediator. C) Gene silencing of specific signaling-related molecules. The figure shows a representative experiment for n=5 or 6 different experiments and experimental conditions. PS, saline. D) Isolated IECs were cultured for 24 h in the presence or absence of specific siRNAs and then irradiated with gamma radiation (same dose as used for in vivo experiments). Cell death was analyzed 12 h after gamma irradiation in isolated and cultured IEC cells as described in Methods. PT (20 μM) and SIL (15 μM) were present in cultures containing IECs isolated from mice treated with PT+SIL. This allowed the effects induced by in vivo treatment to be retained under in vitro conditions.Data are means ± SD for 4 or 5 different experiments per molecule and experimental condition (p<0.01; *comparing treatment with a particular siRNA to control (treatment with vehicle) under PS or under PT+SIL; p<0.01; +comparing data under PT+SIL to their equivalent data under PS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Brief description of the invention In one embodiment, the present invention relates to a composition comprising: a) pterostilbene or a salt thereof; and b) silibinin or a salt thereof. Preferably, the composition comprises: a) NAD + a) a booster; and / or b) at least one radiation mitigating compound selected from fibroblast stimulating lipopeptide-1, or any salt thereof.

[0010] Preferably, the composition further comprises at least two or more radiation mitigation compounds, wherein the at least two or more radiation mitigation compounds are NAD + a booster and fibroblast stimulating lipopeptide-1, or any salt thereof.

[0011] Preferably, the pterostilbene is pterostilbene phosphate disodium salt, and / or the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt. + The booster is nicotinamide riboside.

[0012] Preferably, the composition is a pharmaceutical composition, a nutraceutical composition, a cosmetic composition, or a food composition.

[0013] In a further aspect, the present invention relates to pterostilbene or a pharma- ceutically acceptable salt thereof in combination with silibinin or a pharma- ceutically acceptable salt thereof for use in the prevention, amelioration or alleviation of diseases induced by ionizing radiation in a subject, wherein administration is carried out before, after or during radiation exposure, and wherein the combination is understood to be the administration of a) pterostilbene or a pharma- ceutically acceptable salt thereof and b) silibinin or a pharma- ceutically acceptable salt thereof to said subject, either simultaneously or sequentially in any order.

[0014] Preferably, pterostilbene or a pharmaceutically acceptable salt thereof and silibinin or a pharmaceutically acceptable salt thereof are administered in a single pharmaceutical composition or in separate pharmaceutical compositions.Preferably, the administration of pterostilbene or a pharmaceutically acceptable salt thereof and silibinin or a pharmaceutically acceptable salt thereof is topical, oral, or parenteral.

[0015] Preferably, pterostilbene or a pharma- ceutically acceptable salt thereof and silibinin or a pharma-ceutically acceptable salt thereof are administered simultaneously or sequentially several times before and / or after radiation exposure, or both. Preferably, the combination is present in a dosage capable of providing a radioprotective effect.

[0016] Preferably, the use further comprises administration in combination with at least one or more radiation mitigating compounds, where combination is understood as simultaneous or sequential administration to the subject of a) pterostilbene or a pharma- ceutically acceptable salt thereof, b) silibinin or a pharma- ceutically acceptable salt thereof, and c) at least one or more radiation mitigating compounds or any pharma- ceutically acceptable salt thereof.

[0017] Preferably, the at least one radiation mitigating compound is NAD + booster and / or fibroblast stimulating lipopeptide, or any pharma- ceutically acceptable salt thereof.

[0018] Preferably, the use further comprises administering in combination with at least two or more radiation mitigation compounds, wherein the at least two or more radiation mitigation compounds are NAD + and a fibroblast stimulating lipopeptide, or any pharma- ceutically acceptable salt thereof. + The booster is nicotinamide riboside.

[0019] Preferably, the disease induced by ionizing radiation is one associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage, more preferably acute radiation syndrome and / or chronic radiation syndrome.

[0020] Preferably, the ionizing radiation is gamma radiation or X-rays.

[0021] Preferably, the pterostilbene is pterostilbene phosphate disodium salt and / or the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

[0022] In a further aspect, the present invention relates to a non-therapeutic cosmetic use of a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, where the combination is understood to be a) pterostilbene or a salt thereof and b) silibinin or a salt thereof, administered simultaneously or sequentially in any order to said subject.Preferably, the use is for protecting, improving and / or reducing the harmful effects of sunlight on human skin and lips, such as age spots, sunburn, UV spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, pimples, blemishes, skin fragility, dryness, mottling, rough texture, cracking, sagging, thinning, enlarged pores, etc.

[0023] In a further aspect, the present invention relates to a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, which is used to increase the intracellular level of sirtuin in a patient in need of such an increase, and said increase is compared to the intracellular level of sirtuin in the same patient before treatment with the combination of pterostilbene or a salt thereof and silibinin or a salt thereof.Preferably, the patient in need of such an increase is a patient suffering from a neurodegenerative disorder, a cardiovascular disorder, a pulmonary disorder, a metabolic disorder, or an age-related disorder.Preferably, the disorder is selected from the group consisting of Alzheimer's dementia or other dementias, or age-related cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

[0024] In a further aspect, the present invention relates to a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, which is used as a nutraceutical composition, and the combination is understood to be a) pterostilbene or a salt thereof and b) silibinin or a salt thereof, which is administered to said subject simultaneously or sequentially in any order.Preferably, the use as a nutraceutical composition is for treating or preventing diseases induced by ionizing radiation, for treating or preventing neurodegenerative disorders, cardiovascular disorders, pulmonary disorders, metabolic disorders, or age-related disorders, and / or for protecting or repairing the harmful effects of sunlight on human skin or lips.Preferably, the diseases induced by ionizing radiation are related to DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage. Preferably, the neurodegenerative, cardiovascular, pulmonary, metabolic, or age-related disorder is selected from the group consisting of Alzheimer's or other dementias or age-associated cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes. Preferably, the harmful effects of sunlight on human skin or lips are selected from the group consisting of age spots, sunburn, sun spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discolouration, uneven skin tone, dullness, freckles, pimples, blemishes, fragile skin, dryness, mottling, roughness to the touch, cracking, sagging, thinning, enlarged pores, cellulite formation, acne formation, rosacea, psoriasis and eczema.

[0025] In a further aspect, the present invention relates to a kit of parts comprising at least two receptors comprising at least pterostilbene or a salt thereof, silibinin or a salt thereof, and optionally at least one or more radiation mitigation compounds. Preferably, the optional at least one or more radiation mitigation compounds are a) NAD + and / or b) a fibroblast stimulating lipopeptide or any pharma- ceutically acceptable salt thereof. +The booster is nicotinamide riboside. Preferably, the pterostilbene is pterostilbene phosphate disodium salt, and / or the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

[0026] Description of the Invention General definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0027] The term "about" when applied to a given amount or quantity is intended to include a deviation of plus or minus 5 percent.

[0028] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable except for the second element. The second option refers to the second element being applicable except for the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. It is also understood that two or more options may be simultaneously applicable and thus meet the requirements of the term "and / or."

[0029] Throughout this specification and the appended claims, unless the context otherwise requires, the word "comprise", as well as variants such as "comprises" and "comprising", will be understood to mean the inclusion of the specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced with the term "containing" or "including", or, as used herein, sometimes with the term "having". Any of the above terms (comprising, containing, including, having) may, but is less preferred, be replaced with the term "consisting of" whenever used herein in the context of an aspect or embodiment of the invention.

[0030] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0031] As used herein, the term "disease" is intended to be synonymous with, and is used interchangeably with, the terms "pathology," "disorder," and "pathology" (e.g., medical condition), in that all of these reflect an abnormal condition of the body or one of its parts that impairs normal functioning and is typically manifested by characteristic signs and symptoms.

[0032] A "therapeutically effective amount" as referred to herein is defined as that amount of a compound sufficient to effect such treatment of a conditional condition when administered to a subject. A "therapeutically effective amount" may vary depending on the compound and the condition being treated, the age and relative health of the subject, the route and form of administration, the judgment of the skilled artisan, and other factors.

[0033] The term "treatment" as used herein refers to medical care given to a patient for a disease or injury. This term includes not only curing the disease, but also improving, alleviating, or reducing the symptoms of said disease. Thus, the term "therapeutic treatment" or "treatment" as used herein refers to returning the body from a pathological condition or disease to its normal health state. The term "therapeutic treatment" or "treatment" as used herein also refers to alleviating, ameliorating, or reducing the harmful effects of radiation in a subject, or reducing in any way the symptoms associated with such effects. The term "prophylactic treatment" as used herein refers to preventing a pathological condition.

[0034] "Prevention" means preventing the onset of a disease. "Amelioration" means making a patient's disease better, or taking actions that endeavor to correct said disease or at least make it more tolerable. "Alleviation" or "palliation" means reducing the severity, severity, or pain of a disease.

[0035] The term "subject" as used herein refers to a mammalian subject. Preferably, the subject is selected from humans, companion animals, non-domestic animals, or zoo animals. For example, the subject may be selected from humans, dogs, cats, cows, pigs, sheep, horses, bears, etc. In a preferred embodiment, the mammalian subject is a human subject.

[0036] "Ionizing radiation" means radiation consisting of particles, x-rays, or gamma rays that have sufficient energy to cause ionization in the medium through which they pass. "Gamma rays" or "gamma radiation" means penetrating electromagnetic radiation resulting from the radioactive decay of atomic nuclei. It is composed of electromagnetic waves with the shortest wavelengths, and therefore gives the highest photon energy. "X-rays" or x-radiation means penetrating high-energy electromagnetic radiation. Most x-rays have wavelengths in the range of 10 picometers to 10 nanometers, which corresponds to frequencies in the range of 30 petahertz to 30 exahertz, and energies in the range of 124 eV to 124 keV.

[0037] "Pterostilbene" or PT means 3,5-dimethoxy-4'-hydroxystilbene, or its salts, solvates, isomers (enantiomers, diastereomers), hydrates, or any known derivatives, synthetic variants, and acceptable formulations (pharmaceuticals, cosmetics, nutraceuticals, etc.) thereof. Pterostilbene (PT) is a natural antioxidant and a natural analogue of resveratrol, but is an approximately 60-100 times more potent antifungal agent that exhibits similar anticarcinogenic properties. Pterostilbene is found primarily in blueberries and red grapes, although quantitative studies have revealed that only 1-2 parts t-pterostilbene is present for every 10 parts t-resveratrol. Pterostilbene has the formula: [ka] Corresponds to.

[0038] "Silibinin" or SIL, also known as silybin or silymarin (both from Silybum (milk thistle)), is a flavonoid obtained from two thistle species in the Asteraceae family. Silibinin includes its salts, solvates, isomers (enantiomers, diastereomers), hydrates, or any of its known derivatives, synthetic variants, and acceptable formulations (pharmaceuticals, cosmetics, nutraceuticals, etc.). The plant is native to the Mediterranean region of Europe, North Africa, and the Middle East. Silibinin (SIL) is the main active component of silymarin, a standardized extract of milk thistle seeds that contains a mixture of flavonolignans consisting of silibinin, isosilibinin, silychristin, silydianin, etc. Silibinin itself is a mixture of two diastereomers, silybin A and silybin B, in approximately equal molar ratios. SIL is also known as 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one, which has the following formula: [ka] has.

[0039] Nicotinamide Adenine Dinucleotide Booster, NAD + Precursor, or NAD + The booster is an NAD precursor, an activator of NAD synthesis, or an NAD booster, as specifically disclosed in the literature by Rajman et al., Cell Metab. 2018 March 06; 27(3): 529-547. doi:10.1016 / j.cmet.2018.02.011. + By nicotinamide is meant any compound, such as an inhibitor of the decomposition of nicotinamide, its salts, solvates, isomers (enantiomers, diastereomers), hydrates, or any of its known derivatives, synthetic variants, and acceptable formulations (pharmaceuticals, cosmetics, nutraceuticals, etc.).

[0040] "Fibroblast stimulating lipopeptide" or FSL-1 means a Toll-like receptor 2 / 6 agonist, or a salt, solvate, isomer (enantiomer, diastereomer), hydrate, or any known derivative, synthetic variant, and acceptable formulation (pharmaceutical, cosmetic, nutraceutical, etc.) thereof, having the formula: [ka] Corresponds to.

[0041] Nicotinamide riboside is nicotinamide adenine dinucleotide, or NAD + NR is a pyridine nucleoside similar to vitamin B3 that functions as a precursor to [ka] It is.

[0042] As used herein, the term "kit of parts" refers to a combination preparation in which the active ingredients are physically separated for use in combination therapy by simultaneous or sequential administration to a patient.

[0043] Detailed Description Some natural compounds are known to have radioprotective effects when administered to subjects prior to radiation exposure. As shown in Figure 2A, compounds such as pterostilbene (PT), gallic acid, EGCG, etc., result in nearly 100% survival rate 6 days after radiation exposure in Swiss albino mice. However, some compounds are unable to protect mice for a long period of time. For example, after 30 days (Figure 2B), the survival rate percentage of luteolin dropped to about 40%, and after 60 days (Figure 2C), the survival rate percentage was 0%. Therefore, compounds that can exert a high level of protection for a long period of time are desired.

[0044] Although these compounds can be combined and administered together, the radioprotection provided by the combination cannot be predicted. In some cases, the combination of one compound with another does not change the radioprotective properties of the compound. For example, as shown in FIG. 2C, administration of PT results in a 40% mouse survival rate 60 days after radiation. Delphinidin results in approximately 12% survival rate. However, when PT is combined with delphinidin, the survival rate obtained remains approximately 40%, which is almost the same as the survival rate provided by the PT compound alone. In this case, the effect of delphinidin is not added to the effect of PT.

[0045] In other cases, and surprisingly, the radioprotective effect of one compound can be adversely affected by the coadministration of another compound. This is shown in Figure 2C, where the radioprotective effect of PT results in a 40% survival rate after 60 days, while gallic acid provides approximately 5% protection. However, combining both compounds results in an overall survival rate of approximately 35%, providing a reduced radioprotective effect compared to administration of PT alone.

[0046] In other cases, when two compounds are combined, their radioprotective effects may add up, resulting in a protection that is the sum of their individual effects. For example, in Figure 2C, it is shown that the protection provided by PT is approximately 40%, and the protection provided by genistein is approximately 7%. Therefore, when both compounds are combined, the protection obtained is approximately 45%, which means that both compounds are exerting their effects and the overall protection is more or less the sum of the effects of both compounds.

[0047] More interestingly, the authors of the present invention have surprisingly found that not only can the combination of two compounds not produce altered or harmful or cumulative protection, but also that in very rare cases, the combination of two compounds can produce enhanced radioprotective properties beyond the mere accumulation of their effects. That is, a specific combination of certain compounds produces enhanced protection that cannot be explained by the accumulation of their individual effects, which means that there is a synergistic effect between them. This is shown in Figure 2B and Figure 2C. In Figure 2C, silibinin (SIL) produces approximately 10% survival rate and PT produces approximately 40% survival rate, but the combination of both of them produces a survival rate of over 70%. This also clearly shows that the combination of SIL and PT does not simply produce an additive effect of their radioprotective properties, but produces a synergistic effect that produces enhanced radioprotection.

[0048] These findings suggest that care should be taken with the compounds that are combined to achieve enhanced radioprotection, as some combinations may enhance radioprotection while others may be unfavourable and may suggest the recommendation to administer the compounds individually.

[0049] Therefore, to achieve enhanced radioprotection, compounds with different radioprotective properties cannot simply be combined randomly, since the combination will not necessarily result in enhanced overall radioprotection. Depending on which compounds are co-administered, the resulting radioprotection may be worse, the same, or the sum of the two compounds, but combining some compounds together will result in synergistic effects, providing enhanced protection that would not be derived from the simple summation of their radioprotective properties.

[0050] Another observation made by the present inventors is that certain combinations of PT and SIL provide long-term protection. This is surprising because, as mentioned above, not all compounds exert radioprotective effects beyond 30 days. See, for example, EGCG, quercetin, naringerin, or phlorizin compounds (30 days) in FIG. 2B. Here, their radioprotective effects drop to 0% after 60 days (FIG. 2C). However, as shown in FIG. 3, the combination of PT and SIL provided approximately 25% survival rate (5 mice out of 20 alive) at 360 days, i.e., one year. Thus, the combination of PT and SIL not only provides synergistic enhancement of protective effect, but this effect is also continuous. Furthermore, as shown in FIG. 11B, when PT and SIL were combined with other radiomitigation compounds such as nicotinamide riboside (NR) or fibroblast-stimulating lipopeptide-1 (FSL-1), the survival rate after 1 year was further increased by up to 50%-60%, and notably, when four compounds (PT, SIL, NR, and FSL-1) were combined, they provided almost complete protection (90%) of the animals after 360 days.

[0051] In conclusion, the present invention provides two main findings: on the one hand, it has been found that the simultaneous administration of PT and SIL, two natural compounds with radioprotective properties, results in an enhanced, sustained and synergistic radioprotection that does not result from the mere accumulation of their corresponding radioprotective effects, and on the other hand, it has been found that the combination of these two compounds with radiomitigating compounds such as NR and / or FSL-1 can further enhance the protection against radiation over a long period of time, even up to 90% survival rate over one year.

[0052] To study the synergistic effect of SIL and PT in detail, the authors further investigated the potential signaling pathways that may be involved in the protective effect caused by the combination of PT+SIL. As shown in Figure 15a, compared to the control, both PT and SIL increased nuclear Nrf2 while decreasing nuclear NF-κB levels. Overall, these effects suggest a cooperative effect of promoting antioxidant defense while downregulating NF-κB-dependent proinflammatory responses. pPGC-1α was also increased by PT (Figure 15a). Interestingly, SIL alone had no effect on Sirt1 levels, but the combination of the two polyphenols increased Sirt1 levels more than PT alone (Figure 15a). These results lead to the conclusion that the synergistic effect of PTE and SIL is based on the cooperative effect that both compounds have in mitigating oxidative stress and DNA damage occurring in cells, paving the way for exploiting compositions containing both compounds for use in the fields of beauty, nutrition, nutraceuticals, and medicine.

[0053] Pterostilbene for use in the prevention, amelioration, or alleviation of diseases induced by ionizing radiation - Patents.com A first aspect of the present invention relates to a stilbenoid, preferably resveratrol or pterostilbene or a pharma- ceutically acceptable salt or derivative thereof, in combination with silibinin or a pharma- ceutically acceptable salt or derivative thereof, for use in the treatment, prevention, amelioration, or alleviation of a disease induced by ionizing radiation in a subject. Preferably, the stilbenoid used is pterostilbene or a pharma- ceutically acceptable salt thereof. Thus, in a first aspect, the present invention provides pterostilbene or a pharma- ceutically acceptable salt thereof in combination with silibinin or a pharma- ceutically acceptable salt thereof for use in treating, preventing, improving, or alleviating a disease induced by ionizing radiation in a subject, wherein administration is performed before, after, or during irradiation, and the combination is understood to be administration of a) pterostilbene or a pharma- ceutically acceptable salt thereof and b) silibinin or a pharma- ceutically acceptable salt thereof to said subject, either simultaneously or sequentially, in any order. In a particular embodiment, the disease induced by ionizing radiation is associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage. In a preferred embodiment, the disease induced by ionizing radiation is acute radiation syndrome and / or chronic radiation syndrome. In another embodiment, the disease is selected from the group consisting of skin disease, acute radiation syndrome, cancer, and cardiovascular disease.

[0054] In a first aspect of the present invention, pterostilbene is used in combination with silibinin. PT and SIL may be administered in a single composition or in separate compositions.

[0055] In a preferred embodiment, the pterostilbene is pterostilbene phosphate disodium salt. In another preferred embodiment, the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

[0056] As described above, PT and SIL, or pharma- ceutically acceptable salts or derivatives thereof, are administered in combination to prevent, ameliorate, or reduce ionizing radiation-induced disease in a subject. In a preferred embodiment, the subject is a mammal. More preferably, the subject is a human.

[0057] Dosage regimen and route of administration The administration can be simultaneous (at the same time) or sequential. As used herein, "sequential administration" refers to administration of the SIL or a composition comprising it shortly before or after administration of the PT or a composition comprising it. As used herein, "shortly before or after" is understood to mean within an interval of 1 hour to 24 hours, preferably 1 hour to 12 hours, preferably 1 hour 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, the PT is administered first, followed by the SIL. In another embodiment, the SIL is administered first, followed by the PT. In a preferred embodiment, administration of the PT and the SIL is simultaneous. In one embodiment, the PT and the SIL are administered simultaneously in combination, either as part of a single composition or as part of two different compositions.

[0058] In one embodiment, the pharma- ceutically acceptable compounds and compositions are formulated to be compatible with the intended route of administration. Methods for achieving administration are known to those skilled in the art. This includes, for example, parenteral injection, such as intravascular, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, etc., as well as oral, topical, nasal, intraocular, rectal, or local. In particular, sustained release administration, such as by means of depot injection or erodible implants, is also contemplated. The preferred route of administration is parenteral, which is understood herein as administration in one or more blood vessels, typically including intravenous or intraarterial administration. Currently, the most preferred routes of administration are parenteral, oral, or topical.

[0059] Administration can be performed once or can be performed more than once. In preferred embodiments, the compounds are administered several times. In certain embodiments, administration can include at least one, two, three, four, five, six, seven or more doses.

[0060] Administration can be performed before, during, or after radiation. For example, administration can be performed at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days before radiation is administered. In another embodiment, administration is performed after radiation. In one embodiment, administration after radiation is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 a particular embodiment, administration is performed either on the same day radiation is administered, before radiation is administered, or after radiation is administered. In one embodiment, administration consists of several doses administered before radiation is administered, on the same day radiation is administered, and / or within a few days after radiation is administered. In a preferred embodiment, pterostilbene and silibinin, or any pharma- ceutically acceptable salt thereof, are administered several times, either simultaneously or sequentially, before and / or after radiation, or both.

[0061] Dosage The optimal dose will be selected according to the route of administration, treatment plan, and / or administration plan, taking into account existing toxicity and efficacy data. In a preferred embodiment, the PT and SIL used according to the first aspect of the present invention are administered at a dose that can produce a radioprotective effect. As defined above, "radioprotection" is understood as the ability to reduce the direct damage caused by radiation. Therefore, in the present invention, the dose of the PT and SIL is not particularly limited.

[0062] Furthermore, it will be clear to those skilled in the art to convert the doses applied in the mouse model disclosed herein into equivalent doses applied in humans. To this end, those skilled in the art can refer to well-known general practice guides for dose conversion between animals and humans. For example, see Nair AB, Jacob S., "A simple practice guide for dose conversion between animals and humans." J Basic Clin Pharma 2016;7:27-31. Briefly, the therapeutic dose in the mouse model can be converted to humans by dividing it by about 12.3. That is, if a dose of 100 mg / kg exhibits a therapeutic effect in mice, the human equivalent dose is 8.13 mg / kg.

[0063] In one embodiment, PT or a pharma- ceutically acceptable salt thereof is administered at a dosage of up to 500 mg / kg. PT can be administered at a dosage ranging from 300 mg / kg to 30 mg / kg. Preferably, PT is administered at a dosage of 200 mg / kg to 40 mg / kg, or 150 mg / kg to 40 mg / kg. More preferably, PT is administered at a dosage of 100 mg / kg. In one embodiment, PT or a pharma- ceutically acceptable salt thereof is administered to a human at a dosage of up to 40.65 mg / kg. PT can be administered to a human at a dosage ranging from 24.39 mg / kg to 2.43 mg / kg. Preferably, PT is administered to a human at a dosage of 16.2 mg / kg to 3.25 mg / kg, or 12.19 mg / kg to 3.25 mg / kg. More preferably, PT is administered to a human at a dosage of 8.13 mg / kg.

[0064] In another embodiment, SL or a pharma- ceutically acceptable salt thereof is administered at a dose of up to 500 mg / kg. SIL may be administered at a dose ranging from 200 mg / kg to 35 mg / kg. Preferably, SIL 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 its human equivalent. In another embodiment, SL or a pharma- ceutically acceptable salt thereof is administered to a human at a dose of up to 40.65 mg / kg. SIL may be administered to a human at a dose ranging from 16.26 mg / kg to 2.84 mg / kg. Preferably, SIL 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 its human equivalent.

[0065] Administration of Further Compounds In one embodiment of the first aspect, the use described in any of the above embodiments further comprises administration in combination with at least one further radiation mitigating compound, where the use described herein is understood as herein as the simultaneous or sequential administration to said subject of a) pterostilbene or a pharma- ceutically acceptable salt thereof, b) silibinin or a pharma-ceutically acceptable salt thereof, and c) one or more radiation mitigating compounds. In a preferred embodiment, the at least one radiation mitigating compound is a nicotinamide adenine dinucleotide booster (hereinafter, NAD + The term "FSL-1" refers to a fibroblast stimulating lipopeptide (hereinafter referred to as booster), and / or fibroblast stimulating lipopeptide-1 (hereinafter referred to as FSL-1), or any pharma- ceutically acceptable salt thereof. In the context of the present invention, FSL-1 is preferred, but it should be noted that since FSL-1 is a type of fibroblast stimulating lipopeptide, it can be replaced by any other fibroblast stimulating lipopeptide. Thus, the term "FSL-1" used in any of the embodiments of the present invention can be replaced by any other suitable fibroblast stimulating lipopeptide.

[0066] As defined above, a "radiation mitigating agent" is a compound that can minimize toxicity even after radiation has been administered. Thus, in one embodiment of the first aspect, the administration of a combination of PT and SIL further comprises the administration of one or more radiation mitigating agents. The combination between PT, SIL, and one or more radiation mitigating agents is not limited, i.e., PT can be administered together with SIL and one or more radiation mitigating agents in a single composition, or they can be administered separately in different compositions. All possible combinations of the three (or more) compounds are included herein.

[0067] The above administration routes and dosing schedules also apply when PT and SIL are administered together with one or more radiomitigating compounds.

[0068] In a preferred embodiment, the use of a combination of PT and SIL, their salts or derivatives thereof in the prevention, amelioration, or alleviation of a disease induced by ionizing radiation in a subject is based on the incorporation of NAD + It further includes administration of a booster.Thus, in this embodiment, at least three compounds are administered together, which may be in the same or different compositions, and which may be administered sequentially or simultaneously.

[0069] In another preferred embodiment, the use of a combination of PT and SIL, their salts or derivatives thereof in preventing, ameliorating or reducing ionizing radiation induced disease in a subject further comprises the administration of FSL-1.Thus, in this embodiment, at least three compounds are administered together, which may be contained in the same or different compositions, and which may be administered sequentially or simultaneously.

[0070] In an even more preferred embodiment, the use of a combination of PT and SIL, their salts or derivatives thereof in preventing, ameliorating or alleviating a disease induced by ionizing radiation in a subject is in combination with NAD +It further includes administration of both a booster and FSL-1.Thus, in this embodiment, the four compounds may be contained in the same or different compositions and may be administered sequentially or simultaneously.

[0071] In a more preferred embodiment, NAD + The booster is nicotinamide riboside or 1-(β-D-ribofuranosyl)nicotinamide (hereinafter referred to as NR), a pharma- ceutically acceptable salt or derivative thereof.

[0072] In an even more preferred embodiment, the use of a combination of PT and SIL, their salts or derivatives thereof in the prevention, amelioration or reduction of ionizing radiation induced disease in a subject further comprises the administration of NR and FSL-1.Thus, in this embodiment, the four compounds are administered together in one or more doses, contained in the same or different compositions, administered sequentially or simultaneously before, during or after radiation.

[0073] In a preferred embodiment, PT and SIL are administered once a day for several days before and / or several days after radiation. More preferably, PT and SIL are administered daily for 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably 5 days before radiation. In another embodiment, PT and SIL are administered daily from before radiation until up to 5, 4, 3, 2, 1 days after radiation. Preferably, PT and SIL are administered daily for the preceding 5 days before radiation and 2 days after radiation.

[0074] In another embodiment, NAD + The booster is administered on the same day of radiation and once daily for the next month following radiation. + Boosters are administered at least 60 minutes after radiation, with further administrations during the next 30 days following radiation.

[0075] In another embodiment, FSL-1 is administered only once, preferably after radiation, more preferably at least one day after radiation.

[0076] In one embodiment, NAD + The booster or a pharma- ceutically acceptable salt thereof is administered at a dose of up to 500 mg / kg. + The booster or a pharma- ceutically acceptable salt thereof is administered at a dose of 400 mg / kg to 100 mg / kg. + The booster is administered at a dose of 300 mg / kg to 150 mg / kg, or 200 mg / kg to 150 mg / kg. More preferably, NAD + The booster is administered at a dosage of 185 mg / kg. + The booster or a pharma- ceutically acceptable salt thereof is administered to a human at a dose of up to 40.65 mg / kg. + The booster or a pharma- ceutically acceptable salt thereof is administered to a human at a dose of 32.52 mg / kg to 8.13 mg / kg. + The booster 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 booster 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. + The booster is administered to humans at a dose of 15.04 mg / kg.

[0077] In another embodiment, NAD + The booster is nicotinamide riboside or any pharma- ceutically acceptable salt thereof, administered at a dose of 400 mg / kg to 100 mg / kg. Preferably, nicotinamide riboside is administered at a dose of 300 mg / kg to 150 mg / kg, or 200 mg / kg to 150 mg / kg. More preferably, nicotinamide riboside is administered at a dose of 185 mg / kg. In another embodiment, NAD +The booster is nicotinamide riboside or any pharma- ceutically acceptable salt thereof, which is administered to the human at a dose of 32.52 mg / kg to 8.13 mg / kg. Preferably, the nicotinamide riboside is administered to the 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 nicotinamide riboside is administered to the human at a dose of 15.04 mg / kg.

[0078] In one embodiment, FSL-1 or a pharma- ceutically acceptable salt thereof is administered at a dose of up to 1 mg / kg. In another embodiment, the FSL-1 compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof is administered at a dose of up to 0.08 mg / kg. In another embodiment, the FSL-1 compound or a pharma- ceutically 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 at a dose of 0.020 mg / kg.

[0079] In one embodiment, the ionizing radiation is gamma (γ) radiation or X-rays.

[0080] Composition containing PT and SIL In a second aspect, the present invention relates to a composition comprising a) a stilbenoid, preferably resveratrol or pterostilbene or a salt or derivative thereof, and b) silibinin or a salt or derivative thereof. Preferably, the stilbenoid is pterostilbene or a salt thereof. Thus, the second aspect of the present invention provides a composition comprising a) pterostilbene or a salt thereof, and b) silibinin or a salt thereof. Preferably, the composition is a pharmaceutical composition, a nutraceutical composition, a cosmetic composition, or a food composition. For the purposes of the present invention, the term nutraceutical refers to a "food-drug", i.e. a health food that associates nutritional ingredients with the healing properties of natural active ingredients (in this case PT and SIL) whose efficacy has been proven and recognized.

[0081] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like, and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. If peptides, proteins, or other similar molecules are to be included in the vaccine composition, it is also preferred that the formulation includes pharma-ceutically acceptable excipients that act as stabilizers, especially for these. Examples of suitable carriers include, but are not limited to, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like. 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 thorough discussion of pharma- ceutically acceptable excipients, additives, and carriers is available in Remington's Pharmaceutical Sciences (22nd ed., 2012).

[0082] Various options for pharmaceutical formulations, structural modifications, and delivery systems can help facilitate oral administration of PTEs and SILs, and are also included herein. Some examples include prodrugs (carboxy esters, sulfonates, sulfates, phosphates, acetals, or carbamates or carbonates), nanoemulsions, polyionic / polymer shells encapsulating nanoparticles, liposomes, or exosomes. In particular, any pharma- ceutically acceptable formulation of PTEs and SILs is included herein.

[0083] The one or more compositions according to the second aspect comprise PT and SIL in dosage amounts as defined in the first aspect of the invention and are administered according to the routes of administration and dosing regimens as defined in the first aspect of the invention. Further details and preferred embodiments regarding the dosage amounts, routes of administration and dosing regimens of the compositions are also provided in the first aspect of the invention.

[0084] The composition comprises PT and SIL, or any salt or derivative thereof, and may further comprise one or more radiomitigating compounds as defined in the first aspect of the invention. As mentioned above, in one embodiment, the radiomitigating compound is NAD + It may be a booster and / or FSL-1. In a preferred embodiment, the composition comprising PT and SIL further comprises at least two radiation mitigating compounds, preferably NR and FSL-1.

[0085] Preferably, the one or more compositions are in a form suitable for parenteral, oral or topical administration.In a preferred embodiment, the composition is an aqueous composition, more preferably a stable aqueous composition.As used herein, "stable composition" may refer to a formulation in which the active ingredient therein essentially retains its physical stability and / or chemical stability and / or biological activity during storage.

[0086] The one or more compositions of the second aspect may be a food composition. The food composition may be a liquid composition or a solid composition. In the context of the present invention, such liquid compositions include, but are not limited to, any beverage selected from the group consisting of animal or plant milk and any derivatives thereof, such as milkshakes, yogurt, kefir, etc., fruit juices and / or vegetable juices, still or carbonated water, or flavored or sweetened water or beverages (with nutritive sweeteners (sucrose, fructose, etc.) or artificial sweeteners), seasonings, any type of alcoholic beverage, tea, coffee, and any type of refreshing or soft drink or nutritional supplement beverage.

[0087] The food composition may also be a solid composition. Such a solid composition may be selected from the group consisting of, for example, but not limited to, cheese, butter, margarine, and animal or vegetable milk derivatives such as tofu, bread of any kind, including fresh, packaged or frozen bread, sliced ​​bread, wholemeal bread, spiced bread, sweet bread, savory bread, etc., pasta (macaroni, spaghetti, noodles, etc.) made from any flour, such as wheat flour or semolina, baked goods, loose or sachet infusions for the preparation of beverages, tea or coffee, jellies, candies, including gummy candies, better known as "soft fruit candies", and solid seasonings of any kind, or mixtures thereof.

[0088] Finally, the food composition may also be a nutritional, dietary or food supplement or supplement. These terms are usually used for compositions taken orally that contain ingredients intended to supplement the diet, in the present case PT and SIL, or any of their salts or derivatives. They never replace conventional foods or become the sole component of a diet or meal. The composition may also be a nutraceutical composition. This composition may further be used in the preparation of functional foods or foods that have additional functionality upon the addition of a combination of PTE and SIL, as defined throughout this specification. The nutritional, dietary or nutraceutical composition, or food supplement or functional food, may be found in various expressions such as pastilles, pills, tablets, capsules, soft gelatin capsules, gelatin capsules, wafers, effervescent tablets, liquids (solutions, suspensions, syrups), granules and powders. All of these are included as specific embodiments within the scope of the present invention. Nutritionally or pharmaeutically acceptable excipients will be clear to the skilled person for obtaining any of the above expressions, and these are included within the scope of the present invention.

[0089] The present invention further relates to a composition as defined above for use as defined in the first aspect of the invention.

[0090] Kit of parts including PT and SIL In a third aspect, the present invention relates to a kit of parts comprising at least a) at least two receptors comprising a stilbenoid, preferably resveratrol or pterostilbene and silibinin or any salt or derivative thereof, and optionally at least one or more radiation mitigating compounds as defined above. Preferably, the stilbenoid comprised in the kit of parts is pterostilbene.

[0091] Typically, the two or more components are formulated and provided as a composition and provided in two or more separate receptacles. For example, the PT and SIL can be combined in one receptacle and the radiation mitigating agent in another receptacle. In one embodiment, each compound is contained in a separate receptacle. In a further embodiment, the radiation mitigating agent compound is present in the same receptacle as the radiation mitigating agent compound.

[0092] Preferably, said composition is used as defined in the previous aspect of the invention, preferably in the therapeutic treatment of diseases induced by ionizing radiation.

[0093] Methods for preventing, ameliorating, or reducing disease induced by ionizing radiation In a fourth aspect, the present invention relates to a method for preventing, treating (including ameliorating or alleviating) a disease induced by ionizing radiation in a subject, comprising administering to the subject a combination of a stilbenoid, preferably resveratrol or pterostilbene or a pharma-ceutically acceptable salt or derivative thereof, and silibinin or any pharma-ceutically acceptable salt or derivative thereof. Preferably, the stilbenoid is pterostilbene. Thus, in a fourth aspect, the present invention provides a method for preventing, treating (including ameliorating or alleviating) a disease induced by ionizing radiation in a subject, comprising administering a combination of pterostilbene and silibinin or any pharma- ceutically acceptable salt thereof before, after or during radiation exposure, where combination is understood to mean administering to said subject simultaneously or sequentially a) pterostilbene or a pharma- ceutically acceptable salt thereof, b) silibinin or any pharma-ceutically acceptable salt thereof, and optionally c) at least one or more radiation mitigating compounds, as defined throughout the present specification. As described above in the first and second aspects of the present invention, the at least one or more radiation mitigating compounds may be NAD + It may be a booster and / or FSL-1.

[0094] This fourth aspect is related to the first and second aspects above. Accordingly, the compounds and pharmaceutical compositions, dosages, routes of administration and dosing regimens provided in the previous aspects of the invention apply here.

[0095] Combination of pterostilbene and silibinin for use in the nutraceutical field or as a nutraceutical composition In a fifth aspect, the present invention further relates to the use of a combination of stilbenoid, preferably resveratrol or pterostilbene, and silibinin or any salt or derivative thereof, or the use of the composition according to the second aspect, in the field of nutraceuticals or as nutraceutical compositions.Preferably, the stilbenoid used is pterostilbene.Thus, the fifth aspect provides a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, which is used in the field of nutraceuticals or as nutraceutical compositions, wherein the combination is understood to be a) pterostilbene or a salt thereof and b) silibinin or a salt thereof, which is administered to said subject simultaneously or sequentially in any order.

[0096] In a preferred embodiment the use as a nutraceutical composition 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 disorders and / or for the protection or repair of the harmful effects of sunlight on the human skin or lips. The term "repairing the harmful effects of sunlight on human skin or lips" is used herein to refer to preventing, reversing, improving, reducing and / or alleviating defects, imperfections or aesthetically undesirable conditions of the skin, including, but not limited to, age spots, sun spots, sun spots, UV spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, pimples, blemishes, fragile skin, dryness, mottling, roughness to the touch, cracking, sagging, thinning, enlarged pores, cellulite formation, acne formation, rosacea, psoriasis and eczema. The term "skin" includes the skin of the face or body.

[0097] Preferably, the ionizing radiation induced disease is associated with DNA damage, radiation induced cytotoxicity, genotoxicity, and / or oxidative cell damage. More preferably, the ionizing radiation induced disease is selected from the group consisting of acute radiation syndrome and / or chronic radiation syndrome.

[0098] Preferably, the nutraceutical composition is used in the treatment of a disorder selected from the group consisting of Alzheimer's or other dementias or age-associated cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

[0099] This fifth aspect is related to the first and second aspects above. Accordingly, the compounds and pharmaceutical compositions, dosages, routes of administration and dosing regimens provided in the previous aspects of the invention apply here.

[0100] Combination of pterostilbene and silibinin for non-therapeutic use as a cosmetic composition In the sixth aspect, the present invention further relates to the non-therapeutic cosmetic use of the combination of stilbenoid, preferably resveratrol or pterostilbene, and silibinin 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 stilbenoid used is pterostilbene.Therefore, the sixth aspect of the present invention provides a combination of pterostilbene or a salt thereof and silibinin or a salt thereof for non-therapeutic cosmetic use or as a cosmetic composition, wherein the combination is understood to be a) pterostilbene or a salt thereof and b) silibinin or a salt thereof administered to said subject simultaneously or sequentially in any order.

[0101] As used herein, the expression "non-therapeutic cosmetic use" refers to a method used to improve the appearance of a person, i.e. to beautify the appearance. Cosmetic use does not include surgical or therapeutic treatment. Cosmetic care includes topical application of PTE and SIL to the skin area to be treated. Preferably, the cosmetic use of the composition has a non-therapeutic purpose of cleaning, beautifying, adding attractiveness, changing the appearance, or maintaining or promoting good condition of the skin or hair. The purpose may also be whitening, minimizing the appearance of wrinkles on the face and body, and protecting against sunlight and solar radiation. In one embodiment, the non-therapeutic cosmetic use is to prevent, reduce, and / or improve the natural process of skin aging.

[0102] Preferably, the non-therapeutic use is to protect against, improve and / or reduce the harmful effects of sunlight on human skin and lips, including, but not limited to, age spots, sun spots, sun spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, pimples, blemishes, fragile skin, dryness, mottling, roughness to the touch, cracking, sagging, thinning, enlarged pores. The term "skin" includes the skin of the face or body.

[0103] This sixth aspect relates to the first and second aspects above. Thus, the compounds and compositions, dosages, routes of administration and dosing schedules suitable for cosmetic use and provided in the previous aspects of the invention also apply herein.

[0104] Combination of pterostilbene and silibinin used to increase intracellular sirtuin levels Based on the data reported in Figures 5-8, it was found that PT+SIL exerted protection against induced oxidative damage. Moreover, as shown in Figure 15, this mechanism was found to be directly related to the synergistic increase caused by PTE+SIL in Sirtuin (Sirt) levels, especially Sirt1. Sirtuins are a family of signaling proteins involved in metabolic regulation to control many important functions and also involved in several pathologies such as metabolic, cardiovascular, pulmonary, neurodegenerative, and age-related disorders, as well as cancer. Sirtuins have been found to be modulators of inflammation and autophagy. A growing body of evidence suggests that compounds that activate or increase sirtuins show promise as therapeutic approaches to treat metabolic dysfunction, cancer, and age-related diseases, strongly supporting the use of a combination of PTE and SIL for use in the treatment or prevention of the above diseases. In particular, several studies have revealed the role of sirtuins in neuroprotection, especially in neurodegenerative disorders such as Alzheimer's dementia or other dementias or age-related cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), amyotrophic lateral sclerosis, and other neuro-related disorders (Kim D, et al., "SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis." EMBO J. 2007;26(13):3169-3179; Donmez G et al., "SIRT1 protects against α-synuclein aggregation by activating molecular chaperones." 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 specific HDACs 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 activation confers neuroprotection in experimental optic neuritis." Invest Ophthalmol Vis Sci. 2007;48(8):3602-3609; Montie HL, et al., "SIRT1 modulates aggregation and toxicity through deacetylation of the androgen receptor in cell models of SBMA." J Neurosci. 2011;31(48):17425-17436.

[0105] Considering this, in the seventh aspect, the present invention is a combination of stilbenoid, preferably resveratrol or pterostilbene, and silibinin or any salt or derivative thereof, or a composition according to the second aspect, used to increase the intracellular level of sirtuin in a patient who needs to increase, and said increase is compared with the intracellular level of sirtuin in the same patient before treatment with the combination of stilbenoid, preferably resveratrol or pterostilbene, and silibinin or any salt thereof, or before treatment with the composition according to the second aspect.Preferably, the stilbenoid is pterostilbene.Preferably, the sirtuin whose intracellular level is increased is Sirt1, Sirt2, or Sirt3, most preferably Sirt1. The increase in intracellular sirtuin levels compared to untreated patients is preferably a statistically significant increase and can be measured by any suitable technique aimed at measuring the amount of a protein, such as, but not limited to, the Bradford assay, antibody-based assays such as colorimetric protein measurement BCA, Western blot or ELISA, the Lowry protein assay, fluorescence-based assays, protein mass spectrometry-based assays, nanoparticle and nanopore-based methods, etc.

[0106] A "statistically significant increase in the intracellular level of a sirtuin" is herein considered as a determination by an analyst that the increase in the expression or activation level of the protein (in this case, a sirtuin) cannot be explained by chance alone. Statistical hypothesis testing is the way in which a person skilled in the art makes this determination. The test produces a p-value, which is the probability of observing a result as extreme as the data result, assuming that the result is indeed due to chance alone. 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 the intracellular level of a sirtuin is statistically significant when a statistical test is performed to compare the intracellular level of the sirtuin with the basal intracellular level of the sirtuin in a reference cell or an untreated cell, and the resulting p-value of the statistical test is 0.1 or less, preferably 0.05, 0.01, 0.001 or less.

[0107] Preferably, the patient in need thereof suffers from a disease or disorder characterized by a decreased intracellular level of sirtuins, preferably a decreased intracellular level of Sirt1. Preferably, the patient in need thereof suffers from a neurodegenerative, cardiovascular, pulmonary, metabolic, or age-related disorder characterized by a decreased intracellular level of sirtuins, preferably Sirt1, compared to the intracellular level of sirtuins in healthy individuals. Preferably, the disorder is selected from the group consisting of Alzheimer's dementia or other dementias, or age-associated cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

[0108] Preferably, the combination of pterostilbene or a salt thereof and silibinin or a salt thereof is used in the treatment, prevention, improvement, or alleviation of neurodegenerative diseases, where the combination is understood to be a) pterostilbene or a salt thereof and b) silibinin or a salt thereof administered to the subject simultaneously or sequentially in any order. Preferably, the disorder is characterized by a decrease in intracellular levels of sirtuins, preferably Sirt1, compared to healthy individuals. In one embodiment, the neurodegenerative disorder is selected from the group consisting of Alzheimer's dementia or other dementias, or age-related cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes. In one embodiment, the cell that increases the intracellular level of sirtuins, preferably Sirt1, upon treatment is derived from nervous tissue. Preferably, the cell is a neuron or a neuroglial cell.

[0109] Furthermore, sirtuins are known to be potential therapeutic targets in the treatment of metabolic disorders such as diabetes. Several studies suggest that overexpression of Sirt1 in pancreatic cells can be used to control blood glucose levels 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, based on the findings given herein, the combination of stilbenoid, preferably resveratrol or pterostilbene, and silibinin, or the composition according to the second aspect, can also be used to treat, prevent, improve or alleviate diabetes.Preferably, stilbenoid is pterostilbene.Therefore, in a preferred embodiment, the present invention also provides the combination of pterostilbene or its salt and silibinin or its salt used in the treatment, prevention, improvement or alleviation of diabetes, wherein the combination is understood to be a) pterostilbene or its salt and b) silibinin or its salt administered to said subject simultaneously or sequentially in any order.

[0110] This seventh aspect is related to the first and second aspects above. Accordingly, the compounds and pharmaceutical compositions, dosages, routes of administration and dosing regimens provided in the previous aspects of the invention apply here.

[0111] As defined in the first aspect, and also included as an embodiment in the fifth, sixth and seventh aspects, the combination of PT and SIL can be administered in a single composition or in separate compositions. In an embodiment of the fifth, sixth and seventh aspects, pterostilbene is pterostilbene phosphate disodium salt. In another embodiment of the above aspects, silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt. It is noted that the use described in any of the above aspects further comprises administering a combination of PTE and SIL with at least one further radiation mitigation compound, where the use described herein is understood as the simultaneous or sequential administration to said subject of a) pterostilbene or a salt thereof, b) silibinin or a salt thereof, and c) one or more radiation mitigation compounds. In a preferred embodiment, the at least one or more radiation mitigation compounds is a nicotinamide adenine dinucleotide booster (hereinafter referred to as NAD + Booster), and / or fibroblast stimulating lipopeptide-1 (hereinafter referred to as FSL-1), or any salt thereof.

[0112] As defined in the first aspect, and also included as an embodiment in the fifth, sixth and seventh aspects, PT and SIL, or any salt or derivative thereof, are administered to a subject. In a preferred embodiment, the subject is a mammal. Preferably, the mammal is a human or a domestic animal such as a dog, cat or horse. More preferably, the mammal is a human.

[0113] The present invention further provides a method for increasing intracellular levels of sirtuins in a subject to prevent, treat (including ameliorate or alleviate) a disease characterized by low levels of sirtuins or reduced sirtuin activation, as described in the previous aspect. In one embodiment, the method comprises administering a combination of pterostilbene and silibinin or any salt thereof, where combination is understood to mean the simultaneous or sequential administration to the subject of a) pterostilbene or a salt thereof, b) silibinin or a salt thereof, and optionally c) at least one or more radiation mitigation compounds, as defined throughout the present specification. As described above, the at least one or more radiation mitigation compounds may be NAD + It may be a booster and / or FSL-1. Importantly, the dosages and routes of administration defined in the first aspect also apply to the uses according to the fifth, sixth and seventh aspects.

[0114] It should be noted herein that the present invention also covers the following items or embodiments:

[0115] 1. Pterostilbene or a pharmaceutically acceptable salt thereof in combination with silibinin or a pharmaceutically acceptable salt thereof for use in the prevention, amelioration, or alleviation of diseases induced by ionizing radiation in a subject, wherein administration is carried out before, after, or during radiation exposure, and wherein the combination is understood to be the administration of a) pterostilbene or a pharmaceutically acceptable salt thereof and b) silibinin or a pharmaceutically acceptable salt thereof to the subject, either simultaneously or sequentially in any order.

[0116] 2. The use of pterostilbene according to item 1, wherein pterostilbene and silibinin or a pharma- ceutically acceptable salt thereof are administered in a single pharmaceutical composition or in separate pharmaceutical compositions.

[0117] 3. The use of pterostilbene according to item 1 or 2, wherein the administration of pterostilbene and silibinin or a pharma- ceutically acceptable salt thereof is topical, oral, or parenteral.

[0118] 4. The use of pterostilbene according to any one of items 1 to 3, wherein pterostilbene and silibinin or a pharma- ceutically acceptable salt thereof are administered simultaneously or sequentially several times before and / or after radiation exposure, or both.

[0119] 5. The use of pterostilbene according to any of items 1 to 4, wherein the combination is present in a dosage capable of providing a radioprotective effect.

[0120] 6. Pterostilbene for use according to any of items 1 to 5, wherein pterostilbene or a pharma- ceutically acceptable salt thereof is administered at a dosage of 300 mg / kg to 30 mg / kg, preferably 100 mg / kg, and silibinin or a pharma-ceutically acceptable salt thereof is administered at a dosage of 200 mg / kg to 35 mg / kg of silibinin, preferably 70 mg / kg, or an equivalent dose converted to a human dose.

[0121] 7. The use of pterostilbene according to any of items 1 to 6, further comprising administration in combination with at least one or more radiation mitigating compounds, where combination is understood as simultaneous or sequential administration to said subject of a) pterostilbene or a pharma- ceutically acceptable salt thereof, b) silibinin or a pharma- ceutically acceptable salt thereof, and c) at least one or more radiation mitigating compounds or any pharma- ceutically acceptable salt thereof.

[0122] 8. At least one radiation mitigating compound is NAD + 8. The use of pterostilbene according to item 7, which is a booster and / or a fibroblast stimulating lipopeptide, or any pharma- ceutically acceptable salt thereof.

[0123] 9. The use further includes administering in combination with at least two or more radiation mitigation compounds, wherein the at least two or more radiation mitigation compounds are NAD + 8. The use of pterostilbene according to item 7, which is a booster and a fibroblast stimulating lipopeptide, or any pharma- ceutically acceptable salt thereof.

[0124] 10. NAD + 10. The use of pterostilbene according to any of items 7 to 9, wherein the booster or any pharma- ceutically acceptable salt thereof is administered at a dose of 400 mg / kg to 100 mg / kg, preferably 185 mg / kg, and / or the fibroblast-stimulating lipopeptide or any pharma-ceutically acceptable salt thereof is administered at a dose of 1 mg / kg to 0.1 mg / kg, preferably 0.25 mg / kg, or a human equivalent dose.

[0125] 11. NAD + 11. The use of pterostilbene according to any of items 8 to 10, wherein the booster is nicotinamide riboside.

[0126] 12. The use of pterostilbene according to any of items 1 to 11, wherein the disease induced by ionizing radiation is associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage.

[0127] 13. The use of pterostilbene according to any of items 1 to 12, wherein the disease induced by ionizing radiation is acute radiation syndrome and / or chronic radiation syndrome.

[0128] 14. The use of pterostilbene according to any one of items 1 to 13, wherein the ionizing radiation is gamma rays or X-rays.

[0129] 15. Pterostilbene for use according to any of items 1 to 14, wherein pterostilbene is pterostilbene phosphate disodium salt, and / or silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

[0130] 16. A pharmaceutical composition comprising a) pterostilbene or a pharma- ceutically acceptable salt thereof, and b) silibinin or a pharma- ceutically acceptable salt thereof.

[0131] 17. The pharmaceutical composition according to item 16, wherein the dosage of pterostilbene or a pharma- ceutically acceptable salt thereof present in the composition is 300 mg / kg to 30 mg / kg, preferably 100 mg / kg, and the dosage of silibinin or a pharma- ceutically acceptable salt thereof present in the composition is 200 mg / kg to 35 mg / kg of silibinin, preferably 70 mg / kg, or an equivalent dose converted to a human dose.

[0132] 18. The pharmaceutical composition preferably comprises: a.NAD + Boosters and / or b. fibroblast-stimulating lipopeptide-1, or any pharma- ceutically acceptable salt thereof; 18. The pharmaceutical composition according to item 16 or 17, further comprising at least one or more radiation mitigating compounds selected from:

[0133] 19. The pharmaceutical composition further comprises at least two or more radiation mitigation compounds, wherein the at least two or more radiation mitigation compounds are NAD + 19. The pharmaceutical composition according to item 18, which is a booster and fibroblast stimulating lipopeptide-1, or any pharma- ceutically acceptable salt thereof.

[0134] 20. NAD + 20. The pharmaceutical composition according to item 18 or 19, wherein the booster or a pharma- ceutically acceptable salt thereof is present in said composition in an amount of 400 mg / kg to 100 mg / kg, preferably 185 mg / kg, and / or the fibroblast-stimulating lipopeptide-1 or a pharma-ceutically acceptable salt thereof is present in said composition in an amount of 1 mg / kg to 0.1 mg / kg, preferably 0.25 mg / kg, or a human equivalent dose.

[0135] 21. NAD+ 21. The pharmaceutical composition according to any of items 18 to 20, wherein the booster is nicotinamide riboside.

[0136] 22. The pharmaceutical composition according to any one of items 16 to 21, wherein the composition further comprises a pharma- ceutically acceptable carrier, additive, and / or excipient.

[0137] 23. The pharmaceutical composition according to any of items 16 to 22, wherein pterostilbene is pterostilbene phosphate disodium salt and / or silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

[0138] 24. A kit of parts comprising at least two receptors comprising at least pterostilbene and silibinin or any pharma- ceutically acceptable salt thereof and, optionally, at least one or more radiation mitigating compounds.

[0139] 25. A kit of parts according to item 24, wherein pterostilbene or a pharma- ceutically acceptable salt thereof is present in a dosage of 300 mg / kg to 30 mg / kg, preferably 100 mg / kg, and silibinin or a pharma- ceutically acceptable salt thereof is present in a dosage of 200 mg / kg to 35 mg / kg of silibinin, preferably 70 mg / kg, or a human equivalent dose.

[0140] 26. Any one of at least one radiation mitigating compound is a.NAD + Boosters and / or b. fibroblast stimulating lipopeptides, or any pharma- ceutically acceptable salt thereof; 26. The kit of parts according to item 24 or 25, selected from the group consisting of

[0141] 27. NAD +27. The kit of parts according to item 26, wherein the booster or a pharma- ceutically acceptable salt thereof is present in a dosage of 400 mg / kg to 100 mg / kg, preferably 185 mg / kg, and / or the fibroblast-stimulating lipopeptide or a pharma-ceutically acceptable salt thereof is present in a dosage of 1 mg / kg to 0.1 mg / kg, preferably 0.25 mg / kg, or a human equivalent dose.

[0142] 28. NAD + 28. The kit of parts according to item 26 or 27, wherein the booster is nicotinamide riboside.

[0143] 29. A kit of parts according to any of items 24 to 27, wherein pterostilbene is pterostilbene phosphate disodium salt and / or silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

[0144] 30. A method for preventing, ameliorating, or alleviating a disease induced by ionizing radiation in a subject, comprising administering a combination of pterostilbene and silibinin or a pharma- ceutically acceptable salt thereof, said administration being carried out before, after, or during radiation exposure, wherein the combination is understood to mean the simultaneous or sequential administration of a) pterostilbene or a pharma- ceutically acceptable salt thereof, and b) silibinin or a pharma- ceutically acceptable salt thereof to the subject.

[0145] 31. The method further comprising administering at least one radiation mitigation compound, wherein the at least one radiation mitigation compound is preferably: a.NAD + Boosters and / or b. fibroblast stimulating lipopeptides, or any pharma- ceutically acceptable salt thereof; 31. The method according to claim 30, wherein the method is selected from the group consisting of

[0146] 32. NAD + 32. The method of claim 31, wherein the booster is nicotinamide riboside.

[0147] It is understood that the specific embodiments described in this example are presented by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are encompassed by the claims. EXAMPLES

[0148] Example 1: General Materials and Methods The general materials and methods used in the present invention are disclosed herein, and detailed procedures are further described in Examples 2 to 4, respectively.

[0149] Mice and treatment procedures Swiss albino mice (male, 9-10 weeks old, 30-32 g) were obtained from Charles River Laboratories (Wilmington, MA). This strain is one of the most commonly used strains in radiation studies (Williams 2010). Mice were fed a nutritionally complete diet (standard chow, SD) prior to irradiation. This was A03 from Panlab (Barcelona, ​​Spain), with 3200 kcal / kg and the following nutritional composition: 23.5% crude protein, 4.3% crude fat, 3.7% crude fiber, and 51.1% carbohydrates, with vitamins and minerals provided similar to the NIH-7 open formula for rats and mice. This diet meets the nutritional requirements recommended by the American Institute of Nutrition for adult mice. Animals were fed ad libitum and housed in individual metabolic cages, where weight and food intake were monitored daily. The animal room was maintained at 22°C with a 12-h light / 12-h dark cycle. Drinking water was allowed ad libitum. Care of the irradiated mice followed the recommendations for total body irradiation of mice (Policy 15) from Robert Wood Johnson Medical University (Piscataway, NJ). This included a) use of antibiotics in the drinking water (134 mg ampicillin / kg / day, 40 mg Baytril / kg / day, and 220 mg / 42 mg sulfamethoxazole / trimethoprim / kg / day), b) having drinking water readily available, c) Napa nectar (Systems Engineering Lab Group Inc., Napa, CA) placed on the bottom of the cage for the first 14 days and changed daily, d) feeding softened food in small petri dishes on the floor of the cage, and e) a completely sterile environment (cage, food, and water) to avoid possible infections associated with immunosuppression. The cage was also equipped with a free-standing wheel inside to promote physical exercise of the animals. All experiments were started at 10:00 to minimize possible circadian variations.Animal procedures were in accordance with international law and policy (EEC Directive 86 / 609, OJ L 358.1, 12 December 1987, and the NIH Guide for the Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). Mice were irradiated with γ-radiation (. 137Cs) (total body irradiation, TBI). Single fraction radiotherapy was administered at a rate of approximately 5.0 Gy / min with total doses ranging from approximately 5.0 Gy to 10.0 Gy. The irradiation distance from the source was limited to 12 cm. Radiation dosimetry was controlled with a dosimetric variation of ±0.09 Gy using an alanine dosimeter from Bruker (Billerica, Massachusetts). For oral administration, PTER and SIL (Merck Chemicals GmbH, Darmstadt, Germany) were solubilized in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethylcellulose. For IP administration, PTER salt (pterostilbene phosphate disodium salt, SYNCOM, Groningen, The Netherlands) and SIL salt (silibinin-C-2',3-dihydrogen succinic acid disodium salt, Rottapharm / Madaus, Cologne, Germany) were dissolved in vaccination grade sterile water. All other polyphenolic phytochemicals assayed (Merck Chemicals GmbH and Cayman Chemical Co., Ann Arbor, MI) represented major flavonoids and nonflavonoids (Estrela 2017), namely gallic acid (phenolic acid), caffeic acid (hydroxycinnamic acid), curcumin (curcuminoid), epigallocatechin gallate (flavanol), genistein (isoflavone), quercetin (flavonol), luteolin (flavone), naringenin (flavanone), delphinidin (anthocyanidin), and phlorizin (chalcone), which were solubilized as in PTER and SIL and administered orally after administration (Wani 2016). NR (nicotinamide riboside, 3-aminocarbonyl-1-β-D-ribofuranosyl-pyridinium, Elysium Health, Inc., New York, NY) was dissolved in vaccination-grade water and administered orally (185 mg / kg × day, Obrador 2020) after neutralizing the pH of the solution (pH 7.0).FSL-1 (InvivoGen, San Diego, CA) was resuspended in vaccination grade sterile water and administered IP (0.25 mg / kg × day, dosing volume was 50 mL–60 mL depending on mouse weight as recommended by Kurkjian 2017). Pharmaceutical grade amifostine (Ethyol) was obtained as 500 mg sterile lyophilized powder vials from Cumberland Pharmaceuticals (Nashville, TN) and used after reconstitution with saline.

[0150] pathology Any mice that died, were moribund, or were scheduled for sacrifice were excluded from the study. All mice were necropsied and approximately 30 tissues or organs as well as 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.

[0151] Mice were euthanized 14 days after irradiation, and femurs were then excised. Bone marrow cells were flushed from the excised femurs using a 26-gauge needle with pre-chilled phosphate-buffered saline. Single-cell suspensions were prepared by gently pipetting the flushed cells several times, and the number of viable bone marrow nucleated cells (BMNCs) was counted using a hemocytometer (Neubauer, Marienfeld, Germany) and determined as × 10 6 Expressed as / femur.

[0152] Rotarod test The functions of the test include the assessment of the subjects' balance, grip strength, and motor coordination, especially in testing the effects of experimental drugs. In this test (Rotarod, Harvard Apparatus, Holliston, Massachusetts), each animal was given three trials to measure the maximum period (seconds) that they could stay on a rotating rod (diameter 3.5 cm, rotation speed: 15 rpm) without falling. Each mouse was given a maximum of three trials within an arbitrary limit of 1200 seconds, and the longest period was recorded. Changes over time in the different groups were monitored weekly.

[0153] Pterostilbene Levels Liquid chromatography and mass spectrometry (LC-MS / MS) were performed using a TSQ Vantage™ triple quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a SIL-10ADvp autoinjector together with a Shimadzu LC-10ADvp pump and SLC-10Avp controller system as previously described (Ferrer 2005).

[0154] Silibinin level The measurements were based on the methodology described by Song et al. (2019). Briefly, samples were analyzed via HPLC (Ultimate 3000, Thermo Fisher Scientific) and MS / MS (TQ detector triple quadrupole from Waters) system on a Hypurity™ C18 column (50 mm × 4.6 mm) using an injection volume of 5 μL. The mobile phase consisted of water (A) and acetonitrile (B). The gradient elution system was optimized as follows: 0 min–3.0 min 20%→100% B, 3.0 min–5.0 min 100%→100% B, 5.0 min–5.5 min 100%→20% B, 5.5 min–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 mode was used to monitor the SIL with precursor to product ion transitions at m / z 481.3 / 125.0. The fragmentor voltage was 150 V and the collision energy was 15 eV. Preparation of tissue samples was as described for the determination of PTER levels (above).

[0155] Blood NAD + level NAD in whole blood +Measurement of levels was performed by HPLC according to the methodology described by Yoshino and Imai (Yoshino et al. 2013). The procedure used an HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a Supelco LC-18-T column (15 cm × 4.6 cm, Sigma Aldrich) and a Hypercarb column (15 cm × 4.6 cm, Thermo Fisher Scientific, Waltham, MA), respectively. Whole blood samples (0.5 ml) were collected in sodium citrate tubes at 4 °C. Then, 0.1 ml aliquots of whole blood were transferred to cryovials containing 0.9 ml of 0.5 M ice-cold perchloric acid to lyse all blood cells. Samples were centrifuged at 10000 g × 10 min (4 °C). The supernatants were stored at -80 °C until analysis.

[0156] Assessment of therapy-induced in vivo toxicity This included blood cell counts and chemistry reactions based on standard NIH methodologies.

[0157] Chromosomal aberrations and double-stranded DNA breaks We followed the technique described by M'kacher et al. (2015), where staining of telomeres and centromeres facilitates the simple detection of dicentric, circular and acentric chromosomes of premature chromosome condensation in lymphocytes using optimized hybridization conditions and fusion capture of interphase chromosomes. This approach has great advantages, since damage can be observed within a few hours after blood collection. Blood was collected 8 hours after irradiation, as suggested in the procedure (M'kacher 2015), thus allowing time for DNA repair and the emergence of dicentric, circular and acentric chromosomes.

[0158] Peripheral blood lymphocytes were isolated as described herein below. Premature chromosome condensation (PCC) was performed using CHO cells (ATCC) as previously described (Pantelias 1983). Condensed human chromosomes and CHO chromosomes were differentiated according to their morphological characteristics (M'kacher 2015). The procedure took approximately 4 hours.

[0159] Telomeres and centromeres of PCC fusions were stained using Q-FISH technology with a Cy-3-labeled peptide nucleic acid (PNA) probe specific for telomeric TTAGGG and a fluorescein isothiocyanate-labeled PNA probe specific for centromeric sequences (M'kacher 2015) (both from ThermoFisher Scientific, Waltham, MA). Telomere- and centromere-stained fusions were automatically recorded using Autocapt software from Metasystems (Altlusheim, Germany) and a high-resolution CCD camera (C91002-23B, Hamamatsu Photonics K.K., Herrsching, Germany).

[0160] On the same slide, two different manual procedures were performed as described by M'kacher et al. (2015). Briefly, reverse 4',6-diamidino-2-phenylindole is first used to score ring chromosomes and excess acentric chromosomes. Then, telomere and centromere staining is used to score dicentric, ring, and acentric chromosomes from interstitial deletions without telomere 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 the formation of dicentric or circular fragments. The resulting double-stranded DNA breaks (DSBs) were calculated (M'kacher 2015).

[0161] Micronucleus formation For this purpose, femoral bone marrow was collected from sacrificed mice, smears were made, and fixed using methanol. After drying, double staining was performed using May-Grünwald-Giemsa stain (Schmid 1975). According to this method, polychromatic erythrocytes (PCE) were stained reddish blue, normochromatic erythrocytes (NCE) were stained orange, whereas nuclear material was deep purple. Micronucleated polychromatic (MnPCE) and normochromatic (MnNCE) cells were counted from 2000 cells under oil immersion.

[0162] Isolation and incubation of lymphocytes, hepatocytes, and intestinal epithelial cells Hepatocyte isolation was performed according to previously reported methodology (Berry 1969). Blood mononuclear cells were obtained from heparinized blood by Accuspin-Histopaque (Sigma-Aldrich, St. Louis, MO) gradient centrifugation. Mononuclear cells were washed twice and diluted at 20 × 10 per ml in Krebs-Henseleit bicarbonate medium (pH 7.4) containing 0.3% bovine serum albumin. 6 Cells were resuspended at a concentration of 100x100 cells. Further positive selection of lymphocyte subsets with monodispersed immunomagnetic Dynabeads (Dynal) was performed at 4°C as described elsewhere (Thornton 2003). Hepatocyte and lymphocyte cell volumes were obtained as previously described (Estrela 1992).

[0163] The isolated hepatocytes were incubated in flasks (approximately 10 mg dry weight per ml) in Krebs-Henseleit bicarbonate medium (KHBM, pH 7.4) containing 1.3 mM CaCl2, 5% non-fat bovine serum albumin in the presence of glucose (5 mM) at 37°C. The gas atmosphere was O2 / CO2 (19:1). The isolated lymphocytes were incubated (10 per flask) in Krebs-Ringer medium containing 5% non-fat bovine serum albumin in the presence of glucose (5 mM) and glutamine (2 mM) at 37°C. 6After incubation, 0.2 ml of 25% perchloric acid was added to disrupt the cells. Proteins were removed by centrifugation, and the supernatant was neutralized with 20 ml of 40% KOH solution and tris-(hydroxymethyl)aminomethane / KOH (0.5 M to 2.0 M) solution to measure metabolic products.

[0164] Primary intestinal epithelial cells (IECs) were isolated using a protocol adapted from Graves et al. (2014). The longitudinal muscle layer was removed and IECs were cultured in ice-cold MgCl2 containing 100 U penicillin, 100 μg / ml streptomycin, 25 μg / ml gentamicin, and 0.5 mM dithiothreitol (DTT). 2+ and Ca 2+ Colon tissue was prepared by washing with Hank's Balanced Salt Solution (HBSS) without DMSO. The tissue was cut into small pieces, suspended in 50 ml of HBSS washing solution, shaken, and the contents allowed to settle for 2 min. The supernatant was removed and the settled contents were washed three times. The settled contents were minced and suspended in HBSS. The resulting suspension was filtered through a 1000 μm filter. 2 The remaining tissue was digested in a buffer containing 75 U / ml collagenase type XI (Sigma-Aldrich), 25 μg / ml dispase neutral protease II (ThermoFisher Scientific, Waltham, MA), 0.5 mM DTT, and 2% (vol / vol) fetal bovine serum (FBS) (ThermoFisher Scientific) in Dulbecco's modified Eagle's 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 h. The resulting digestion mixture was again passed through a 1000 μm mesh filter. 2The tissue was then passed through a 100-μl filter and the tissue fragments on the filter were washed with 25 ml of complete growth medium (DMEM, 8.5 g / l sodium pyruvate, 2% (vol / vol) 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% (wt / vol) D-sorbitol). The tissue debris remaining on the filter was discarded and the effluent containing the proliferative crypt structures was centrifuged at 200 g for 5 min at 4° C. The remaining pellet containing isolated intestinal crypts was suspended in DMEM. Crypts were suspended in complete growth medium and plated at a density of approximately 2000 crypts / ml / well in 12-well collagen type I-coated culture dishes (ThermoFisher Scientific) and incubated at 37°C with O2 / CO2 (19:1).

[0165] Glucose and glutamine utilization was measured as previously described (Newsholme 1987).

[0166] cell culture Isolated hepatocytes were cultured at 10 per mL in William's complete medium. 6 The isolated C2D cells were cultured at a concentration of 100 μg / mL. + Lymphocytes were cultured at 2 × 10 per mL in PB-MAX medium (GIBCO, ThermoFisher Scientific). 6 The IECs were cultured at a concentration of 10 cells per mL. 6 Cells) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 300 mg / ml L-glutamine. All cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2.

[0167] DNA oxidation Tissue samples were homogenized in 10 mM Tris (pH 7.0) + 1 mM EDTA + 150 mM NaCl. They were then first incubated with RNase A for 30 min at 37°C, then with proteinase K overnight at 55°C. This was followed by a chloroform / isoamyl alcohol extraction. Samples of 100 μg of isolated DNA were digested to nucleosides with nuclease P1 and alkaline phosphatase (Crain 1990). Analysis of the modified DNA base 8-hydroxy-2'-deoxyguanosine (8OHdG) was performed by UPLC-MS / MS.

[0168] Lipid peroxidation For isoprostanes measurements, tissue samples were homogenized in 0.1 M phosphate buffer (pH 7.4) + 1 mM EDTA + 0.005% butylated hydroxytoluene. Isoprostanes were measured using an 8-isoprostane EIA kit (Cayman Chemical Co.) according to the manufacturer's protocol.

[0169] Protein carbonylation Protein carbonylation, an irreversible oxidative damage, was measured using a Protein Carbonyl Fluorescence Assay Kit (Cayman Chemical Co.) according to the manufacturer's protocol.

[0170] Enzyme activities and metabolite levels The isolated cells were homogenized in 0.1 M phosphate buffer (pH 7.2) at 4°C. The activities of γ-glutamylcysteine ​​ligase (GCL), glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase 1 and superoxide dismutase 2 (SOD1 and SOD2) were measured as previously described in detail (Benlloch 2016). Protein concentrations were measured with the Pierce BCA protein assay (Thermo Fisher Scientific). Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) was measured by LC / MS as previously reported (Obrador 2014). Cell processing was performed using rapid N-ethylmaleimide derivatization to prevent GSH autoxidation according to published methodology (Asensi 1994). NAD in isolated cells was determined by the Pierce BCA protein assay (Thermo Fisher Scientific). + and NADH were quantified using assay kits from MyBiosource (San Diego, CA).

[0171] RT-PCR and mRNA detection Total RNA was isolated using the TRIzol kit from Invitrogen (San Diego, CA) according to the manufacturer's instructions. cDNA was obtained using random hexamer primers recommended by the manufacturer and the MultiScribe reverse transcriptase kit (Taq-Man RT Reagents; Applied Biosystems, Foster City, CA). PCR master mix and AmpliTaq Gold DNA polymerase (Applied Biosystems) were added to the primers as previously reported (Benlloch 2016). Real-time quantification of mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed as previously reported (Benlloch 2016).

[0172] Comet assay This assay is a single cell gel electrophoresis assay for simple assessment of cellular DNA damage. We used the assay kit (ab238544) from Abcam (Cambridge, UK). We followed the manufacturer's instructions step by step.

[0173] Nicotinamide phosphoribosyltransferase activity assay 14 C-nicotinamide (NAM) from GE Healthcare Life Sciences, Björkgatan, Uppsala, Sweden 14 The formation of C-nicotinamide mononucleotide (NMN) was measured by soaking in acetone on filter paper. 14 C-NMN can be measured by precipitation and scintillation counting. Dispersion of cells was determined by trypsinization (0.2% trypsin, 0.5 mM EDTA, and 5 mM glucose supplemented with Mg 2+ and Ca 2+ The assay was carried out by centrifugation (800g x 5 min at 4°C in PBS) for 3 min at 37°C. After obtaining the cell pellet by centrifugation (800g x 5 min at 4°C), 100 μl of phosphate buffer [0.01 M Na2HPO4 / NaH2PO4 buffer (pH 7.4) containing protease inhibitors (Sigma-Aldrich)] was added to each cell sample. The samples were further processed and NAMPT activity was determined for 30 μg of protein as previously described (Elliott GC, et al. 1980, and Schuster S, et al. 2014). Radioactivity (disintegrations per minute) was measured using a Perkin-Elmer Tri-Carb™ liquid scintillation counter. Background disintegrations per minute from the scintillation fluid were subtracted from each measurement.

[0174] Colony-forming cells in bone marrow Mice were sacrificed under aseptic conditions and femurs were removed 7 days after irradiation. Bone marrow cells were flushed from the removed 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 strainer to remove debris and clumps. Cells were washed twice with phosphate-buffered saline (4°C) and counted using a hemocytometer. 5 × 10 cells were cultured per 35 mm cell culture dish in Methocult GF M3434 (Stemcell Technologies, Vancouver, Canada) growth medium according to the manufacturer's protocol. 4 Cells were plated ex vivo at a concentration of 100x100 cells and incubated at 37°C, 5% CO2, and 95% humidity for 14 days. On day 14 of culture, colonies were visualized under polarized light using a light microscope (AxioCam MRc5, Zeiss, Oberkochen, Germany) and scanned in 60 mm gridded scoring Petri dishes (Stemcell Technologies). Hematopoietic colony forming cells CFU-GM and CFU-GEMM were quantified.

[0175] Preparation of cell, nuclear, and mitochondrial extracts Cell extracts were prepared using the Sigma Aldrich Whole Cell Extraction Kit. The NE-PER™ Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific) allows for the stepwise isolation and preparation of cytoplasmic and nuclear extracts from mammalian cells or tissues. Mitochondrial extracts were prepared according to the standardized cell fractionation and organelle isolation procedure by ThermoFisher Scientific.

[0176] Gene silencing Long-term gene silencing was achieved using the PSilencer 3.1-H1 linear vector from Ambion Inc. (Austin, TX). To target mouse Nfκbie, siRNA oligoduplexes (Locus ID 18037) from Origen (Rockville, MD) were used. siRNAs targeting mouse Nfe2l2 (reference sequence NM_010902.3), Ppargc1 (reference sequence NM_008904.2), Sirt1 (reference sequence NM_019812.2), Sirt3 (reference sequence MN_022433.2), Parp1 (reference sequence NM_007415.2), SOD2 (reference sequence NM_013671.3), and GPX1 (reference sequence NM_008160.6) were from Invitrogen. In all cases, the silencing procedures followed the manufacturer's technical recommendations. Control experiments were performed using equal amounts of corresponding sense and scrambled oligonucleotides with the same base composition and randomized sequence. Silencing was confirmed by immunoblotting. siRNA was delivered using Invitrogen's Lipofectamine RNAiMAX transfection reagent and the manufacturer's protocol.

[0177] Cell death analysis Apoptotic and necrotic cell death were differentiated by using fluorescence microscopy. For this purpose, isolated MNs were incubated with Hoescht 33342 (10 mM; stains all nuclei) and propidium iodide (10 mM; stains nuclei of cells with disrupted plasma membrane) for 3 min and analyzed using a Diaphot 300 fluorescence microscope (Nikon, Tokyo, Japan) with excitation at 360 nm. The nuclei of viable, necrotic, 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.

[0178] Western blot Western blot analysis was performed as previously described (Benlloch M, et al. 2016). Proteins were transferred to nitrocellulose membranes and subjected to Western blotting with specific anti-human monoclonal antibodies (OriGene, Rockville, MD, and Abcam). Blots were developed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL system; GE HealthCare Life Sciences). Protein bands were quantified using laser densitometry.

[0179] Tumor xenografts Human A549 and MDA-MB-231 cells were from ATCC (Manassas, VA). Cells were grown in DMEM (Invitrogen, San Diego, CA) (pH 7.4) supplemented with 10% heat-inactivated FCS (Biochrom KG, Berlin, Germany), 100 units / mL penicillin, and 100 μg / mL streptomycin. Cells were plated (1 cm 2The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 at 20,000 cells / well (20,000 cells per well). Cells were harvested by incubation with 0.05% (wt / vol) trypsin (Sigma Aldrich, St. Louis, MO) in PBS (pH 7.4) containing 0.3 mM EDTA for 5 min, followed by inactivation of trypsin by adding 10% FCS. Cells were allowed to attach for 12 h before any treatment was added. Cell numbers and viability were determined using a TC20 automated cell counter from BioRad (Hercules, CA). Nude (nu / nu) mice (male, 9-10 weeks old, Charles River Laboratories, Wilmington, MA) were fed standard chow (Letica, Rochester Hills, MI) ad libitum and housed at room temperature of 22°C with a 12-h light / 12-h dark cycle. The procedures were in accordance with international law and policy (EEC Directive 86 / 609, OJ L 358.1, 12 December 1987, and the NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Tumor cells were harvested from the culture flasks by exposure to 0.02% EDTA (5 min at 37°C), washed twice in DMEM, resuspended in the same culture medium, and injected subcutaneously into the backs of the animals (5 × 10 per mouse). 6 Local tumor growth was measured using calipers every 2 days starting on the second day of treatment.

[0180] statistics Data are expressed as mean ± SD for a number of different experiments. Statistical analysis was performed using Student's t-test, and P < 0.05 was considered significant.

[0181] G * Sample size was determined using Power 3.1.9.2 software (ANOVA test, effect size = 0.21, α = 0.05, β = 0.80, and groups as indicated). Survival data were analyzed using Kaplan-Meier curves and LogRank (Mantel-Cox) tests.

[0182] References Asensi, M. et al. A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Anal Biochem 217, 323-328 (1994). Benlloch M, et al. Pterostilbene Decreases the Antioxidant Defenses of Aggressive Cancer Cells In Vivo: A Physiological Glucocorticoids- and Nrf2-Dependent Mechanism. Antioxid Redox Signal. 2016;24:974-90 Berry, M. N., and Friend, D. S. (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 43, 506-520. Benlloch, M., Obrador, E., Valles, S. L., Rodriguez, M. L., Sirerol, J. A., Alcacer, J., Pellicer, J. A., Salvador, R., Cerda, C., Saez, G. T., and Estrela, J. M. (2016) Pterostilbene Decreases the Antioxidant Defenses of Aggressive Cancer Cells In Vivo: A Physiological Glucocorticoids- and Nrf2-Dependent Mechanism. Antioxid. Redox Signal. 24, 974-990. Boehning, D., Sedaghat, L., Sedlak, T. W. & Snyder, S. H. Heme Oxygenase-2 Is Activated by Calcium-Calmodulin. J. Biol. Chem. 279, 30927-30930 (2004). Canto C, Sauve AA, Bai P. Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes. Mol Aspects Med. 2013 Dec;34(6):1168-201. Crain, P. F. Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol. 193:782-790; 1990. Elliott GC, et al. A rapid procedure for assaying nicotinamide phosphoribosyltransferase. Anal Biochem. 1980;107:199-205; Estrela JM, Hernandez R, Terradez P, Asensi M, Puertes IR, and Vina J. Regulation of glutathione metabolism in Ehrlich ascites tumour cells. Biochem J 286 (Pt1): 257-262, 1992. Estrela JM, Mena S, Obrador E, Benlloch M, Castellano G, Salvador R, Dellinger RW. Polyphenolic Phytochemicals in Cancer Prevention and Therapy: Bioavailability versus Bioefficacy. J Med Chem. 2017 Dec 14;60(23):9413-9436. Ferrer P, Asensi M, Segarra R, Ortega A, Benlloch M, Obrador E, Varea MT, Asensio G, Jorda L, and Estrela JM. Association between PTERostilbene and quercetin inhibits metastatic activity of B16 melanoma. Neoplasia 7: 37-47, 2005. Fischer N, Seo EJ, Efferth T. Prevention from radiation damage by natural products. Phytomedicine. 2018 Aug 1;47:192-200. Graves CL, Harden SW, LaPato M, Nelson M, Amador B, Sorenson H, Frazier CJ, Wallet SM. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function. J Immunol Methods. 2014 Dec 1;414:20-31. Groen, A. K., Sips, H. J., Vervoorn, R. C., and Tager, J. M. (1982) Eur. J. Biochem. 122, 87-93. Klimberg VS, Souba WW, Dolson DJ, Salloum RM, Hautamaki RD, Plumley DA, Mendenhall WM, Bova FJ, Khan SR, Hackett RL, Bland KI, Copeland III EM. Prophylactic glutamine protects the intestinal mucosa from radiation injury. Cancer. 1990 Jul 1;66(1):62-8. Kurkjian CJ, Guo H, Montgomery ND, Cheng N, Yuan H, Merrill JR, Sempowski GD, Brickey WJ, Ting JP. The Toll-Like Receptor 2 / 6 Agonist, FSL-1 Lipopeptide, Therapeutically Mitigates Acute Radiation Syndrome. Sci Rep. 2017 Dec 11;7(1):17355. Martens CR, Denman BA, Mazzo MR, Armstrong ML, Reisdorph N, McQueen MB, Chonchol M, Seals DR. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults. Nat Commun. 2018 Mar 29;9(1):1286. Mavragani IV, Laskaratou DA, Frey B, Candeias SM, Gaipl US, Lumniczky K, Georgakilas AG. Key mechanisms involved in ionizing radiation-induced systemic effects. A current review. Toxicol Res (Camb). 2015 Aug 11;5(1):12-33. M'kacher R, El Maalouf E, Terzoudi G, Ricoul M, Heidingsfelder L, Karachristou I, Laplagne E, Hempel WM, Colicchio B, Dieterlen A, Pantelias G, Sabatier L. Detection and automated scoring of dicentric chromosomes in nonstimulated lymphocyte prematurely condensed chromosomes after telomere and centromere staining. Int J Radiat Oncol Biol Phys. 2015 Mar 1;91(3):640-9. Newsholme, P., Gordon, S., and Newsholme, E. A. (1987) Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem. J. 242, 631-636. Obrador, E., Valles, S. L., Benlloch, M., Sirerol, J. A., Pellicer, J. A., Alcacer, J., Coronado, J. A.-F., and Estrela, J. M. (2014) Glucocorticoid receptor knockdown decreases the antioxidant protection of B16 melanoma cells: an endocrine system-related mechanism that compromises metastatic cell resistance to vascular endothelium-induced tumor cytotoxicity. PloS One. 9, e96466. Obrador E, Salvador R, Marchio P, Lopez-Blanch R, Jihad-Jebbar A, Rivera P, Valles SL, Banacloche S, Alcacer J, Colomer N, Coronado JA, Alandes S, Drehmer E, Benlloch M, Estrela JM. Nicotinamide Riboside and Pterostilbene Cooperatively Delay Motor Neuron Failure in ALS SOD1G93A Mice. Mol Neurobiol. 2020 Nov 10. doi: 10.1007 / s12035-020-02188-7. Pantelias GE, Maillie HD. A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol. Somat Cell Genet 1983;9:533-547. Patyar RR, Patyar S. Role of drugs in the prevention and amelioration of radiation induced toxic effects. Eur J Pharmacol. 2018 Jan 15;819:207-216. Riklis E, Kol R, Marko R. Trends and developments in radioprotection: the effect of nicotinamide on DNA repair. Int J Radiat Biol. 1990 Apr;57(4):699-708. Qi Z, Whitt I, Mehta A, Jin J, Zhao M, Harris RC, et al. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. Am J Physiol Renal Physiol. 2004;286:F590-596. Reisz JA, Bansal N, Qian J, Zhao W, Furdui CM. Effects of ionizing radiation on biological molecules-mechanisms of damage and emerging methods of detection. Antioxid Redox Signal 2014, 21: 260-92. Riley, P.A., 1994. Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int. J. Radiat. Biol. 65, 27-33. Schmid W. 1975. The micronucleus test. Mutat Res Mutagen Relat Subj. 31: 9-15. Sirerol JA, Feddi F, Mena S, Rodriguez ML, Sirera P, Aupi M, Perez S, Asensi M, Ortega A, Estrela JM. Topical treatment with pterostilbene, a natural phytoalexin, effectively protects hairless mice against UVB radiation-induced skin damage and carcinogenesis. Free Radic Biol Med. 2015 Aug;85:1-11. Song Y, Sun H, Xiao J, Wang F, Ding Y, Zhao J, Bai J, Cheng L, Gao K, Liu M, Guo Q, Zhang Y, Gao W, Jia Y, Wen A. Development of a liquid chromatography-tandem mass spectrometric (LC-MS / MS) method for simultaneous determination of epigallocatechin-3-gallate, silibinin, and curcumin in plasma and different tissues after oral dosing of Protandim in rats and its application in pharmacokinetic and tissue distribution studies. J Pharm Biomed Anal. 2019 Jun 5;170:54-62. Schuster S, et al. Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and 833 primary human hepatocytes. PLoS One. 2014;9:e91045 Thornton, A. M. (2003) Fractionation of T and B cells using magnetic beads. Curr. Protoc. Immunol. ChaPTER 3, Unit 3.5A. Wani TA, Shah AG, Wani SM, Wani IA, Masoodi FA, Nissar N, Shagoo MA. Suitability of Different Food Grade Materials for the Encapsulation of Some Functional Foods Well Reported for Their Advantages and Susceptibility. Crit Rev Food Sci Nutr. 2016 Nov 17;56(15):2431-2454 Williams JP, Brown SL, Georges GE, Hauer-Jensen M, Hill RP, Huser AK, Kirsch DG, Macvittie TJ, Mason KA, Medhora MM, Moulder JE, Okunieff P, Otterson MF, Robbins ME, Smathers JB, McBride WH. Animal models for medical countermeasures to radiation exposure. Radiat Res. 2010 Apr;173(4):557-78. Yoshino J, Imai SI. Accurate measurement of nicotinamide adenine dinucleotide (NAD+) with high-performance liquid chromatography. Methods Mol Biol. 2013; 1077: 203-215.

[0183] Example 2: PT and radioprotective effects First, we studied the effect of gamma radiation and survival of mice. Mice that received total body gamma irradiation were studied (Figure 1A). Mice were given total body gamma irradiation (one single dose; 137Cs, 0.5 Gy / min–0.6 Gy / min; Biobeam 8000, GSM GmbH). 1 Gy = 1 J of absorbed energy per kg of material. LD50 / 30 of approximately 7.2 Gy (n=20 per radiation dose).

[0184] Next, the 30-day survival rate of mice treated with γ-rays (LD50 / 30) and pterostilbene (PT) was analyzed (Figure 1B). To this end, Swiss albino mice (Charles River, n = 20 per group) were treated with amifostine (2-(3-aminopropylamino)ethylsulfanylphosphonic acid) (dissolved in saline, administered orally once 30 min before irradiation, at a dose of 200 mg / m2, which is the dose recommended for humans). 2 Mice were treated with either 100 mg / kg / day of total body γ-irradiation (7.2 Gy at 0.5 Gy / min to 0.6 Gy / min) or pterostilbene (dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethylcellulose; given orally once daily for 5 days before and 2 days after irradiation). 137 A single dose of 1000 1Cs was administered (equipment = Biobeam 8000, GSM GmbH). 1 Gy = 1 J of absorbed energy per kg of material. Data were analyzed by the LogRank (Mantel-Cox) test ( * (All groups are compared to vehicle-treated controls.) The results in FIG. 1B show that the best effect on improving survival (60 days) was exerted by a dose of 100 mg pterostibene per kg.

[0185] The survival rates of mice treated with gamma radiation (LD50 / 30) and resveratrol (Resv) were also analyzed, and these results are shown in Figure 1C. Swiss albino mice (n=20 per group) were treated with Resvl (dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethylcellulose; orally administered once daily for 5 days before and 2 days after irradiation). Mice received total body gamma irradiation (7.2 Gy at 0.5 Gy / min to 0.6 Gy / min). 137A single dose of 10Cs was given (Mortazavi SMJ et al. Dose-Response 11: 281-292, 2013; instrument = Biobeam 8000, GSM GmbH). 1 Gy = 1 J of absorbed energy per kg of material. These results revealed that the radioprotective effect of Resv is lower than that of PT.

[0186] Plasma and extravascular levels of pterostilbene were studied in mice after oral administration. Mice were treated with 200 mg Pter / kg body weight. Groups of 4 or 5 animals were sacrificed at each time point. Pter was dissolved in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethylcellulose. Not detectable = nd. * P<0.1, data obtained from 10 min to 1440 min are compared to data seen at 5 min. These results are shown in Table 1 below.

[0187] [Table 1]

[0188] These results show that pterostilbene levels peaked 60 minutes after administration in all tissues studied.

[0189] Taken together, the above results support the potential of PTER as a radioprotector.

[0190] Example 3: PT+SIL combination To elucidate whether pterostilbene can be combined with other polyphenols to achieve better radioprotective effects, Swiss albino mice (n=20 per group) were treated with pterostilbene and / or other polyphenols. For oral administration, PT was solubilized in 2-hydroxypropyl-β-cyclodextrin and then suspended in 0.3% carboxymethylcellulose. All other polyphenolic phytochemicals assayed represented the major flavonoids and non-flavonoids, namely gallic acid (phenolic acid), caffeic acid (hydroxycinnamic acid), curcumin (curcuminoid), epigallocatechin gallate (flavanol), genistein (isoflavone), quercetin (flavonol), luteolin (flavone), naringenin (flavanone), delphinidin (anthocyanidin), and phloridzin (chalcone), which were solubilized as PT and then administered orally. The doses of each phytochemical were selected from the published maximum non-toxic doses. Data were analyzed with the LogRank (Mantel-Cox) test ( * All groups are compared to vehicle-treated controls. + All groups are compared to a group treated with PT alone) (Figure 2).

[0191] These results reveal that the combination of PT with various polyphenols has different effects: a) In some cases, the combination resulted in a lower survival rate than that achieved by administration of PT alone. This is seen, for example, in the combination of PT and gallic acid 60 days after irradiation. b) In some cases, the survival rate obtained by the combination of the two compounds was similar to that obtained by administering PT alone, indicating that the addition of the other compound had little effect on overall survival rate. This is seen, for example, in the combination of PT and delphinidin 60 days after irradiation. c) In some other cases, the survival rate achieved is approximately equal to the sum of the survival rates exerted by administering the two compounds separately, as seen, for example, in the combination of PT and genistein 60 days after irradiation. d) Finally, and most importantly, the survival rate obtained as a result of combining the two compounds produced a synergistic effect, resulting in an exceptional increase in survival rate that could not be derived from the sum of the effects of the two compounds administered individually. This last case only occurred with the combination of silibinin (SIL) and PT, and occurred both 30 days and 60 days after irradiation.

[0192] Taken together, these results indicate that the combination of SIL+PT exerts a synergistic radioprotective effect, thus achieving a high survival percentage at 30 days and 60 days after gamma irradiation.

[0193] Considering these results, improved survival and enhanced radioprotection over a longer period of time were studied. Swiss albino mice (n=20 per group) were treated IP with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (5 days before and 2 days after gamma irradiation). For IP administration, PT salt (pterostilbene phosphate disodium salt, SYNCOM, Groningen, The Netherlands) and SIL salt (silibinin-C-2',3-dihydrogen succinic acid disodium salt, Rottapharm / Madaus, Cologne, Germany) were dissolved in vaccination grade sterile water. Figure 3A shows the Kaplan-Meier curves and the LogRank (Mantel-Cox) test ( * Figure 3A shows the results of the study (comparing PT+SIL treated groups to vehicle treated controls). IP administration was chosen to facilitate regular dosing to a large number of mice. As can be seen in Figure 3A, the combination of PT+SIL provided long-term protection with a survival rate of approximately 25% (5 / 20) at 1 year post-irradiation. These results support the synergistic enhancing effect of the combined administration of these two compounds and demonstrate that their combined radioprotective effect is long-lasting.

[0194] Furthermore, Figure 3B shows a comparison between PT+SIL administration before or after gamma irradiation: Swiss albino mice (n=20 per group) were treated IP with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (5 days before and 2 days after LD50 / 30 gamma irradiation, light grey bars, or 7 days after gamma irradiation, dark grey bars). Data were analysed by LogRank (Mantel-Cox) test ( * (Comparing groups treated after irradiation to groups treated before irradiation.) These results indicate that survival rates are higher when polyphenols are administered before irradiation.

[0195] Next, the effect of pterostilbene (PT) and silibinin (SIL) on survival of γ-irradiated (LD50 / 30) mice, and on body weight: Swiss albino mice (n=20 per group) were treated IP 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 to vehicle-treated controls). These results are shown in FIG. 4A, where it can be seen that polyphenols attenuate radiation-induced weight loss.

[0196] The effect of combined administration of pterostilbene (PT) and silibinin (SIL) on whole body radiation-induced changes in neuromotor function in mice was also studied: Swiss albino mice (n=20 per group) were treated IP with PT (100 mg / kg) and SIL (70 mg / kg) for 7 days (5 days before and 2 days after gamma irradiation). To study neuromotor function, a standardized rotarod test was performed as follows: the function of the test includes the assessment of the balance, grip strength, and motor coordination of the subjects, especially in the testing of the effects of experimental drugs. In this test (rotarod, Harvard Apparatus, Holliston, MA), three trials were performed for each animal, and the maximum period (in seconds) that they could stay on the rotating mandrel (diameter of 3.5 cm, rotation speed: 15 rpm) without falling was measured. Each mouse was given a maximum of three trials within an arbitrary limit of 1200 seconds, and the longest period was recorded. The changes over time in the different groups were monitored weekly. As shown in Figure 4B, these results revealed that neuromotor function was not significantly altered by either radiation and / or administration of PT and SIL.

[0197] Then, the expression analysis of various antioxidant defense-related enzyme activities in the isolated cells was studied by RT-PCR. Statistical analysis was performed using Student’s t test ( P < 0.01; * Mice treated with PT+SIL+γ-rays are compared to mice treated with γ-rays alone; + (PT+SIL treated mice are compared to vehicle treated controls). The results are shown in Figure 5. It was concluded that radiation reduced the expression of all antioxidant defense related molecules studied, whereas PT and SIL counteracted the radiation-induced effects.

[0198] Furthermore, the protective effect of PT and SIL against γ-radiation-induced oxidative damage was studied by assessing various molecular markers. For that, PT, SIL, and γ-radiation were administered at the previously indicated doses (n=5 per molecule, cell type, and treatment) and statistical analysis was performed using Student's t-test (P<0.01; * All groups are compared against controls; + (Mice treated with PT+SIL+γ-rays are compared to mice treated with γ-rays alone.) As shown in Figure 6, PT and SIL counteracted the radiation-induced increase in various oxidative stress-related biomarkers.

[0199] To provide a complete analysis of the radioprotective effects of PT and SIL administered in combination, antioxidant defenses mediated by GCL and GSH were also measured. Therefore, PT, SIL, and γ-irradiation were administered at the doses / doses previously indicated (n=5 per molecule, cell type, and treatment) and statistical analysis was performed using Student's t test (P<0.01; * All groups are compared against controls; + (Mice treated with PT+SIL+γ-rays are compared to mice treated with γ-rays alone.) As shown in Figure 7, PT and SIL counteracted the radiation-induced decrease in various glutathione-related biomarkers.

[0200] In Figure 8, catalase and glutathione peroxidase levels (Figures 8A and 8B) and superoxide dismutase levels (Figures 8C and 8D) were measured following PT, SIL, and γ-irradiation at the doses / doses previously indicated (n=5 per molecule, cell type, and treatment). Statistical analysis was performed using Student's t-test (P<0.01; * All groups are compared against controls; +(Mice treated with PT+SIL+γ-rays were compared to mice treated with γ-rays alone.) These results revealed that PT and SIL counteracted the radiation-induced decrease in the activities of various antioxidant enzymes.

[0201] Finally, circulating CD2 + The effect of PT and SIL on gamma radiation-induced chromosomal aberrations and micronucleus formation in lymphocytes was studied. Telomeres and centromeres of premature chromosome condensation fusions were stained using Q-FISH technique. Telomere and centromere staining was used to score dicentric, circular, and acentric chromosomes from interstitial deletions without telomere 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 the formation of dicentric or circular fragments. The resulting double-stranded DNA breaks (DSBs) were calculated. In bone marrow, double staining was performed using May-Grünwald-Giemsa stain. According to this method, polychromatic erythrocytes (PCE) were stained reddish blue and normochromatic erythrocytes (NCE) were stained orange, whereas nuclear material was dark purple. Micronucleated polychromatic (MnPCE) and normochromatic (MnNCE) cells were counted. Statistical analysis was performed using Student's t test (P<0.01; * All groups are compared against controls; + (Mice treated with PT+SIL+gamma radiation are compared to mice treated with gamma radiation alone.) These results are shown in Table 2 below.

[0202] [Table 2]

[0203] As can be seen from Table 2, PT and SIL reduce all radiation-induced cytogenetic changes, ie chromosomal alterations and micronucleus formation.

[0204] Example 4: Combination of PT and SIL with radiation mitigation agents Next, we investigated whether a combination of radioprotectors (molecules that can reduce the direct damage caused by radiation) and radiomitigators (molecules that minimize toxicity even after radiation has been administered) might favor longer survival. To study this, we combined PT and SIL with NAD + It was combined with the booster nicotinamide riboside (NR) and the Toll-like receptor 2 / 6 agonist FSL-1 lipopeptide (FSL-1).

[0205] First, the effect of both molecules alone and in combination was studied to confirm the radiation mitigation properties. To that end, NR (nicotinamide riboside, 3-aminocarbonyl-1-β-D-ribofuranosyl-pyridinium, Elysium Health, Inc., New York, NY) was dissolved in vaccination grade water and administered orally (185 mg / kg x day) after making the pH of the solution neutral (pH 7.0). FSL-1 (fibroblast stimulating lipopeptide 1, InvivoGen, San Diego, CA) was resuspended in vaccination grade sterile water and administered IP (0.25 mg / kg x day, administration volume was 50 mL-60 mL depending on the mouse weight). NR was administered daily for 30 days in a single dose once a day starting after irradiation. FSL-1 was administered only once, 24 hours after irradiation. The total number of mice used was 20. These results are shown in Figure 9. Here, it is observed that administration of each of these molecules alone resulted in survival rates 60 days after gamma irradiation of 0% for NR, 5% for FSL-1, and 10% for the combination of NR+FSL-1.

[0206] Swiss albino mice (n = 6 per group) were then treated with PT, SIL, or NR at the doses indicated above as previously planned, and statistical analysis was performed using Student's t test (P < 0.01; * You are comparing 120 minutes against 30 minutes; + You are comparing day 2 against day 0; #(3 or 15 days are compared to 0 days). These results are shown in Figure 10. As can be seen from this figure, PT and SIL levels were higher 30 minutes after their administration, but NR-induced NAD + Levels were found to be similar at the two time points measured (30 min and 120 min).

[0207] Four compounds, PT, SIL, NR, and FSL-1, were then administered in groups of n=20 mice at the doses indicated above. Figure 11A shows the dosing schedule followed. Figure 11B shows the Kaplan-Meier curves and the LogRank (Mantel-Cox) test ( * All groups are compared to vehicle-treated controls; + (Comparing groups treated with PT+SIL+NR+FSL-1 to groups treated with PT+SIL+NR or groups treated with PT+SIL+FSL-1). Surprisingly, a synergistic effect was also observed when NR and FLS1 were combined with PT and SIL, as the survival of mice was extended for up to one year. Importantly, the combination of the four components provided protection and survival of up to 90% of treated mice, indicating that the combination of the four components achieves long-term survival of irradiated mice.

[0208] The cause of death in γ-irradiated mice (LD50 / 30) and the effect of administering the four compounds in combination were studied as shown in Figure 12. Swiss albino mice (n=20 per group) were treated with PT, SIL, NR, and FSL-1 at the doses shown above as planned, and the results showed that the complete combination (PT+SIL+NR+FSL-1) prevented all deaths related to a) gastrointestinal and hematopoietic ARS, and b) nephropathy. The complete combination also minimized deaths related to myeloid leukemia and infection / inflammation.

[0209] Next, gamma radiation-induced toxicity and the protection provided by the combination of PT, SIL, NR, and FSL-1 (abbreviated as PSNF) were studied. The results are shown in Table 3 below: [Table 3] TIFF2024504645000008.tif81170

[0210] As can be seen from Table 3, treatment with the full combination (PT+SIL+NR+FSL-1) helps ameliorate radiation-induced toxicity.

[0211] Finally, to elucidate whether treatment with the four combined compounds (PT+SIL+NR+FSL-1; PSNF) would interfere with the therapeutic efficacy of anticancer radiotherapy (X-rays) in tumor xenografts, female nu / nu nude mice (6-8 weeks old) (n=5 mice per group) were treated with 5 × 10 6 A549 (lung adenocarcinoma) cells or MDA-MB-231 (triple-negative breast cancer) cells were inoculated subcutaneously. Tumor volume was calculated based on two dimensions measured using a caliper and calculated using the formula: V = 0.5a × b 2 The tumor volume was expressed in cubic millimeters according to the formula: (where a and b are the long and short diameters of the tumor, respectively). Tumors were irradiated with X-rays (10 Gy) using a Philips 6-keV SL75 linear accelerator. Single-fraction radiation therapy was administered at a rate of 5.0 Gy / min. The protocol for PSNF administration was the same as described above, with radiation therapy administered on day 21. Statistical analysis was performed using Student's t-test (P<0.01; * All groups are compared against controls; + Groups treated with gamma radiation + PSNF are compared to groups treated with gamma radiation alone).

[0212] These results are shown in Figure 13, where it is seen that the combination of the four compounds does not reduce the effectiveness of anticancer treatment with X-rays, thus demonstrating the feasibility of using these compounds in patients undergoing the above-mentioned radiotherapy treatment.

[0213] Taken together, these results support the use of compositions comprising at least PT and SIL, and optionally NR and FSL-1, to protect against and mitigate damage caused by radiation. It is important to note that the radioprotective effects of these compounds were extended for an extended period of time (more than one year), as shown in Figures 3A and 11B.

[0214] Example 5: Pterostilbene, silibinin, and nicotinamide riboside reduce cellular DNA damage and FSL-1 lipopeptide promotes hematopoietic recovery As shown in Figure 14a, NR treatment increased NAD + Increases the content of NAD induced by gamma radiation + PT+SIL administered alone also prevented the decrease of NAD in all cells studied. + Administration of FK866, a specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), did not affect NAD levels in mice treated with PT+SIL+NR or PT+SIL+NR+γ-irradiation, nor did it affect the increase induced by NR (results not shown). + The levels of NAD induced by FK866 were reduced (Fig. 14a). + Depletion was associated with a dramatic decrease in NAMPT activity (Fig. 14b) (PT, SIL, NR, FSL-1, and / or γ-radiation did not affect the kinetics of FK866-induced inhibition of NAMPT activity (not shown)). Importantly, as shown in Fig. 14c, based on the DNA damage comet assay, PT+SIL or NR favored DNA repair, with their combination being the most effective (Fig. 14c). Possibly, the effect of polyphenols is an antioxidant-related mechanism, whereas NR-induced NAD +The increase in FSL-1 directly promotes DNA repair activity. As shown in Figure 14d, FSL-1 increases hematopoietic colony forming cells CFU-GM (colony forming unit-granulocyte, macrophage) and CFU-GEMM (colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte). Thus, confirmation of hematopoietic recovery after irradiation is suggested by the hematology-based data in Table 3 above.

[0215] Interaction of Nrf2, NF-κB, PGC-1α, and PARP1 in radioprotection Using isolated intestinal epithelial cells, we investigated potential signaling pathways that may be involved in the protective effect caused by the PT+SIL combination. Based on the data reported in Figures 5-8, PT+SIL exerts protection against γ-radiation-induced oxidative damage. Presumably, the observed protection is due to the induction of Nrf2 (nuclear factor erythroid 2-related factor 2)-dependent antioxidant defense (Figures 5-8). Anti-inflammatory properties have been reported for both polyphenols, suggesting the involvement of NF-κB (nuclear factor kappa light-chain-enhancer of activated B cells)-dependent signaling. NR-induced NAD +Increased pPGC-1α would also favor PARP1 (poly[ADP-ribose] polymerase 1)-dependent DNA repair, sirtuin activity, and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1α) activation. Furthermore, PT could induce PGC-1α, Nrf2 activation, and Sirt1 expression, while SIL could affect Sirt3 expression and PARP1 activity. As shown in Fig. 15a, compared with the control, both PT and SIL increased nuclear Nrf2 while decreasing nuclear NF-κB levels. In the case of Nrf2, the effect of PT is more pronounced than that exerted by SIL. Overall, these effects suggest a coordinated effect of promoting antioxidant defense while downregulating NF-κB-dependent proinflammatory responses. pPGC-1α was also increased by PT (Fig. 15a). Sirt1 levels were increased by PT. Interestingly, SIL alone had no effect on Sirt1 levels, but the combination of the two polyphenols increased Sirt1 levels more than PT alone (Fig. 15a). Both PT and SIL increased Sirt3 levels, but the effect of SIL on Sirt3 was stronger (Fig. 15a). Both PT and SIL increased PARP1 (Fig. 15a). Fig. 15b outlines the possible molecular interrelationships between these signaling cascades and the steps at which PT and SIL may exert their effects. To further investigate which specific molecules may be determinants in the radioprotective effect, we used isolated IECs (from mice treated with control or PT+SIL, as in Fig. 11a) and gene silencing-based approaches. As shown in Figure 15c, siRNAs targeting mouse Nfκbie, Nfe2l2, Ppargc1, Sirt1, Sirt3, Parp1, SOD2, or GPX1 gene expression promote an increase in free NF-κB and downregulation of all other molecular targets. Isolated IECs were cultured for 24 h in the absence or presence of PT+SIL at concentrations that ensured preservation of the radioprotective defense induced in vivo (see caption of Figure 15) and in the presence of specific siRNAs.As shown in Figure 15d, PT and SIL dramatically reduced the γ-irradiation-induced cell death observed in control (vehicle-treated) cells. Cell death analysis based on the effect of specific RNAs in cells exposed to γ-irradiation indicates that Nrf2, pPGC-1α, SOD2, and Sirt3 are the main contributors to the radioprotective effect caused by polyphenol treatment (Figure 15d).

[0216] conclusion Here, we investigated the effect of systemically administered PT against gamma radiation and found that combining PT and SIL resulted in a high percentage of survival. Furthermore, the relevance of potential radioprotectors and radiomitigators as a strategy to reduce radiation-induced damage and enhance survival remains an underexplored field. This study shows the efficacy of the combination to achieve long-term survival in mice subjected to lethal (LD50 / 30) gamma radiation. This novel strategy to demonstrate long-term survival may serve as a paradigm going forward. Thus, considering the large number of molecules being tested as radioprotectors and / or radiomitigators, this paradigm may help to open up the field. PT and SIL upregulate antioxidant defenses in various cell types and reduce gamma radiation-induced oxidative damage and cytogenetic changes (chromosomal aberrations and micronuclei formation). As shown in Figure 15, the protection exerted by PT and SIL may involve different interrelated signaling mechanisms. Oxidative stress and inflammation are direct pathophysiological mechanisms activated by exposing cells to harmful ionizing radiation. PT and SIL increase nuclear Nrf2 and antioxidant responses in various cell types (Figure 15), whereas they decrease NF-κB (Figure 15) and possibly radiation-associated inflammatory responses. Furthermore, PT increases Sirt1 levels, which promotes deacetylation of PGC-1α (Figure 15). PGC-1α is an inducer of Sirt3 expression, and as shown in Figure 15, SIL treatment is also associated with increased Sirt3 levels. Mitochondrial SOD2 (deacetylated by Sirt3) and GSH peroxidase (dependent on uptake of GSH from the cytosol) are thought to act to reduce mitochondria-derived ROS and prevent activation of apoptosis. Importantly, as shown in Figures 15c and 15d, the gene silencing-based approach identified a pathway linking Nrf2, Sirt3, and SOD2 as a clue to the radioprotective effect caused by the combined treatment with PT and SIL. Furthermore, NR inhibits NAD +PARP1 activity is also induced by both polyphenols (Figure 15). NR, the pyridine-nucleoside form of vitamin B3, produces NAD with a superior pharmacokinetic profile to other forms of B3 (nicotinic acid and nicotinamide). + This is the optimal NAD + Equivalent to a booster.

[0217] This study demonstrates that the combination of two radioprotectors, pterostilbene and silibinin, with two radiomitigators, nicotinamide riboside and fibroblast-stimulating lipoprotein 1, can provide long-term protection of normal tissues against lethal doses of radiation in mice. The established safety profile of these four molecules also makes it clear that continued research into this combination and its application in humans is warranted. An ideal radioprotector or radiomitigator should be stable, easily administered, have no associated systemic toxicity, and protect normal tissues from radiation-induced damage. The proposed combination meets these criteria while demonstrating high efficacy.

Claims

1. A composition comprising: a) pterostilbene or a salt thereof; and b) silibinin or a salt thereof.

2. The composition of claim 1, further comprising at least one radiation mitigating compound.

3. 3. The composition of claim 1 or 2, further comprising at least one or more radiation mitigation compounds, wherein said at least one or more radiation mitigation compounds is a NAD+ booster and / or fibroblast stimulating lipopeptide-1, or any salt thereof.

4. The composition of any one of claims 1 to 3, wherein the pterostilbene is pterostilbene phosphate disodium salt and / or the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt and the NAD+ booster is nicotinamide riboside.

5. A composition described in any one of claims 1 to 4, which is a pharmaceutical composition, a nutritional supplement food composition, a cosmetic composition, or a food composition.

6. A pharmaceutical comprising pterostilbene or a pharma- ceutical acceptable salt thereof in combination with silibinin or a pharma- ceutical acceptable salt thereof for use in the prevention, amelioration, or alleviation of a disease induced by ionizing radiation in a subject, said pharmaceutical comprising pterostilbene or a pharma- ceutical acceptable salt thereof, said pharmaceutical being administered before, after, or during radiation exposure, said combination being understood to mean the administration to said subject of a) pterostilbene or a pharma- ceutical acceptable salt thereof, and b) silibinin or a pharma-ceutical acceptable salt thereof, either simultaneously or sequentially in any order.

7. The pharmaceutical described in claim 6, administered in a single pharmaceutical composition or in separate pharmaceutical compositions.

8. The pharmaceutical of claim 6 or 7, wherein administration is topical, oral, or parenteral.

9. A pharmaceutical described in any one of claims 6 to 8, administered simultaneously or sequentially several times before and / or after radiation exposure, and wherein the combination is present in a dosage capable of providing a radioprotective effect.

10. The pharmaceutical agent according to any one of claims 6 to 9, which is administered in combination with at least one or more radiation mitigating compounds, where combination is understood as simultaneous or sequential administration to the subject of a) pterostilbene or a pharma- ceutically acceptable salt thereof, b) silibinin or a pharma- ceutically acceptable salt thereof, and c) at least one or more radiation mitigating compounds or any pharma- ceutically acceptable salt thereof.

11. The method of claim 10, wherein the at least one or more radiation mitigating compounds are a NAD+ booster and / or a fibroblast stimulating lipopeptide, or any pharma- ceutically acceptable salt thereof.

12. The pharmaceutical of claim 11, administered in combination with at least two or more radiation mitigation compounds, wherein the at least two or more radiation mitigation compounds are an NAD+ booster and a fibroblast stimulating lipopeptide, or any pharma- ceutical acceptable salt thereof.

13. The pharmaceutical composition of any one of claims 10 to 12, wherein the NAD+ booster is nicotinamide riboside.

14. Diseases induced by ionizing radiation are associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage; The pharmaceutical composition according to any one of claims 6 to 13, wherein the disease induced by ionizing radiation is acute radiation syndrome and / or chronic radiation syndrome.

15. The pharmaceutical according to any one of claims 6 to 14, wherein the ionizing radiation is gamma rays or X-rays.

16. The pharmaceutical according to any one of claims 6 to 15, wherein the pterostilbene is pterostilbene phosphate disodium salt and / or the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

17. Non-therapeutic cosmetic use of a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, wherein the combination is understood to be the administration of a) pterostilbene or a salt thereof, and b) silibinin or a salt thereof, simultaneously or sequentially in any order, to a subject.

18. A non-therapeutic cosmetic use as claimed in claim 17 for protecting against, ameliorating and / or reducing the harmful effects of sunlight on human skin and lips such as age spots, sunburn, sun spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discolouration, uneven skin tone, dullness, freckles, pimples, blemishes, fragile skin, dryness, mottling, roughness to the touch, cracking, sagging, thinning and enlarged pores.

19. A pharmaceutical comprising a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, used to increase the intracellular level of a sirtuin in a patient in need of such an increase, wherein the increase is compared to the intracellular level of sirtuin in the same patient prior to treatment with the combination of pterostilbene or a salt thereof and silibinin or a salt thereof.

20. The method of claim 19, wherein the patient in need of the increase is a patient suffering from a neurodegenerative disorder, a cardiovascular disorder, a pulmonary disorder, a metabolic disorder, or an age-related disorder.

21. The pharmaceutical composition of claim 20, wherein the disorder is selected from the group consisting of Alzheimer's disease or other dementia, or age-associated cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

22. A nutritionally supplemented food composition comprising a combination of pterostilbene or a salt thereof and silibinin or a salt thereof, wherein the combination is understood to mean administration of a) pterostilbene or a salt thereof, and b) silibinin or a salt thereof, simultaneously or sequentially in any order, to a subject.

23. 23. The nutraceutical composition according to claim 22, wherein the use as a nutraceutical composition 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 disorders and / or for the protection or repair of the harmful effects of sunlight on the human skin or lips.

24. 24. The nutraceutical composition of claim 23, wherein the diseases induced by ionizing radiation are associated with DNA damage, radiation-induced cytotoxicity, genotoxicity, and / or oxidative cell damage.

25. 24. The nutritional supplement food composition of claim 23, wherein the neurodegenerative, cardiovascular, pulmonary, metabolic or age-related disorder is selected from the group consisting of Alzheimer's disease or other dementias or age-associated cognitive impairment, Parkinson's disease, Huntington's disease, multiple sclerosis, Kennedy's disease (spinal-bulbar muscular atrophy), kidney disease, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, and diabetes.

26. 24. The nutraceutical composition of claim 23, wherein the harmful effects of sunlight on human skin or lips are selected from the group consisting of age spots, sunburn, sun spots, wrinkles, fine lines, wrinkles, crow's feet, spider veins, stretch marks, dark circles under the eyes, hyperpigmentation, hypopigmentation, discoloration, uneven skin tone, dullness, freckles, pimples, blemishes, fragile skin, dryness, mottling, roughness to the touch, cracking, sagging, thinning, enlarged pores, cellulite formation, acne formation, rosacea, psoriasis, and eczema.

27. A kit of parts comprising at least two receptors comprising at least pterostilbene or a salt thereof and silibinin or a salt thereof.

28. a. NAD+ booster, and / or b. a fibroblast stimulating lipopeptide, or any pharma- ceutically acceptable salt thereof; 30. The kit of parts of claim 27, further comprising at least one radiation mitigating compound selected from the group consisting of:

29. 30. The kit of parts of claim 28, wherein the NAD+ booster is nicotinamide riboside.

30. A kit of parts according to any one of claims 27 to 29, wherein the pterostilbene is pterostilbene phosphate disodium salt and / or the silibinin is silibinin-C-2',3-dihydrogen succinic acid disodium salt.

Images