Use of ergothioneine for inhibiting detrimental effects of aging

Abstract

The present invention relates to a method for inhibiting the detrimental effects of aging in mammals. The method comprises administering to the mammal a composition comprising a therapeutically effective amount of ergothioneine, or a pharmaceutically acceptable salt, acid, ester, analog, or derivative thereof. In some embodiments, the compositions inhibit the detrimental effects of aging by down-regulating p21 and / or SAbetaG.

Description

Background Art

[0001] Aging is the gradual deterioration of an organism's functional characteristics. It is characterized by a decreased ability to respond to stress, increased imbalances in homeostasis, and an increased risk of age-related diseases, including cancer and heart disease. Aging is an inevitable fate for nearly all germ-free multicellular organisms, but it can be delayed.

[0002] Ergothioneine ("Ergo") is a naturally occurring amino acid, a thiourea derivative of histidine, containing a sulfur atom on the imidazole ring. It is derived from relatively few organisms, notably Actinobacteria, Cyanobacteria, and certain fungi. Humans obtain ergothioneine only through the diet, where it is concentrated in red blood cells, bone marrow, liver, kidneys, semen, and eyes.

[0003] Although the effect of ergothioneine in vivo is an active area of ​​research, its physiological role in humans has not yet been determined (Cheah et al., "Ergothioneine; antioxidant potential, physiological function and role in disease", Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 2012, 1822(5):784-93). Summary of the invention

[0004] In a first aspect, the present invention provides a method of inhibiting the deleterious effects of aging in a mammal. The method comprises administering to the mammal a composition comprising a therapeutically effective amount of ergothioneine or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof.

[0005] In some embodiments, the mammal is a human, horse, cattle or other ruminant, pig, or pet.

[0006] In some embodiments, the composition is in the form of a food, a beverage, a nutritional composition, or a pharmaceutical composition.

[0007] In some embodiments, the composition is in the form of a solution, a liquid suspension, a parenteral solution, an injectable solution, a tablet, a pill, a granule, a powder, a film, a (micro)capsule, an aerosol, a tonic, a syrup, a beverage, or a dietary supplement.

[0008] In some embodiments, administration is at least once a day or multiple times a day.

[0009] In some embodiments, administration is by oral, intravenous, intramuscular, intraperitoneal, intranasal, rectal, or sublingual route.

[0010] In some embodiments, administration of the composition is by oral administration at a daily dose of ergothioneine in the range of 2-2,000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg, or 5-50 mg.

[0011] In some embodiments, the daily dose is administered in divided doses or in a single dose.

[0012] In some embodiments, the composition inhibits the deleterious effects of aging by downregulating p21 and / or SAβG.

[0013] In a second aspect, the present invention provides a composition comprising a therapeutically effective amount of ergothioneine or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof for use in inhibiting the deleterious effects of aging in a mammal in need thereof.

[0014] In some embodiments, the mammal is a human, horse, cattle or other ruminant, pig, or pet.

[0015] In some embodiments, the composition is in the form of a food, a beverage, a nutritional composition, or a pharmaceutical composition.

[0016] In some embodiments, the composition is in the form of a solution, a liquid suspension, a parenteral solution, an injectable solution, a tablet, a pill, a granule, a powder, a film, a (micro)capsule, an aerosol, a tonic, a syrup, a beverage, or a dietary supplement.

[0017] In a third aspect, the present invention provides a method of preparing any of the compositions disclosed herein.

[0018] In a fourth aspect, the present invention provides the use of any one of the compositions disclosed herein for inhibiting the deleterious effects of aging in a mammal.

[0019] In some embodiments, ergothioneine is administered in a daily dose of 2-2000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg, or 5-50 mg.

[0020] In some embodiments, administration is at least once a day or multiple times a day.

[0021] In some embodiments, administration is by oral, intravenous, intramuscular, intraperitoneal, intranasal, rectal, or sublingual route.

[0022] In some embodiments, the composition inhibits the deleterious effects of aging by downregulating p21 and / or SAβG.

[0023]

[0011] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0025] Figures 1A-1C is a bar graph showing the percentage of HDFn cells positive for Ki67, p21 or p16 protein treated with different concentrations (0, 0.5, 3.0 and 30 μM) of EGT and H2O2 at passages 22, 24 and 26, respectively. Figure 1D The percentage of HDFn cells positive for β-galactosidase after the same treatment is shown. p22 refers to passage 22, p24 refers to passage 24, and p26 refers to passage 26. Figures 1A-1D For each graph, two-way ANOVA was performed on 0 μM treatment with Bonferroni post hoc analysis. *p<0.05, **p<0.01, ***p<0.001. Figure 1E Representative images of p21 immunostaining of HDFn cells treated with different concentrations (0, 0.5, 3.0 and 30 μM) of EGT at passage 22 are shown. Figure 1F Shown are representative images of SAβG staining of HDFn cells treated with different concentrations (0, 0.5, 3.0, and 30 μM) of EGT at passage 26. Images are magnified 100 times.

[0026] Figure 2A is a bar graph showing cell counts of HDFn cells treated with different concentrations (0, 0.5, 3.0 and 30 μM) of EGT, DMSO, fisetin (FIS) and navitoclax (NAV) at passage 31. Positive controls for senescence elimination function are 600 μM fisetin and 13 μM navitoclax. DMSO is a vehicle control for fisetin. d0 refers to day 0 of inoculation, d0-3 refers to 3 days after treatment of cells inoculated on day 0, d6-9 refers to 3 days after treatment of cells inoculated on day 6, and d9-12 refers to 3 days after treatment of cells inoculated on day 9. Figure 2B Representative images of cell counts for various treatments are shown. Images are magnified 100X.

[0027] Figures 3A-3Cis a bar graph showing the percentage of HDFn cells positive for Ki67, p21 or p16 protein treated with different concentrations (0, 0.5, 3.0 and 30 μM) of EGT, DMSO, fisetin (FIS) and navitoclax (NAV), respectively, at passage 31. Figure 1D The percentage of HDFn cells positive for β-galactosidase after the same treatment is shown. d0-3 refers to 3 days after treatment for cells seeded on day 0, d6-9 refers to 3 days after treatment for cells seeded on day 6, and d9-12 refers to 3 days after treatment for cells seeded on day 9. Figures 3A-3D For each graph, the results of two-way ANOVA for 0 μM treatment were analyzed with Bonferroni post hoc analysis. *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0028] In the above Summary of the Invention section and the following Detailed Description section and in the claims, reference is made to specific features of the present invention. It should be understood that the disclosure of the present invention in this specification includes all possible combinations of these specific features. For example, where a specific feature is disclosed in the context of a specific aspect or embodiment of the present invention or a specific claim, the feature may also be used in combination with and / or in the context of other specific aspects and embodiments of the present invention to the extent possible, and is generally used in the present invention.

[0029] In a first aspect, the present invention provides a method of inhibiting the deleterious effects of aging in a mammal. The method comprises administering to the mammal a composition comprising a therapeutically effective amount of ergothioneine or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof.

[0030] In a second aspect, the present invention provides a composition comprising a therapeutically effective amount of ergothioneine or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof for use in inhibiting the deleterious effects of aging in a mammal in need thereof.

[0031] In a third aspect, the present invention provides a method of preparing any of the compositions disclosed herein.

[0032] In a fourth aspect, the present invention provides the use of any one of the compositions disclosed herein for inhibiting the deleterious effects of aging in a mammal.

[0033] At the macroscopic level, aging is the gradual deterioration of the functional characteristics of living organisms. It is characterized by a decreased ability to respond to stress, increased imbalances in homeostasis, and an increased risk of aging-related diseases, including cancer and heart disease.

[0034] At the microscopic level, senescence refers to the phenomenon in which normal non-malignant cells stop dividing in vitro. Microscopic senescence is also known as replicative senescence. Replicative senescence is induced by telomere shortening. With each round of DNA replication, telomeres gradually shorten, eventually reaching a critical length that prevents further replication, thus stopping cell division. Very short uncapped telomeres trigger a DNA damage response, which triggers senescence.

[0035] Certain proteins have been used as indicators of senescence induction or progression. Ki67 is a standard nuclear marker for cell proliferation. Proliferating cells result in stronger Ki67 staining, whereas senescent and quiescent cells do not show Ki67. P21 and p16 are early and late cell cycle checkpoint proteins, respectively. Upon induction of senescence by various inducers, p21 is first recruited to the nucleus to prevent further proliferation. If the stressor that induces senescence is not alleviated, then the recruitment of p16 strengthens this checkpoint signal. β-Galactosidase is a lysosomal enzyme that is upregulated during periods of cell proliferation arrest, including senescence and contact inhibition. The presence of each marker alone is not sufficient to declare a senescent phenotype. In MRC5 and IMR90 human fibroblast cell lines, the progression of Ki67, p21, p16, and SAβG during senescence development has been mentioned in many publications, but has never been clearly established and shown (e.g., Correia-Melo et al., 2016, Mitochondria are required for the pro-senescence features of the senescence phenotype. Journal of the European Molecular Biology Society, 35(7), 724-742). A consistent feature of these markers during senescence induction is the immediate downregulation of Ki67, followed by nuclear localization of p21 1–3 days after induction, followed by increased recruitment of p16 and SAβG activity 7–10 days after induction.

[0036] Ergothioneine is a naturally occurring amino acid, a thiourea derivative of histidine, containing a sulfur atom on the imidazole ring. It comes from relatively few organisms, particularly actinomycetes, cyanobacteria and certain fungi. Humans can only obtain ergothioneine through diet, and it is enriched in red blood cells, bone marrow, liver, kidney, semen and eyes. In vitro, ergothioneine requires a specific transporter ETT, also known as OCTN1 (gene symbol SLC22A4), to enter cells. Although the role of ergothioneine in vivo is an active research field, its physiological role in the human body has not yet been determined (Cheah et al., "Ergothioneine; Antioxidant capacity, physiological function and role in disease", "Biochimica et Biophysica Sinica (BBA)-Molecular Basis of Disease". 2012, 1822 (5): 784-93).

[0037] As used herein, "therapeutically effective amount" refers to a sufficient amount of ergothioneine for suppressing the harmful effects of mammal aging, with a reasonable benefit / risk ratio applicable to any medical treatment. However, it is understood that the total daily dosage of ergothioneine can be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutic effective dose level of any particular subject will depend on a variety of factors, including the severity of the illness and illness being treated; the specific composition used, the age, weight, general health, sex and diet of the subject; the excretion rate of the time of administration, route of administration and ergothioneine used; the duration of treatment; the medicine used in combination or simultaneously with ergothioneine; and similar factors well-known in the medical field. For example, it is well known to those skilled in the art that the dosage of the compound starts from less than the dosage required to achieve the desired therapeutic effect, and gradually increases the dosage until the desired effect is achieved. In addition, "therapeutically effective amount" refers to the amount of the biological or medical response of the tissue, system or subject sought by the researcher or clinician, particularly the amount of some desired therapeutic or preventive effects for suppressing the harmful effects of mammal aging.

[0038] Those skilled in the art recognize that even if the condition is not completely eradicated or prevented, but it or its symptoms and / or effects are partially ameliorated or alleviated in the subject, the amount can be considered therapeutically "effective." Various indicators for determining the effectiveness of a method for inhibiting the deleterious effects of aging in a mammal are known to those skilled in the art.

[0039] As used herein, the terms "treating / treatment," "therapeutic," or "therapy" do not necessarily refer to a complete cure or elimination of a disease or condition. Any alleviation of any undesirable signs or symptoms of a disease or condition, to any extent, may be considered treatment and / or therapy. In addition, treatment may include actions that may worsen a patient's overall sense of well-being or appearance.

[0040] In some embodiments, the composition comprises about 5 wt % to about 10 wt %, 20 wt %, 30 wt %, 40 wt %, 50 wt %, 60 wt %, 70 wt %, 80 wt %, 90 wt % or 95 wt % ergothioneine, preferably about 30 wt % to about 90 wt % ergothioneine, based on the total weight of the composition as 100 wt %.

[0041] The claimed compositions may contain other ingredients, such as other active agents, preservatives, buffers, salts, pharmaceutically acceptable carriers or other pharmaceutically acceptable ingredients.

[0042] As used herein, "carrier" refers to a compound that facilitates the incorporation of a compound into cells or tissues. For example, but not limited to, dimethyl sulfoxide (DMSO), ethanol (EtOH) or PEG400 are commonly used carriers that facilitate the absorption of many organic compounds into cells or tissues of a subject.

[0043] "Mammal" includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates such as monkeys, chimpanzees and apes, and particularly humans.

[0044] In some embodiments, the mammal is a human, horse, cow or other ruminant, pig, or pet. "Pet" includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, fish, reptiles, and sheep.

[0045] In some embodiments, the composition is prepared in the form of a food, a beverage, a nutritional composition, or a pharmaceutical composition.

[0046] The term "pharmaceutical composition" refers to a mixture of ergothioneine and other chemical components such as a diluent or carrier. The pharmaceutical composition helps to administer the compound to an organism. The pharmaceutical composition can also be obtained by reacting the compound with an inorganic or organic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. The pharmaceutical composition is generally suitable for specific desired routes of administration. The pharmaceutical composition is suitable for human and / or veterinary applications.

[0047] The pharmaceutical compositions described herein can be administered to human patients by themselves, or mixed with other active ingredients in pharmaceutical compositions, such as in combination therapy, or mixed with carriers, diluents, excipients, or combinations thereof. Suitable formulations depend on the selected route of administration. The formulation and administration techniques of the compounds described herein are known to those skilled in the art.

[0048] As used herein, "diluent" refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the volume of an effective drug whose mass is too small for manufacture and / or administration. It can also be a liquid used to dissolve a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate buffered saline that simulates the composition of human blood.

[0049] As used herein, "excipient" refers to an inert substance added to a pharmaceutical composition to provide the composition with, but not limited to, bulk, consistency, stability, binding capacity, lubrication, disintegration capacity, etc. A "diluent" is an excipient.

[0050] The pharmaceutical compositions disclosed herein can be manufactured in a manner that is itself known, such as by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

[0051] If desired, pharmaceutical composition can be presented in packaging or dispenser device, and packaging or dispenser device can contain one or more unit dosage forms containing active ingredient.Packaging can for example include metal or plastic foil, such as blister pack.Packaging or dispenser device can be accompanied by application instructions.Packaging or dispenser can also be accompanied by a notice relevant to the container, and its form is regulated by the government agency of management drug manufacturing, use or sale, and this notice reflects the approval of the agency to the drug form used for human or veterinary administration.For example, this type of notice may be the prescription drug label approved by the U.S. Food and Drug Administration, or the product instructions approved.It is also possible to prepare a composition that can include ergothioneine formulated in a compatible pharmaceutical carrier, place it in a suitable container, and label it for the treatment of indications.

[0052] In some embodiments, the composition is in the form of a solution, a liquid suspension, a parenteral solution, an injectable solution, a tablet, a pill, a granule, a powder, a film, a (micro)capsule, an aerosol, a tonic, a syrup, a beverage, or a dietary supplement.

[0053] As used herein, "parenteral solution" refers to a solution that can be administered to other parts of the body besides the mouth and digestive tract. It is not delivered through the intestines. For example, a parenteral solution can be delivered intravenously.

[0054] As used herein, "tonic" refers to a medicinal substance that gives a person a feeling of vitality or wellness.

[0055] As used herein, "syrup" refers to a viscous liquid derived from sugar-rich plants such as sugar cane, corn, and maple trees.

[0056] In some embodiments, administration is at least once a day or multiple times a day.

[0057] There are multiple techniques for administering the composition in the art, including but not limited to oral, rectal, topical, aerosol, injection and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injection. People can also administer the composition in a local rather than systemic manner, for example, by injecting the compound directly into the infected area, usually in the form of a long-acting or sustained release formulation. In addition, the composition can be administered in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposome will target the organ and be selectively absorbed by the organ.

[0058] In some embodiments, administration is by a route selected from oral, intravenous, intramuscular, intraperitoneal, intranasal, rectal, or sublingual routes.

[0059] As used herein, "intraperitoneal" refers to administration within or through the peritoneum. The peritoneum is a thin, transparent membrane that covers the walls of the abdominal cavity and contains / surrounds the abdominal organs, such as the stomach and intestines.

[0060] As used herein, "sublingual" means located or applied under the tongue.

[0061] In some embodiments, the administration of the composition is carried out by oral administration with a daily dose of 2-2000 mg ergothioneine. In some embodiments, the administration of the composition is carried out by oral administration with a daily dose of 5-500 mg ergothioneine. In some embodiments, the administration of the composition is carried out by oral administration with a daily dose of 5-25 mg ergothioneine.

[0062] The scope of dosage can be very wide, depending on the desired effect and treatment indication. The daily dosage regimen of adult patients can be, for example, ergothioneine between 0.01mg and 3000mg per kilogram of subject body weight, preferably between 1mg and 700mg, for example, 5 to 200mg, or the oral dosage of ergothioneine between about 0.1mg and about 1,000mg. According to the needs of the subject, the dosage can be a single dose or two or more doses given in one or more days. In some embodiments, the compound is applied in a continuous treatment of a period of time, such as a week or longer, or months or years. In some embodiments, ergothioneine or its pharmaceutically acceptable salt can be applied at a frequency lower than the frequency of application of the medicament in the standard of care. In some embodiments, ergothioneine or its pharmaceutically acceptable salt can be applied once a day. In some embodiments, the total time of the treatment regimen using ergothioneine or its pharmaceutically acceptable salt can be less than the total time of the treatment regimen using standard care.

[0063] In cases where human dosages of ergothioneine have been determined for at least some conditions, those same dosages may be used, or dosages between about 0.1% and 500%, more preferably between about 25% and 250%, of the determined human dosages. In cases where human dosages have not been determined, as in the case of newly discovered pharmaceutical compositions, suitable human dosages may be derived from the ED 50 or ID 50 Values ​​may be used, or extrapolated from other appropriate values ​​obtained from in vitro or in vivo studies, as defined by animal toxicity studies and efficacy studies.

[0064] As used herein, the term "ED 50 ” refers to the dose that produces the desired effect in 50% of the population, or the median effective dose.

[0065] In the case of administering a pharmaceutically acceptable salt, the dosage can be calculated as the free base. As will be appreciated by those skilled in the art, in certain cases, in order to effectively and actively treat particularly aggressive diseases or infections, it may be necessary to administer the compounds disclosed herein in an amount exceeding or even far exceeding the above preferred dosage range.

[0066] Dosage and interval can be adjusted individually to provide a plasma level of the active part sufficient to maintain a modulatory effect or minimum effective concentration (MEC). The dosage required to reach the MEC will depend on individual characteristics and route of administration. However, HPLC analysis or bioassay can be used to determine the plasma concentration. The dosage interval can also be determined using the MEC value. The composition should be administered using a regimen that maintains plasma levels above the MEC for 10-90%, preferably between 30-90%, and most preferably between 50-90%. In the case of local administration or selective uptake, the effective local concentration of the drug may be independent of the plasma concentration.

[0067] It should be noted that the attending physician knows how and when to terminate, interrupt or adjust administration due to toxicity or organ dysfunction. On the contrary, if the clinical response is insufficient (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. In the management of the disease of interest, the size of the administered dose will vary with the severity of the condition to be treated and the route of administration. For example, the severity of the condition can be assessed in part by standard prognostic assessment methods. In addition, the dosage and possible dosage frequency will also vary according to the age, weight and response of the individual patient. Programs comparable to those discussed above can be used in veterinary medicine.

[0068] In some embodiments, the daily dose is administered in divided doses or in a single dose.

[0069] In some embodiments, the composition inhibits the deleterious effects of aging by downregulating p21 and / or SAβG.

[0070] Upon induction of senescence by various inducers, p21 is first recruited to the nucleus to prevent further proliferation. β-Galactosidase is a lysosomal enzyme that is upregulated during cessation of cell proliferation. Therefore, downregulation of p21 and / or SAβG could inhibit the deleterious effects of senescence. definition

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. If there are multiple definitions for a term herein, the definition in this section shall prevail unless otherwise stated.

[0072] The terms used herein are intended to describe specific situations only and are not intended to be limiting. As used herein, the singular forms "a / an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, with respect to the terms "include," "comprise," "have," "have," or variations thereof used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0073] As used herein, the terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to supervision (e.g., continuous or intermittent) by a health care worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, nursing assistant, or hospice worker).

[0074] "Replicative senescence" refers to the phenomenon in which normal non-malignant cells stop dividing in vitro.

[0075] "Senostatic" refers to the selective inhibition of the deleterious effects of aging. "Senostatic signature" refers to the expression pattern of certain protein markers in the senostatic process.

[0076] "Senolytic" refers to the selective elimination of senescent cells. "Senolytic signature" refers to the expression pattern of certain protein markers in the senolytic process.

[0077] Control: A control is an individual or sample group used as a standard of comparison for checking the results of a survey or experiment. In some cases, a control is denoted as a reference.

[0078] As used herein, "diluent" refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the volume of an effective drug whose mass is too small for manufacture and / or administration. It can also be a liquid used to dissolve a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate buffered saline that simulates the composition of human blood.

[0079] As used herein, "excipient" refers to an inert substance added to a pharmaceutical composition to provide the composition with, but not limited to, bulk, consistency, stability, binding capacity, lubrication, disintegration capacity, etc. A "diluent" is an excipient.

[0080] As used herein, the term "treat / treatment" refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or preventive benefit. For example, treatment can include administering a system or cell population disclosed herein. Therapeutic benefit can refer to any treatment-related improvement or effect of one or more diseases, conditions, or symptoms being treated. For preventive benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who reports physiological symptoms of one or more diseases, even though the disease, condition, or symptom may not yet be manifested.

[0081] A "therapeutic effect" may occur if there is a change in the condition being treated. This change may be positive or negative. A "change" in the condition being treated may refer to a change of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75%, or 100% in the condition. The change may be based on an improvement in the severity of the condition being treated in an individual, or on a difference in the frequency of improvement in the condition in a population of individuals who receive and do not receive administration of therapy. The term "therapeutically effective" should be understood to have a definition corresponding to "having a therapeutic effect."

[0082] Any headings or sub-headings used herein are for organizational purposes only and should not be used to limit the scope of the embodiments disclosed herein.

[0083] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0084] The following examples are presented to provide a complete disclosure and description of how to make and use the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Example 1

[0085] MitoPrime (L-ergothioneine (EGT); lot number MP20200906) was received as 1 g powder from Nanjing Newbond Biotechnology Co., Ltd. (NNB), Nanjing, China. MitoPrime (lot number MP20200906) was produced by NNB using proprietary fermentation technology and added to the culture medium to achieve the specified final concentrations.

[0086] Pour the powder into a plastic bucket and store at room temperature away from light. Prepare fresh 0.5 mM, 3.0 mM, 30 mM, and 300 mM stock solutions of DMEM weekly and filter sterilize. Store the stock solutions at 4 °C. HDFn (neonatal human dermal fibroblast) cells

[0087] HDFn cells (C0045C, Thermo Scientific, US) were obtained and proliferated in standard DMEM proliferation medium (10% FBS) under standard culture conditions (37°C, 20% O2, 5% CO2) to passage 32 (population doubling 58). At each passage, the cells were cryopreserved and stored. The cells were considered replicatively senescent at passage 32. When necessary, the cryopreserved solution in the cryopreservation tube was gently transferred to 5 ml of pre-warmed 10% FBS medium using a pipette and gradually thawed and centrifuged. The supernatant was discarded, the cell pellet was resuspended in 10% FBS medium, and the cells were counted using a hemocytometer and trypan blue. Senescence inhibition feature analysis

[0088] The treatment conditions tested in this assay were 0, 0.5, 3.0 and 30 μM EGT in standard DMEM (10% FBS) with constant supplementation. HDFn cells were thawed at passage 20 and cultured at 1.5 × 10 5 Cells were seeded. Three culture flasks were seeded for each condition. The medium was changed every three days. At passages 22, 24, and 26, HDFn cells were seeded in 48-well plates at 10,000 cells / well, triplicate / flask, and cultured in DMEM medium containing 0, 0.5, 3.0, and 30 μM EGT, respectively. Cells proliferated to 70% or confluence after 7 days. The cells were then washed with PBS, fixed with 2% PFA, and stored at 4°C until immunostaining. For β-galactosidase staining, the staining solution was added to the cells immediately after fixation and incubated at 37°C for 24 hours. The staining solution was removed and infiltrated with PBS until imaging. As a positive control for the senescent phenotype, a set of wells from an untreated culture flask was seeded. After 70% confluence, the cells were treated with serum-free DMEM containing 600 μM hydrogen peroxide for 2 hours and then incubated with standard DMEM proliferation medium for 5 days to allow the senescent phenotype to develop. Cells were fixed and stained in the same manner as other treatment conditions.

[0089] The cell pellet from each flask was collected for RNA analysis and stored at -80 °C until further action. Analysis of aging elimination characteristics

[0090] The treatment conditions tested in this analysis were standard DMEM (10% FBS) containing 0, 3, 30, and 300 μM EGT as acute supplementation over 3 days. Positive controls for senescence-eliminating activity were 13 μM Navitoclax and 600 μM Fisetin. A 0.1% DMSO condition was introduced as a vehicle control for Fisetin. At passage 31, HDFn cells were seeded in triplicate in 48-well plates at 10,000 cells per well. After 2 days of adaptation, the cells were treated with the above conditions for 3 days. The cells were then washed with PBS and fixed with 2% PFA. The cells were infiltrated with PBS and stored at 4°C until immunostaining. For β-galactosidase staining, the staining solution was added to the cells immediately after fixation and incubated at 37°C for 24 hours. The staining solution was removed and the cells were infiltrated with PBS until imaging. This process was repeated at time points 0 to 3 days, 6 to 9 days, and 9 to 12 days. Histological procedures

[0091] The senescence marker panel Ki67, p16 and p21 were analyzed by immunohistochemical staining using HRP / DAB kit (ab64264, Abcam, UK). 1) Ki67-1:1000 dilution, rabbit antibody, ab92742, Abcam; 2) p16-1:500 dilution, rabbit antibody, ab108349, Abcam; 3) p21-1:100 dilution, rabbit antibody, ab109542, Abcam.

[0092] Cells were counterstained with hematoxylin.

[0093] Senescence-associated β-galactosidase solution and staining were performed according to the method described in Dimri et al., A biomarker that identifies senescent human cells in culture and in aging skin in vivo, Proc. Natl. Acad. Sci. 1995 Sep 26; 92(20): 9363-9367. Cells were counterstained with Nuclear Fast Red.

[0094] All stainings were imaged on an EVOS XL core microscope at 100X magnification. Quantification, analysis, statistics

[0095] Images were white balanced using macro commands on ImageJ. Cells were manually counted and recorded using the cell counter plugin. Graphs and statistics were created using GraphPad Prism 5. For each dataset, two-way ANOVA and Bonferroni post hoc test were performed on untreated controls. P values ​​on the graphs are shown as *p<0.05, **p<0.01, ***p<0.001. result Senescence inhibition feature analysis

[0096] In this analysis, HDFn cells treated with lower EGT concentrations (0, 0.5, 3.0, and 30 μM) for long periods of time were analyzed for senescence markers at two passages when the HDFn cells were still proliferating (passages 22 and 24), and at one passage when the HDFn cells were in replicative senescence (passage 26). Figures 1A-1F ). Passage 26 was chosen for final passage because HDFn cells showed a senescent phenotype for up to one week after this passage, however, replication continued at a slow rate until passage 31 (data not shown).

[0097] HDFn cells showed a general progression toward replicative senescence between passages 22 and 26; baseline (0 μM EGT treatment) Ki67-positive HDFn cells increased from 20% to 40% of the total cell population between passages 22 and 24 and decreased back to 20% at passage 26 ( Figure 1A Between passages 22 and 24, p21-positive HDFn cells increased from 82.5% ± 2.3 to 94.2% ± 0.3 (mean ± SEM) of the total cell population, and baseline p21-positive HDFn cells were high in all three passages ( Figure 1B In contrast, p16-positive HDFn cells showed a generation-dependent increase from 12.5% ​​to 51.2% ± 3.9 to 88.8% ± 9.9 (mean ± SEM) of the total cell population at passages 22, 24, and 26, respectively ( Figure 1C Baseline aging-associated β-galactosidase (SAβG) was low in all three generations (3.6% ± 0.6, 7.1% ± 0.2, and 9.8% ± 1.7) ( Figure 1D ). This was unexpected because even with hydrogen peroxide treatment, SAβG levels remained low.

[0098] For all three passages, EGT treatment did not result in a statistically significant change in the percentage of Ki67-positive HDFn cells when compared to untreated controls ( Figure 1AIn contrast, EGT reduced the percentage of p21-positive HDFn cells in passage 22 by 16.5% (82.5% ± 2.3 to 66.0% ± 2.4, p < 0.001) at 0.5 μM, by 20.1% (p < 0.001) at 3 μM, and by 21.8% (p < 0.001) at 30 μM ( Figure 1B EGT treatment did not result in a statistically significant change in the percentage of p21-positive HDFn cells at passages 24 or 26 ( Figure 1B EGT also did not strongly affect the percentage of p16-positive HDFn cells in all three passages, although p16-positive cells were reduced by 15% in passage 24 after 0.5 μM EGT treatment (p<0.001, Figure 1C All three concentrations of EGT reduced the percentage of SAβG-positive HDFn cells at passage 26: from 9.8% ± 2.7 to 4.8% ± 5.7, 3.5% ± 2.0, and 5.0% ± 2.9 at 0.5 μM, 3.0 μM, and 30 μM, respectively (p < 0.001). Although the absolute levels of SAβG-positive cells were low, the average reduction in SAβG was 50%.

[0099] Upon induction of senescence by various inducers, p21 is first recruited to the nucleus to prevent further proliferation. β-Galactosidase is a lysosomal enzyme that is upregulated during cessation of cell proliferation. Therefore, downregulation of p21 and / or SAβG may suppress the deleterious effects of senescence. In conclusion, it appears that long-term low-dose L-ergothioneine can produce a senescence-suppressive effect in HDFn cells. Analysis of aging elimination characteristics

[0100] In this analysis, HDFn cells acutely treated with higher EGT concentrations were analyzed for cell population counts and senescence markers at passage 31 to determine if EGT was senescence-abating. 13 μM navitoc and 600 μM fisetin were used as positive controls. Three time points were taken for this analysis at days 0, 6, and 9 post-plating, plus three days of EGT exposure, to ensure that senescent HDFn time points could be captured.

[0101] Figure 2A is a bar graph showing the cell counts for various treatments. Figure 2B Representative images of cell counts for various treatments are shown. These data show that cell counts did not change much in the treatments with 0, 3, 30, and 300 μM EGT, whereas in the positive control, cell counts were significantly reduced ( Figure 2A The data indicated that EGT did not exhibit senescence-eliminating activity when HDFn cells were treated starting at passage 31.

[0102] Figures 3A-3DThe percentage of HDFn cells positive for senescence markers at each time point is shown. Figure 3B It was shown that at time point 1 (day 0 to day 3), 30 and 300 μM EGT reduced the percentage of p21-positive HDFn cells from 63.1% ± 9.3 to 29.1% ± 7.4 and 27.0% ± 2.0, respectively. For this time point, the percentage of p16-positive HDFn cells was also reduced from 67.8% ± 4.7 to 7.0% ± 2.8 and 11.7% ± 5.9 ( Figure 3C ), while EGT had no statistically significant effect on the percentage of SAβG-positive HDFn cells at any time point ( Figure 3D ). At the 9-12 day time point, 30 and 300 μM EGT also increased the Ki67-positive population from 3.4% ± 1.5 to 9.2% ± 3.8 and 11.4% ± 2.6, respectively (p < 0.001) ( Figure 3A ). The increase in the percentage of Ki67-positive HDFn cells at later time points could be a result of downregulation of p16 and p21 at earlier time points. Despite the observed decrease in senescent markers, the number of cells present in the analysis remained the same. This means that senolytic activity is not occurring because senolytic activity is being recognized by the selective clearance of senescent cells.

[0103] In summary, this analysis demonstrates that L-ergothioneine is not senolytic up to 300 μM.

[0104] This study aims to investigate whether the compound ergothioneine (EGT) has any aging inhibition or aging elimination activity. Human dermal fibroblast (HDFn) cultures were supplemented with 0.5, 3.0 and 30 μM L-ergothioneine, and the effect of EGT on replicative aging was observed. Biomarkers of cell aging (Ki67, p16, p21 and aging-related β-galactosidase) were analyzed in three selected passages to determine aging inhibition activity. In addition, once the HDFn cultures reached senescence, they were supplemented with 3, 30 and 300 μM EGT for three days, and the biomarkers of aging were analyzed to determine any potential aging elimination effects of EGT.

[0105] The data suggest that EGT has senolytic activity, but does not show senolytic activity. There are no reports of EGT activity in the caspase pathway, through which the best-known senolytics act. Therefore, the data presented here are fairly consistent with previous reports.

[0106] Current publications on EGT suggest that it is an antioxidant that may act through the p38 MAPK pathway. This may indicate that, in addition to other senescence-inhibiting functions through the MAPK pathway (Anerillas et al., 2020, Regulation of aging traits by MAPKs. Gerontology, 42(2), 397-408), EGT also induces senescence inhibition by affecting the NAD cycle (Nacarelli et al., 2019, NAD. Nature Cell Biology, 21(3), 397-407). The data presented here provide a basis for further mechanistic studies of the senescence-inhibiting function of EGT, and it can be safely stated that EGT has senescence-inhibiting properties.

[0107] Based on the above data, L-ergothioneine has a putative senescence-suppressing function but not a senescence-eliminating function in HDFn. References Anerillas, C., Abdelmohsen, K., & Gorospe, M. (2020). Regulation of senescence traits by MAPKs. Geroscience, 42(2), 397-408. Correia-Melo, C., Marques, F. D., Anderson, R., Hewitt, G., Hewitt, R., Cole, J., Passos, J. F. (2016). Mitochondria are required for pro-ageing features of the senescent phenotype. EMBO J, 35(7), 724-742. Nacarelli, T., Lau, L., Fukumoto, T., Zundell, J., Fatkhutdinov, N., Wu, S., Zhang, R. (2019). NAD. Nat Cell Biol, 21(3), 397-407.

Claims

1. A method of inhibiting the deleterious effects of aging in a mammal in need thereof, characterized in that The method comprises administering to the mammal a composition comprising a therapeutically effective amount of ergothioneine or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof.

2. The method according to claim 1, characterized in that The mammal is a human, a horse, a cow or other ruminant, a pig or a pet.

3. The method according to any one of claims 1 to 2, characterized in that: The composition is in the form of a food, a beverage, a nutritional composition or a pharmaceutical composition.

4. The method according to any one of claims 1 to 3, characterized in that The composition is in the form of a solution, a liquid suspension, a parenteral solution, an injectable solution, a tablet, a pill, a granule, a powder, a film, a (micro)capsule, an aerosol, a tonic, a syrup, a beverage or a dietary supplement.

5. The method according to any one of claims 1 to 4, characterized in that The administration is at least once a day or multiple times a day.

6. The method according to any one of claims 1 to 5, characterized in that The administration is carried out by oral, intravenous, intramuscular, intraperitoneal, intranasal, rectal or sublingual routes.

7. The method according to any one of claims 1 to 5, characterized in that The administration of the composition is performed orally at a daily dose of ergothioneine in the range of 2-2,000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg or 5-50 mg.

8. The method according to claim 7, characterized in that The daily dose is administered in divided doses or as a single dose.

9. The method according to any one of claims 1 to 8, characterized in that The composition inhibits the deleterious effects of aging by downregulating p21 and / or SAβG.

10. A composition comprising a therapeutically effective amount of ergothioneine or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof, characterized in that, The compositions are useful for inhibiting the deleterious effects of aging in a mammal in need thereof.

11. The composition according to claim 10, characterized in that The mammal is a human, a horse, a cow or other ruminant, a pig or a pet.

12. The composition according to any one of claims 10-11, characterized in that The composition is in the form of a food, a beverage, a nutritional composition or a pharmaceutical composition.

13. The composition according to any one of claims 10 to 12, characterized in that The composition is in the form of a solution, a liquid suspension, a parenteral solution, an injectable solution, a tablet, a pill, a granule, a powder, a film, a (micro)capsule, an aerosol, a tonic, a syrup, a beverage or a dietary supplement.

14. A method for preparing the composition of any one of claims 10-13.

15. The use of the composition according to any one of claims 10 to 13, characterized in that The compositions are useful for inhibiting the deleterious effects of aging in a mammal in need thereof.

16. The use according to claim 15, characterized in that Ergothioneine is administered in a daily dose of 2-2000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg or 5-50 mg.

17. The use according to claim 16, characterized in that The administration is at least once a day or multiple times a day.

18. The use according to any one of claims 16-17, characterized in that The administration is carried out by oral, intravenous, intramuscular, intraperitoneal, intranasal, rectal or sublingual routes.

19. The use according to any one of claims 15 to 18, characterized in that The composition inhibits the deleterious effects of aging by downregulating p21 and / or SAβG.

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