CN116528854A - Methods and compositions related to the use of ergothioneine - Google Patents

Abstract

Provided herein are compositions comprising ergothioneine (e.g., a natural form of purified L-ergothioneine) and methods of using such compositions, e.g., for improving cell viability, maintaining telomere length, or treating telomere-related disorders. In some embodiments, the ergothioneine has a purity of at least or about 98%.

Description

Methods and compositions related to the use of ergothioneine

RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S. provisional application No. 62/969,607 entitled "methods and compositions related to the use of ergothioneine" (METHODS AND COMPOSITIONS RELATED TO THE USE OF ERGOTHIONEINE) filed on 3/2/2020; united states provisional application 62/969,645 entitled "methods and compositions related to the use of ergothioneine" (METHODS AND COMPOSITIONS RELATED TO THE USE OF ERGOTHIONEINE), filed on 3/2/2020; united states provisional application No. 63/051,211 entitled "methods and compositions related to the use of ergothioneine" (METHODS AND COMPOSITIONS RELATED TO THE USE OF ERGOTHIONEINE) filed on 7/13 in 2020; the contents of each of which are incorporated herein by reference in their entirety.

Disclosure of Invention

Ergothioneine is a naturally occurring histidine metabolite with a thiol group attached to the C2 atom of the imidazole ring. It is synthesized by bacteria and fungi that have antioxidant and anti-inflammatory properties and can help prevent and counter oxidative stress that can trigger inflammatory responses. The present disclosure is based, at least in part, on the surprising discovery of the ability of ergothioneine to maintain telomere length in cells and improve cell viability. Thus, herein are ergothioneine compositions useful for such purposes as well as for treating, preventing, or reducing the risk of diseases or disorders associated with telomere shortening, preventing or delaying the aging process, treating, preventing, or reducing the risk of cognition-related diseases or disorders, and the like. The compositions and methods provided herein are also useful for treating or preventing oxidative stress-related disorders, diseases, or conditions associated with telomere shortening. Methods and compositions for treating, preventing, or reducing the risk of oxidative stress-related disorders, diseases, or conditions are also provided. Methods for achieving the above result are also provided.

Some aspects of the present disclosure provide methods for improving cell viability by contacting a cell with a ergothioneine composition.

Some aspects of the present disclosure provide methods for maintaining telomere length in a cell or inhibiting or preventing telomere shortening in a cell by contacting the cell with a ergothioneine composition.

Some aspects of the disclosure provide methods for reducing the rate of telomere shortening in a cell by contacting the cell with a ergothioneine composition.

Some aspects of the present disclosure provide methods for reducing the amount, e.g., percentage, of short telomeres in a cell by contacting the cell with a ergothioneine composition.

Some aspects of the disclosure provide methods for prolonging telomeres in cells by contacting the cells with a ergothioneine composition.

Some aspects of the disclosure provide methods of treating, preventing, or reducing the risk of a telomere related disorder, disease, or other condition. In some embodiments, the method is a method of treating, preventing, or reducing the risk of premature aging in a subject.

Some aspects of the present disclosure provide methods of treating, preventing, or reducing the risk of a cognition-related disorder, disease, or other condition.

Some aspects of the present disclosure provide methods of treating, preventing, or reducing the risk of oxidative stress-related disorders, diseases, or other conditions. In some embodiments, the oxidative stress-related disorder, disease, or other condition is a viral infection (e.g., a respiratory viral infection). In some embodiments, the oxidative stress-related disorder, disease, or other condition is covd-19.

In some embodiments of any one of the methods or compositions provided herein, the contacting is in vitro. In some embodiments, the contacting is performed by incubating the cells, such as a test cell population, with the ergothioneine composition.

In some embodiments of any one of the methods or compositions provided herein, the contacting is in vivo. In some embodiments, the contacting is performed by administering ergothioneine such that the ergothioneine contacts cells in the subject.

In some embodiments of any one of the methods or compositions provided herein, the composition comprises purified ergothioneine, such as a natural form of purified ergothioneine. In some embodiments of any one of the methods or compositions provided herein, the purified ergothioneine has a purity of about 98% or at least 98%.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine is L-ergothioneine.

In some embodiments of any one of the methods or compositions provided herein, the composition is in the form of a free-flowing powder.

In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine contacted with a cell (e.g., an incubated cell, such as a cell in a test cell population) is about 0.04mg/ml to about 1mg/ml.

In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine contacted with a cell (e.g., an incubated cell, such as a cell in a test cell population) is about 0.04mg/ml or at least 0.04mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine contacted with a cell (e.g., an incubated cell, such as a cell in a test cell population) is about 0.1mg/ml or at least 0.1mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine contacted with a cell (e.g., an incubated cell, such as a cell in a test cell population) is about 0.3mg/ml or at least 0.3mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine contacted with a cell (e.g., an incubated cell, such as a cell in a test cell population) is about 1mg/ml or at least 1mg/ml.

In some embodiments of any one of the methods or compositions provided herein, cell viability is achieved or measured by maintaining the average telomere length of a cell, such as a test cell population, or preventing or inhibiting the shortening of telomere length in a cell, or slowing the rate of shortening of telomere length in a cell, or reducing the amount, such as a percentage, of short telomeres in a cell, or extending telomeres in a cell.

In some embodiments of any one of the methods or compositions provided herein, the average telomere length of a cell, e.g., a test cell population, is maintained as a result of contact (e.g., by incubation or administration) with the ergothioneine composition as compared to the average telomere length of a control cell, e.g., a control cell population, in the absence of such contact. In some embodiments of any one of the methods or compositions provided herein, the average telomere length of a cell, e.g., a test cell population, resulting from contact (e.g., by incubation or administration) with the ergothioneine composition is equal to or greater than the average telomere length of a control cell, e.g., a control cell population, without such contact. In some embodiments of any one of the methods or compositions provided herein, due to contact with the ergothioneine composition (e.g., by incubation or administration), telomere shortening is slowed in cells, such as a test cell population, such as the average telomere shortening rate of the cells is lower than that of control cells without such contact, as shown by the average telomere shortening rate of the control cell population. In some embodiments of any one of the methods or compositions provided herein, the amount, e.g., percentage, of short telomeres in the cells is reduced. In some embodiments of any one of the methods or compositions provided herein, the telomeres of the cells are elongated.

In some embodiments of any one of the methods or compositions provided herein, the telomere length is determined by single cell telomere mapping techniques. In some embodiments of any one of the methods or compositions provided herein, the single cell telomere mapping technique is based on a fluorescent in situ hybridization assay.

In some embodiments of any one of the methods or compositions provided herein, the cell, such as the test cell population, is a skin cell or tissue sample from the skin. In some embodiments of any one of the methods or compositions provided herein, the cell, such as the test cell population, is an intestinal cell or a tissue sample from the small intestine. In some embodiments of any one of the methods or compositions provided herein, the cell, e.g., test cell population, is a testicular cell or a tissue sample from a testicle. In some embodiments of any one of the methods or compositions provided herein, the cell, such as the test cell population, is a keratocyte or tissue sample from the cornea. In some embodiments of any one of the methods or compositions provided herein, the cell, such as the test cell population, is a neural cell or brain cell, or a tissue sample from the brain. In some embodiments of any one of the methods or compositions provided herein, the cell, e.g., the test cell population, is a blood cell. In some embodiments of any one of the methods or compositions provided herein, the cell, e.g., test cell population, is a bone marrow cell or from a bone marrow sample. In some embodiments of any one of the methods or compositions provided herein, the cell, such as the test cell population, is a tumor or cancer cell, or a tissue sample from cancerous or tumor tissue.

In some embodiments of any one of the methods or compositions provided herein, the cell or cell population is from or is an implantable organ or body part comprising tissue, such as heart, lung, heart lung, kidney, liver, pancreas, intestine, skin, face, hand, leg, penis, bone, uterus, thymus, islets, heart valve, or ovary. In some embodiments of any one of the methods or compositions provided herein, the cell or cell population is from or is an amputation or other body part, comprising tissue.

In some embodiments of any one of the methods or compositions provided herein, the cell, such as a control cell, is a cell of an immortalized cell line. In some embodiments of any one of the methods or compositions provided herein, the cells, such as control cells, are Hela 2, heLa, MCF7, heLa S3, 293T, L5178Y-S, MEFs BL 6G 3 Terc-/-, MEFs BL6 wild-type, heLa 1211, MEFs 129Sv/BL6 wild-type, and L5178Y-R cells, or are of the same type as the test cells, or are derived from a tissue sample, such as a tissue sample of the same type as the test cells.

In some embodiments of any one of the methods provided herein, the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising ergothioneine. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 5mg to about 30mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 5mg to about 25mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 5mg per day or at least 5mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 25mg per day or at least 25mg per day.

In some embodiments of any one of the methods provided herein, treating, preventing, or reducing the risk of a disease or disorder associated with telomere shortening comprises maintaining the average telomere length of the cells, preventing or inhibiting telomere length shortening in the cells, reducing the rate of telomere shortening of the cells in the target environment, reducing the amount, e.g., percentage, of short telomeres, or extending telomeres in a subject in need thereof. In some embodiments of any one of the methods provided herein, the disease or disorder associated with telomere shortening is premature aging or associated with premature aging, or is a cognition-related disorder, disease, or other condition. In some embodiments of any one of the methods provided herein, the disease or disorder associated with telomere shortening is or is associated with chronic inflammation. In some embodiments of any one of the methods provided herein, the disease or disorder associated with telomere shortening is caused by or associated with oxidative stress.

In some embodiments of any one of the methods provided herein, the method is for maintaining healthy, a healthy condition, a healthy body, healthy cells, e.g., may slow the aging process, reduce the risk of disease, etc. in some embodiments.

Some aspects of the present disclosure provide compositions, such as pharmaceutical formulations, comprising a therapeutically effective amount of a ergothioneine composition and optionally one or more pharmaceutically acceptable carriers. In one embodiment, any of the compositions provided herein may be used for any of the purposes provided herein.

Drawings

FIG. 1 shows a graph of ergothioneine metabolism and its antioxidant mechanism. The figure was adapted from krey (Kerley) et al, free radical biology and medicine (Free Radical Biology and Medicine), DOI: 10.1016/j.freereadbioid.2017.12.030.

FIG. 2 shows the pathways of ergothioneine biosynthesis in bacteria and fungi. The figure was adapted from Fan Dehuo g (van der Hoek) et al, bioengineering and biotechnology front (front. Bioeng. Biotechnol) (2019).

FIGS. 3A and 3B show the growth curves of primary human fibroblasts treated with ergothioneine. Cells were cultured under (A) standard conditions or (B) oxidative conditions (10. Mu. M H2O 2). Each point on the population curve represents the average of three replicates per cell passage. PD: population doubling.

FIG. 4 shows the effect of ergothioneine on relative telomerase activity. Data are mean ± standard deviation (n=3). * p <0.05, compared to control.

Figures 5A-5D show the effect of ergothioneine on telomere length under standard conditions. (a) median telomere length (50 th percentile), (B) 20 th percentile telomere length, (C) short telomere (< 3 kbp) percent, (D) telomere shortening rate. Median telomere length (initial-final)/population doubling. Data are mean ± standard deviation (n=3). * p <0.05, < p <0.01, < p <0.001, compared to control. Kbp: kilobase pairs; PD: population doubling.

FIGS. 6A-6D show the effect of ergothioneine on telomere length under oxidative conditions. (a) median telomere length (50 th percentile), (B) 20 th percentile telomere length, (C) short telomere (< 3 kbp) percent, (D) telomere shortening rate. Median telomere length (initial-final)/population doubling. Data are mean ± standard deviation (n=3). * p <0.05, < p <0.01, < p <0.001, compared to control. Kbp: kilobase pairs; PD: population doubling.

FIGS. 7A-7D show the effect of ergothioneine, including telomere prolongation (FIG. 7D, dose 0.3 mg/ml).

Detailed Description

Aspects of the present disclosure relate to the use of ergothioneine to improve cell viability in vitro or in vivo and/or by maintaining the telomere length of cells when compared to cells that have not been contacted or treated with ergothioneine. Thus, in some embodiments, the methods and compositions described herein, e.g., pharmaceutical formulations, can be used to treat, prevent, or reduce the risk of a telomere-related disorder, disease, or other related condition.

As used herein, the terms "improvement" and "improvement" refer to a process or action: which produces enhanced results when compared to the corresponding counterpart that is not affected by the process or action. As used herein, the term also refers to a process or action that does not result in a change in result when compared to the corresponding counterpart that is not affected by the process or action.

As used herein, "purified" or "purified" refers to the release of something from foreign contaminated or bad substances. As used herein, a "substantially pure" formulation of something refers to a formulation having a purity of the desired substance of at least 90% (i.e., 90% or greater than 90%). Any of the ergothioneine compositions provided herein can be substantially pure.

As used herein, the terms "incubation" and "incubation" refer to the process of mixing two or more chemical or biological entities (e.g., compounds and cell populations) and allowing them to interact under conditions that favor the cell culture conditions disclosed herein.

As used herein, "cell viability" refers to a measure or determination of the proportion of living, healthy cells within a cell population, which can be measured by assays known or understood by those skilled in the art. Cell viability assays can be used to determine the overall health of cells, optimize culture or experimental conditions, and measure cell viability after treatment with a compound. For example, as described herein, cell viability may be determined by measuring the average telomere length of a cell of interest after any of the ergothioneine compositions provided herein.

The term "free-flowing powder" or "free-flowing powder form" refers to a powder composition having satisfactory flowability, which is the ability of the powder to flow. For example, the free flowing powder may be pourable or easily pourable.

Generally, as used herein, the term "about" when referring to an amount that is measurable, such as weight, time, dose, etc., is intended to encompass a variation from the specified amount of ± 15% or ± 10% in one example, a variation of ± 5% in another example, a variation of ± 1% in another example, and a variation of ± 0.1% in yet another example, as such a variation is suitable for performing the disclosed methods and disclosed compositions, such as pharmaceutical compositions.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Purified ergothioneine compositions

Aspects of the disclosure relate to the use of purified ergothioneine compositions, e.g., to improve cell viability of cells, e.g., test cell populations, or in methods of treating, preventing, or reducing risk provided herein.

Ergothioneine is a naturally occurring histidine metabolite with a thiol group attached to the C2 atom of the imidazole ring, synthesized by bacteria and fungi with antioxidant properties. Ergothioneine can scavenge oxidized species that are not free radicals. It has the action of being a fungal metabolite, a plant metabolite, a xenobiotic metabolite and a chelating agent.

Ergothioneine is ubiquitous in plants and animals, and in many human foods. Actinomycetes, such as Mycobacterium smegmatis (Mycobacterium smegmatis) and filamentous fungi, such as Neurospora crassa (Neurospora crassa), can produce ergothioneine. L-ergothioneine can be present in very small amounts in mushrooms, oats, garlic, animal organs (liver, kidney) and some species of beans. Without wishing to be bound by theory, ergothioneine is biosynthesized by fungi and mycobacteria and is captured by plants through their roots. In general, the highest levels of ergothioneine (0.1 mg/g-1mg/g dry matter) have been found in mushrooms.

Animals and plants are unable to produce ergothioneine endogenously, and therefore, ergothioneine must be obtained from food sources, or from their environment for plants. Ergothioneine is rapidly cleared from the circulation and then remains with minimal metabolism. For example, rats ingest ergothioneine with a systemic half-life of 1 month. The tissue of ergothioneine content varies widely and depends largely on its dietary level. In addition to erythrocytes and bone marrow, high ergothioneine levels are also found in semen, ocular lenses, and skin.

Ergothioneine is a powerful hydroxyl radical scavenger and is iron or copper ion dependent from hydrogen peroxide(H ₂ O ₂ ) Inhibitors of hydroxyl radical production. A specific ergothioneine transporter has been identified (gene symbol SLC22A4-PMID 15795384). SLC22A4 is highly expressed in the kidney, which is thought to contribute to the active secretion of organic cations in the kidney and possibly promote the active reabsorption of ergothioneine. A highly specific transporter of L-ergothioneine, known as OCTN1, has also been produced, which results in high bioavailability and is striven for retention. Furthermore, ergothioneine appears to play a critical protective role in monocytes, as it has recently been found that the occurrence of rheumatoid arthritis and Crohn's disease is associated with a mutated ergothioneine transporter gene (PMID: 15795384).

Ergothioneine is synthesized from one molecule of L-histidine (1), one molecule of L-cysteine (2), and 3 methyl groups provided by S-adenosyl-L-methionine. The biosynthesis of ergothioneine in bacteria is described in Richard-Greenblatet et al, (2015) journal of biochemistry (J Bio Chem) 290 (38): 23064-23076 and incorporated by reference. The biosynthesis of ergothioneine involves at least: (1) EgtB (or iron (II) -dependent oxidoreductase EgtB) which catalyzes the oxidative vulcanization of histidine trimethylinner salt (N-alpha, N-alpha-trimethyl-L-histidine) by adding oxygen and gamma-glutamyl-cysteine to the histidine trimethylinner salt; (2) EgtC (or an amidase) catalyzes the hydrolysis of gamma-glutamyl amide bonds from N- (gamma-glutamyl) - [ N (alpha), (alpha) -trimethyl-L-histidyl ] -cysteine sulfoxide to produce histidine trimethylcysteine sulfoxide; (3) EgtD (or histidine-specific methyltransferase EgtD) catalyzes histidine methylation to form N- α, N- α, N- α -trimethyl-L-histidine (also known as histidine trimethylinner salt); and (4) EgtE (or pyridoxal phosphate dependent protein EgtE) is believed to catalyze the removal of pyruvate, ammonia and oxygen to produce ergothioneine.

In some embodiments, microbial ergothioneine biosynthesis can be performed in vitro using engineered host cells. In some embodiments, suitable host cells may be, for example, bacterial cells and yeast cells. In some embodiments, a suitable yeast cell may be Saccharomyces (Saccharomyces), preferably Saccharomyces cerevisiae (Saccharomyces cerevisiae). In some embodiments, a suitable yeast cell may be Pichia (Pichia), preferably Pichia pastoris. In some embodiments, microbial ergothioneine biosynthesis may involve a fermentation process. Methods for producing ergothioneine are described in PCT/US2015/027977 and incorporated by reference.

The ergothioneine composition may be purified and in a native form. In some embodiments of any one of the methods or compositions provided herein, the native form may be derived from a bacterium. In some embodiments of any one of the methods or compositions provided herein, the native form may be derived from a plant. In some embodiments of any one of the methods or compositions provided herein, the native form may be derived from an animal. In some embodiments of any one of the methods or compositions provided herein, the native form may be derived from sources including, but not limited to, liver, kidney, black beans, oat bran, bolete, and oyster mushrooms. In some embodiments of any one of the methods or compositions provided herein, the native form may be derived from any source suitable for producing the compositions described herein.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine can be derived from an in vitro bioengineered cell host.

In some embodiments of any one of the methods or compositions provided herein, the form of ergothioneine for use in any one of the methods or compositions provided herein is(Blue California, san ma gerita farm, california). In some embodiments of any one of the methods or compositions provided herein, the ergothioneine comprises L-ergothioneine. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine comprises ergothioneine in any configuration suitable for the methods and compositions described herein. For example, a suitable configuration of ergothioneine is one that improves cell viability and/or maintains telomere length.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine in the compositions described herein is substantially pure. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine in the composition has a purity greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%, greater than about 76%, greater than about 77%, greater than about 78%, greater than about 79%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%, including all ranges and subranges therebetween. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine in the compositions described herein has a purity of 98%, 99%, or more.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be in the form of a free-flowing powder. Without wishing to be bound by theory, the flow of the powder may be affected by factors such as particle shape, particle surface, and the presence of static charges. In some embodiments of any one of the methods or compositions provided herein, to improve flowability, a flow enhancer may be used in the composition to reduce inter-particle friction. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be in any form suitable for use in the methods or compositions described herein.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be in various forms for human consumption or animal consumption. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be used in a dietary supplement, including a human or animal supplement. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition can be used in foods, including human foods, pet foods, animal feeds (e.g., feeds for horses, cattle). In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be used in a beverage. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be used in cosmetics. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be used in any dietary product suitable for use in the methods described herein. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be used in any pharmaceutical product for humans or animals suitable for use in the methods described herein.

In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 0.04mg/ml, about 0.05mg/ml, about 0.1mg/ml, about 0.2mg/ml, about 0.3mg/ml, about 0.4mg/ml, about 0.5mg/ml, about 0.6mg/ml, about 0.7mg/ml, about 0.8mg/ml, about 0.9mg/ml, about 1.0mg/ml, about 1.1mg/ml, about 1.2mg/ml, about 1.3mg/ml, about 1.4mg/ml, about 1.5mg/ml, about 1.6mg/ml, about 1.7mg/ml, about 1.8mg/ml, about 1.9mg/ml, about 2.0mg/ml, about 2.2mg/ml, about 2.4mg/ml, about 3.3mg, about 3.4mg, about 3.5mg, about 3.3mg/ml, about 3.4mg, about 3.5mg/ml, about 3.5mg/ml, about 3.3mg/ml, about 3.4mg, about 3.5mg, about 3.4mg, about 3.5mg, about 3mg, about 3.3mg, about 3mg, and about 4.5 mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 0.04mg/ml to about 0.1mg/ml, about 0.1mg/ml to about 0.5mg/ml, or about 0.5mg/ml to about 1.0mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 0.04mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 0.1mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 0.3mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 1mg/ml.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be present in an amount of about 0.04mg/g, about 0.05mg/g, about 0.1mg/g, about 0.2mg/g, about 0.3mg/g, about 0.4mg/g, about 0.5mg/g, about 0.6mg/g, about 0.7mg/g, about 0.8mg/g, about 0.9mg/g, about 1mg/g, about 1.1mg/g, about 1.2mg/g, about 1.3mg/g, about 1.4mg/g, about 1.5mg/g, about 2mg/g, 2.5mg/g, 3mg/g, about 3.5mg/g, 4mg/g, 4.5mg/g, about 5mg/g, about 6mg/g, about 7mg/g, about 8mg/g, about 9mg/g, about 10mg/g, about 11mg/g, about 12mg, about 13mg, about 14mg, about 15mg, about 45mg, about 25mg, about 50mg, about 45mg, about 30mg, and the whole range therebetween. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 1mg/g to about 10mg/g, about 5mg/g to about 25mg/g, or about 25mg/g to about 50mg/g.

In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is suitable for use in a human subject in need thereof. In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine composition is suitable for use in any one of the methods described herein. For example, the amount of ergothioneine composition is suitable for improving cell viability and/or maintaining telomere length. For example, the ergothioneine composition is in a therapeutic amount suitable for treating, preventing, or reducing the risk of any one of the disorders, diseases, or conditions provided herein, such as a telomere-related disorder, disease, or other condition. For example, the ergothioneine composition is in a therapeutic amount suitable for treating, preventing, or reducing the risk of premature aging. For example, the ergothioneine composition is in a therapeutic amount suitable for treating, preventing, or reducing the risk of a cognition related disorder, disease, or other condition. For example, the ergothioneine composition is in a therapeutic amount suitable for treating, preventing, or reducing the risk of oxidative stress-related disorders, diseases, or other conditions.

Application method

Aspects of the present disclosure provide, at least in part, methods of improving cell viability. In some embodiments, cell viability is improved by maintaining the average telomere length of cells, such as a test cell population.

Telomeres are regions of repeated nucleotide sequences at each end of the chromosome that protect the chromosome ends from nucleolytic degradation, unnecessary recombination, repair, and intershromosomal fusion. For vertebrates, the nucleotide sequence in the telomere is TTAGGG. As a normal cellular process, a small portion of telomeric DNA is lost each time the cell divides. Human telomere shortening can induce replicative senescence that blocks cell division. Progressive shortening of telomeres can lead to somatic aging, apoptosis, or oncogenic transformation, affecting the health and longevity of an individual. Thus, telomere length can act as a biological clock that determines the life of cells and organisms. Telomere length is also known as a marker for a variety of diseases (e.g., aging and cancer). Regarding aging, it is known that telomere length decreases with age, because telomerase activity in adult human tissues is insufficient to prevent telomere shortening, thereby compromising cell viability. Without wishing to be bound by theory, for cancer cells, telomere length is maintained due to overexpression of telomerase or activation of alternative mechanisms that promote telomerase elongation.

Telomeres are dynamic structures that are affected by both genetic and medical history of individuals and numerous environmental influences. Various forms of stress can alter the cellular environment and promote telomere shortening. Oxidative stress can lead to shortening of telomeres in vitro [11]. In vivo, a significant correlation between various markers of oxidative stress and telomere length and/or shortening was reported, and experimental studies to date appear to also indicate that oxidative stress affects telomere shortening [12]. Furthermore, it has been shown that telomeres are shorter in women with higher living or psychological stress than women with lower stress [13]. The exact mechanism that links psychological stress to telomere length is not clear, but without wishing to be bound by theory, it is believed that oxidative stress may be a potential link. Glucocorticoids secreted in response to stress are known to cause oxidative damage to nerves, and self-reported afflictions are associated with greater oxidative DNA damage [14,15]. Oxidative stress is also associated with the pathogenesis of many chronic diseases, which are also associated with short telomere length, including cardiovascular disease, diabetes and cancer [16].

It has surprisingly been found that ergothioneine can exert a protective effect on telomeres under oxidising conditions. Oxidative stress can lead to shortening of telomeres, and it is demonstrated herein that ergothioneine treatment can confer a beneficial effect by reducing the rate of telomere shortening and preserving telomere length under oxidative stress conditions. Under oxidative conditions, ergothioneine treatment, for example, significantly increases median telomere length and 20 th percentile telomere length, and significantly reduces the percentage of short telomeres (< 3 kilobase pairs) while reducing the telomere shortening rate. Thus, in some aspects, the present disclosure provides methods of treating, preventing, or reducing the risk of disorders, diseases, or other conditions associated with oxidative stress. In some embodiments, the present disclosure provides methods of improving cell viability by protecting telomeres under oxidative conditions (e.g., oxidative stress conditions). In some embodiments, cell viability is improved by maintaining cells (e.g., oxidative stress conditions), such as testing the average telomere length of a population of cells.

In some embodiments of any one of the methods provided herein, cells contacted or incubated with ergothioneine as described herein, such as a test cell population, may be maintained for average telomere length. In some embodiments of any of the methods provided herein, the average telomere length of cells, e.g., test cell populations, incubated with or in contact with ergothioneine as described herein can be maintained under oxidative conditions (e.g., oxidative stress conditions). In some embodiments of any one of the methods provided herein, the average telomere length of a cell, e.g., a test cell population, incubated with ergothioneine is maintained when compared to the average telomere length of a control cell, e.g., a control cell population, without contact or incubation as described herein. For example, the control cell or control cell population may be the same type of cell or cell population tested but not a cell or cell population that has been contacted or incubated with ergothioneine. Such control cells or cell populations may be incubated under standard cell culture conditions known to those skilled in the art. In some embodiments, the control cell or control cell population may be a different cell type than the test cell or test cell population. For example, such control cells or control cell populations may be cells having specifically known and stable telomere lengths. The test cell or population of test cells treated with ergothioneine as described herein can be compared to a control cell or population of control cells having a known and stable telomere length to determine whether the test cell or population of test cells has a retained or maintained telomere length. In this case, a constant or relatively constant telomere length of the test cell or test cell population indicates maintenance or maintenance of telomere length and/or improvement in cell viability. In some embodiments of any one of the methods provided herein, "maintaining" or "maintaining" telomere strength refers to the lack of or minimization of an indication of a reduction in telomere length that does not negatively affect cell viability.

In some embodiments of any one of the methods provided herein, the cell viability of a cell contacted with ergothioneine, e.g., a test cell or a test cell population, can be increased by 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, including all ranges and subranges therebetween, as compared to the cell viability of a control cell or control cell population not contacted with ergothioneine.

In some embodiments of any of the methods provided herein, the cell, such as a test cell, can be a skin, small intestine, testis, cornea, blood, bone marrow, brain or tumor tissue cell, or a tissue sample that belongs to skin, small intestine, testis, cornea, bone marrow, brain or tumor tissue. In some embodiments of any one of the methods provided herein, the cell, such as a test cell or population of test cells, can be a sample derived from any tissue or organ suitable for use in the methods described herein. In some embodiments of any one of the methods provided herein, the control cells may be derived from Hela 2, hela, MCF7, hela S3, 293T, L5178Y-S, MEFs BL 6G 3 tec-/-, MEFs BL6 wild-type, hela 1211, MEFs129Sv/BL6 wild-type, or L5178Y-R cell lines. In some embodiments of any one of the methods provided herein, the control cell can be derived from any cell suitable for use in the methods disclosed herein. In some embodiments of any one of the methods or compositions provided herein, the cell or cell population is from or is an implantable body part, including whole organs, parts of organs, and tissues such as heart, lung, heart lung, kidney, liver, pancreas, intestine, skin, face, hand, leg, penis, bone, uterus, thymus, islets, heart valves, or ovaries. In some embodiments of any one of the methods or compositions provided herein, the cell or cell population is from or is an amputation or portion thereof, comprising tissue.

In some embodiments, telomere length can be determined by various methods or assays known in the art. Methods or assays may include, but are not limited to, single cell telomere mapping techniques, telomere restriction fragment assays, flow-fluorescence in situ hybridization (flow-FISH), hybridization assays, primer in situ labeling (PRIMS), and PCR-based methods, such as STELA and quantitative PCR. With respect to real-time PCR assays, telomere length can be determined by assessing the ratio of telomeres to single copy genes (T/S). In some embodiments, single cell telomere mapping techniques are based on fluorescent in situ hybridization assays. Methods for determining telomere length are described in US8084203 and incorporated by reference.

The compositions of the present disclosure may also be used in methods of preserving cells, such as organ transplants or methods of preserving amputations or other body parts (including tissues) as may be desired. Accordingly, aspects of the present disclosure provide methods of preserving an organ for organ transplantation or preserving an amputation or other body part (including tissue). In some embodiments, the organ used for organ transplantation is any implantable organ or body part (including tissue) such as, but not limited to, heart, lung, heart lung, kidney, liver, pancreas, intestine, skin, face, hand, leg, penis, bone, uterus, thymus, islets, heart valves, or ovaries. In some embodiments, the method comprises treating an organ or limb segment or other body part (including tissue) for organ transplantation with an effective amount of a composition comprising ergothioneine.

Aspects of the present disclosure provide methods of treating, preventing, or reducing the risk of a telomere related disorder, disease, or other condition. In some aspects, the telomere-related disorder, disease, or other condition is a cognition-related disorder. Other aspects of the disclosure provide methods of treating, preventing, or reducing the risk of oxidative stress-related disorders, diseases, or other conditions. In some embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of a composition comprising ergothioneine.

As used herein, a "therapeutically effective amount" refers to an amount of a ergothioneine composition that, when administered to a subject in need thereof, can treat, prevent, or alleviate a disorder, disease, or other condition provided herein, such as a symptom of a telomere-related disorder, disease, or other condition, and can achieve at least a therapeutic result or effect as understood by one of skill in the art. A therapeutically effective amount also refers to an amount of ergothioneine composition that may be used for any of the purposes provided, e.g., for maintaining or supporting health, slowing the aging process, reducing the risk of a disease, disorder or condition, etc.

The dosage of the ergothioneine composition required to achieve a particular "therapeutic result or effect", e.g., milligrams of purified ergothioneine per milliliter of administration solution, will vary depending on several factors including, but not limited to: the route of administration, the weight of the subject in need thereof, the level of ergothioneine required to achieve a therapeutic effect in various organs or tissues, the particular disease, disorder or condition being treated, the stability of the ergothioneine composition, and the like. The dosage range of the ergothioneine composition for treating a subject in need thereof having a particular disease, disorder or condition can be readily determined by one of skill in the art based on the factors described above, as well as other factors related and desired. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be in any amount suitable for use in a human subject in need thereof as described herein. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 0.04mg/ml to about 0.1mg/ml, about 0.1mg/ml to about 0.5mg/ml, or about 0.5mg/ml to about 1.0mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition may be about 0.04mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition may be about 0.1mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition may be about 0.3mg/ml. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition may be about 1mg/ml.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be present in an amount of about 0.04mg/g, about 0.05mg/g, about 0.1mg/g, about 0.2mg/g, about 0.3mg/g, about 0.4mg/g, about 0.5mg/g, about 0.6mg/g, about 0.7mg/g, about 0.8mg/g, about 0.9mg/g, about 1mg/g, about 1.1mg/g, about 1.2mg/g, about 1.3mg/g, about 1.4mg/g, about 1.5mg/g, about 2mg/g, 2.5mg/g, 3mg/g, about 3.5mg/g, 4mg/g, 4.5mg/g, about 5mg/g, about 6mg/g, about 7mg/g, about 8mg/g, about 9mg/g, about 10mg/g, about 11mg/g, about 12mg, about 13mg, about 14mg, about 15mg, about 45mg, about 25mg, about 50mg, about 45mg, about 30mg, and the whole range therebetween. In some embodiments of any one of the methods or compositions provided herein, the amount of the ergothioneine composition is about 1mg/g to about 10mg/g, about 5mg/g to about 25mg/g, or about 25mg/g to about 50mg/g.

In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of: about 5mg daily, about 6mg daily, about 7mg daily, about 8mg daily, about 9mg daily, about 10mg daily, about 11mg daily, about 12mg daily, about 13mg daily, about 14mg daily, about 15mg daily, about 16mg daily, about 17mg daily, about 18mg daily, about 19mg daily, about 20mg daily, about 21mg daily, about 22mg daily, about 23mg daily, about 24mg daily, about 25mg daily, about 26mg daily, about 27mg daily, about 28mg daily, about 29mg daily, or about 30mg daily, including all ranges and subranges therebetween. In some embodiments, ergothioneine is administered at a dose of about 5mg to about 30mg per day. In some embodiments, ergothioneine is administered at a dose of about 5mg to about 25mg per day. In some embodiments, ergothioneine is administered at a dose of about 5mg to about 10mg per day, about 10mg to about 15mg per day, about 15mg to about 20mg per day, about 20mg to about 25mg per day, or about 25mg to about 30mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 5mg per day or at least 5mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 10mg per day or at least 10mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 15mg per day or at least 15mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 20mg per day or at least 20mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 25mg per day or at least 25mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 30mg per day or at least 30mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 40mg per day or at least 40mg per day. In some embodiments of any one of the methods provided herein, the method comprises administering ergothioneine at a dose of about 50mg per day or at least 50mg per day.

Non-limiting examples of telomere-related disorders, diseases, or other conditions may include, but are not limited to, aging-related diseases such as atherosclerosis and cardiovascular diseases, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and alzheimer's disease, age-related macular degeneration, benign prostatic hyperplasia, telomere disease, bone marrow failure, congenital dysplasia, acquired aplastic anemia, pulmonary fibrosis, and liver disease. In some embodiments of any one of the methods provided herein, the telomere related disorder, disease, or other condition is or is associated with any one of the foregoing.

In some embodiments of any one of the methods provided herein, the telomere related disorder, disease, or other condition is premature aging or is associated with premature aging.

In some embodiments of any one of the methods provided herein, the telomere-related disorder, disease, or other condition is or is associated with a cognitive-related disorder, disease, or other condition.

In some embodiments of any one of the methods provided herein, the telomere related disorder, disease, or other condition is or is associated with chronic inflammation.

In some embodiments of any one of the methods provided herein, the telomere-related disorder, disease, or other condition is caused by or associated with oxidative stress.

Non-limiting examples of cognition-related disorders, diseases, or other conditions include cognitive impairment, mild cognitive impairment, frontotemporal dementia, vascular dementia, dementia with lewy bodies, presenile dementia, senile dementia, friedriich's ataxia, down's syndrome, huntington's chorea, hyperkinesia, mania, tourette's syndrome, alzheimer's disease, progressive supranuclear palsy, cognitive impairment, including attention, orientation, learning impairment, memory (e.g., memory impairment, amnesia, memory loss, transient complete amnesia syndrome, and age-related memory impairment), and language functioning; cognitive impairment resulting from stroke, huntington's disease, pick's disease, aids-related dementia or other demented states, such as multi-infarct dementia, alcoholic dementia, hypothyroidism-related dementia and dementia associated with other degenerative disorders, such as cerebellar atrophy and amyotrophic lateral sclerosis; other acute or subacute conditions that may lead to cognitive decline, such as delirium or depression (pseudo-dementia state) trauma, head trauma, age-related cognitive decline, stroke, neurodegeneration, drug-induced states, neurotoxic agents, age-related cognitive impairment, autism-related cognitive impairment, down's syndrome, psychosis-related cognitive deficits, and post-electroconvulsive therapy-related cognitive disorders; cognitive disorders due to substance abuse or withdrawal, including nicotine, cannabis, amphetamine, cocaine, attention Deficit Hyperactivity Disorder (ADHD) and movement disorders such as parkinson's disease, neuroleptic-induced parkinsons disease and tardive dyskinesia, schizophrenia, schizophreniform disorders, psychotic depression, mania, acute mania, paranoid, hallucinations and delusions, personality disorders, obsessive-compulsive disorders, schizophreniform disorders, delusions, malignant psychotic disorders, metabolic disorders, endocrine disorders or narcolepsy, substance abuse or withdrawal-induced psychoses, bipolar disorders and schizoaffective disorders. In some embodiments, the cognition-related disorder, disease or other condition is alzheimer's disease, frontotemporal dementia, schizophrenia, parkinson's disease, huntington's disease, lewy body disease, vascular dementia, traumatic Brain Injury (TBI), parkinson's disease, prion disease, dementia, amnesia, or delirium.

Non-limiting examples of disorders, diseases, or other conditions associated with oxidative stress include cancer, neurodegenerative diseases (e.g., parkinson's disease, alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis), ocular disorders, cardiovascular disease, atherosclerosis, sickle cell disease, thrombotic thrombocytopenic purpura, sepsis, cystic fibrosis, chronic fatigue syndrome, kidney disease, diabetes, acute respiratory distress syndrome, gout, arthritis, and other inflammatory diseases. Oxidative stress is related to several age-related disorders (e.g., cardiovascular disease, chronic obstructive pulmonary disease, chronic kidney disease, neurodegenerative disease, and cancer), including sarcopenia and frailty. Oxidative stress also involves viral infections (e.g., HCV infection, HIV infection, etc.). In some embodiments, the viral infection is a respiratory viral infection (e.g., an infection caused by influenza virus, influenza (IV), human Respiratory Syncytial Virus (HRSV), human Rhinovirus (HRV), human Metapneumovirus (HMPV), parainfluenza virus, and adenovirus and coronavirus (CoV) (e.g., SARS-CoV, MERS, and SARS-CoV-2). In some embodiments, the viral infection is COVID-19 caused by SARS-CoV-2. Without wishing to be bound by theory, it is believed that ergothioneine will improve the symptoms of covd-19 by exerting a protective effect on telomeres under oxidative conditions. For example, ergothioneine is believed to treat or prevent inflammation and/or cytokine storm, protect against acute respiratory distress syndrome, alleviate oxidative stress, lung injury, and other long-term conditions associated with covd-19, and the like.

Non-limiting examples of inflammatory diseases or disorders include Alzheimer's disease, arthritis, asthma, atherosclerosis, crohn's disease, colitis, cystic fibrosis, dermatitis, diverticulitis, hepatitis, irritable Bowel Syndrome (IBS), lupus erythematosus, muscular dystrophy, nephritis, parkinson's disease, rheumatoid arthritis, shingles, and ulcerative colitis. Inflammatory diseases also include, for example, stroke, cardiovascular disease, chronic Obstructive Pulmonary Disease (COPD), bronchiectasis, chronic cholecystitis, tuberculosis, hashimoto's thyroiditis, renal fibrosis, sepsis, sarcoidosis, silicosis and other pneumoconiosis.

In some embodiments of any one of the methods provided herein, the disease, disorder, or condition is preeclampsia, overfill hereditary lipstick cytopenia (overhydrated hereditary stomatocytosis), lung injury, fertility (e.g., sperm motility, embryo development, embryo quality), cataract.

In some embodiments of any one of the methods provided herein, treating, preventing, or reducing the risk of a telomere-related, oxidative stress-related, inflammatory-related, or cognition-related disorder, disease, or other condition comprises maintaining the average telomere length of cells in the target environment of a subject in need thereof. In some embodiments of any one of the methods provided herein, maintaining the average telomere length of cells in the target environment of a subject in need thereof improves cell viability and/or stability.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine is L-ergothioneine. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine is

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine is purified. In some embodiments of any one of the methods or compositions provided herein, the purified ergothioneine has a purity of about or at least 98% or 99% or more. In some embodiments of any one of the methods or compositions provided herein, the purified ergothioneine can have any purity described herein.

In some embodiments of any one of the methods or compositions provided herein, the amount of ergothioneine is any one of the amounts provided herein. In some embodiments of any one of the methods or compositions provided herein, ergothioneine can be in any amount suitable for use in the methods described herein.

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be in the form of a free-flowing powder. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be in any of the forms described herein.

Pharmaceutical composition

In some embodiments, aspects of the present disclosure provide a composition comprising a therapeutically effective amount of ergothioneine, such as L-ergothioneine, e.g., a purified natural form of L-ergothioneine. The composition may be a pharmaceutical composition. In some embodiments of any one of the methods or compositions provided herein, the pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers. The purified ergothioneine may have a purity of about 98%, 99%, or greater, or any other purity level suitable for use in the compositions, such as methods and formulations of pharmaceutical compositions, as disclosed herein.

One skilled in the art can readily select a suitable carrier in view of the type of antioxidant or dietary supplement. For example, one suitable carrier comprises saline, which may be formulated with a variety of buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of the present disclosure.

In some embodiments of any one of the methods or compositions provided herein, a pharmaceutical composition comprising a therapeutically effective amount of ergothioneine may include other pharmaceutical ingredients, such as a preservative or chemical stabilizer. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin. In some embodiments of any one of the methods or compositions provided herein, a composition, such as a pharmaceutical composition, comprising a therapeutically effective amount of ergothioneine may comprise a flavoring or sweetener.

The methods described herein comprise administering a composition, such as a pharmaceutical composition, comprising a therapeutically effective amount of ergothioneine in an amount sufficient to treat or prevent any one of the disorders, diseases, or other conditions provided herein without undue adverse effects. Routes of administration are known to those skilled in the art and may be combined if desired.

As described herein, the dosage or amount of ergothioneine required to achieve a particular "therapeutic result or effect" will vary based on several factors. For example, a therapeutically effective amount of ergothioneine is an amount sufficient to alleviate one or more symptoms of any one of the diseases, disorders, or conditions provided herein. The therapeutically effective amount will depend on factors such as the age, sex, weight, health and associated tissues of the subject. In some embodiments of any one of the methods or compositions provided herein, the therapeutically effective amount of ergothioneine is any one of the amounts provided herein, e.g., in mg/ml, mg/g, or mg/d.

In one example, a therapeutically effective amount of ergothioneine may generally range from about 0.1mg/ml to 100mg/ml, from about 1mg/ml to 90mg/ml, from about 0.04mg/ml to about 1mg/ml, and the like. In some embodiments, a therapeutically effective amount of ergothioneine may be 0.04mg/ml. In some embodiments, a therapeutically effective amount of ergothioneine may be 0.1mg/ml. In some embodiments, a therapeutically effective amount of ergothioneine may be 0.3mg/ml. In some embodiments, a therapeutically effective amount of ergothioneine may be 1mg/ml.

In some embodiments, the therapeutically effective amount of ergothioneine is about 5mg to about 50mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 5mg to about 25mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 5mg to about 10mg per day, about 10mg to about 15mg per day, about 15mg to about 20mg per day, about 20mg to about 25mg per day, or about 25mg to 30mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 5mg per day or at least 5mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 10mg per day or at least 10mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 15mg per day or at least 15mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 20mg per day or at least 20mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 25mg per day or at least 25mg per day. In some embodiments, the therapeutically effective amount of ergothioneine is about 30mg per day or at least 30mg per day.

In some embodiments, ergothioneine is administered to a subject at a desired frequency to treat or prevent a disorder, disease, or other condition. In some embodiments, ergothioneine is administered to a subject at a desired frequency to improve cell viability and/or maintain telomere length. In some embodiments, ergothioneine is administered daily. In some embodiments, ergothioneine is administered at least once weekly (1, 2, 3, 4, 5, or 6 times weekly), weekly, biweekly, or monthly. In some embodiments, ergothioneine is administered to a subject for a desired period of time to improve cell viability and/or maintain telomere length. In some embodiments, ergothioneine is administered to a subject for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or a year, including all ranges and subranges therebetween.

The formulation of pharmaceutically acceptable excipients and carrier solutions is known to those skilled in the art, as are the development of suitable dosing and therapeutic regimens using the specific compositions described herein. The amount of active compound in each therapeutically useful composition can be prepared in the following manner: suitable dosages will be obtained in any given unit dose of compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological factors, and as such, various dosages and treatment regimens may be desirable.

Forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent microbial growth. In many cases, the form is sterile and fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and/or vegetable oils. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Microbial action may be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

The ergothioneine composition may be formulated or prepared in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. After formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is known in the art. Supplementary active ingredients may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to a host.

Food/beverage and supplement products

In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be formulated or prepared as a food supplement for oral consumption. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be formulated or prepared for cosmetic applications. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be formulated or prepared for use in dietary products, such as food products, medical foods, and the like. In some embodiments of any one of the methods or compositions provided herein, the ergothioneine composition may be formulated or prepared for use in a beverage.

The food composition according to the invention comprises any formulation or composition suitable for consumption and for nutritional or enjoyment purposes. They are generally products intended to be consumed by humans or animals and introduced into the body through the mouth, where they stay for a period of time and then are consumed (e.g. ready-to-eat foods or feeds, see also below) or removed (e.g. chewing gum). Such products include any substance or product that is to be ingested by a human or animal in a processed, partially processed or unprocessed state. It also comprises substances added to the orally consumable product during production, preparation or treatment and intended to be introduced into the oral cavity of a human or animal.

The food composition according to the invention further comprises a substance to be swallowed by a human or animal and then digested in an unaltered, therapeutic or prepared state; in this regard, the orally consumable product according to the present invention also comprises a shell, coating, or other encapsulation that will be swallowed simultaneously or that is intended to be swallowed. The expression "food composition" covers ready-to-eat foods, beverages and feeds, i.e. foods, beverages or feeds that have been completed in terms of substances important in terms of taste. The expressions "ready-to-eat food" and "ready-to-eat feed" also include beverages and solid or semi-solid ready-to-eat food or feed. Examples which may be mentioned are frozen products, which must be thawed and heated to the eating temperature before they are consumed. The instant food or feed of the present invention also comprises products such as yoghurt or ice cream, chewing gum or hard caramel.

Non-limiting examples of food and beverage products include carbonated soft drinks, ready-to-drink beverages, energy beverages, isotonic beverages, low calorie beverages, zero calorie beverages, sports drinks, tea, fruit and vegetable juices, fruit juice beverages, dairy beverages, yogurt beverages, alcoholic beverages, powdered beverages, baked goods, cookies, biscuits, baked goods, cereals, confection, candy, toffee, chewing gum, dairy products, flavored milk, yogurt, flavored yogurt, fermented milk, soy sauce and other soy-based products, salad dressings, mayonnaise, vinegar, frozen desserts, meat products, fish products, bottled and canned foods, tabletop sweeteners (tabletop sweetener), fruits and vegetables.

The composition may be used "as is" or in combination with sweeteners, flavoring agents and food ingredients. Dry compositions, such as powders, granules or tablets, are stable indefinitely when stored under dry conditions at room temperature. The composition in the form of an aqueous solution is stable indefinitely when frozen. If a preservative, such as benzoic acid or a salt thereof, sulfur dioxide or sodium metabisulfite, is added to such a composition, it can be stored at room temperature for almost indefinite periods.

Non-limiting examples of flavoring agents include lemon, orange, fruit, banana, grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla flavoring.

Non-limiting examples of other food ingredients include flavors, acidulants, organic and amino acids, colorants, fillers, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents.

Examples

Example 1: microbial ergothioneine biosynthesis

Ergothioneine or L-ergothioneine (e.g) Is produced in an engineered microbial system. Briefly, E.coli (E.coli) was transformed with the pConB7A vector and the pConA5K vector encoding EgtB, egtC, egtD and EgtE. To co-express the four genes in the E.coli system (EgtB, C, D, E), transformants were grown in LB medium containing 100mg/L ampicillin and 50mg/L kanamycin at 37℃until an OD 600-0.6 was reached. Expression was induced by the addition of 0.2mM-0.5mM isopropyl β -D-l-thiopyran galactoside (IPTG) and the cultures were further grown at 30℃or 37℃for 16-24 hours. Cells were harvested by centrifugation and supernatant and cell pellet were collected separately. The supernatant was centrifuged at 16,000Xg for 5 minutes for HPLC analysis. The pellet was resuspended in 1ml 50% methanol and sonicated for 1 min (3 x20 sec). After centrifugation at 16,000Xg for 5 minutes, 5. Mu.l of the sample was analyzed by HPLC as described below. Coli transformed with the empty vector was treated in the same manner and analyzed by HPLC. 20mg/L ergothioneine was incorporated into samples obtained from IPTG-induced E.coli harboring the EgtB, egtC, egtD, egtE gene and analyzed by HPLC.

Samples were analyzed using Dionex UPLC Ultimate 3000 (senyveromyces, california). The compounds were separated on an Atlantis HILIC silica gel column (particle size 3.0 μm, diameter x length=2.1 x 100mm; waters) and detected at 264 nm. The mobile phase consisted of 0.1% formic acid/water (a) and 0.1% formic acid/acetonitrile (B). The gradient program was 95% b at 1 min, 40% b at 8 min, 95% b at 8.1 min, and stopped at 11 min. The flow rate was 0.6 ml/min and the sample loading was 5. Mu.l.

Furthermore, the retention time of ergothioneine from the E.coli strain containing EgtB, egtC, egtD and EgtE overlapped with the retention time of the ergothioneine standard. In addition to retention time, the ultraviolet spectrum of the ergothioneine peak also matched the ergothioneine standard. Other methods of microbial ergothioneine biosynthesis are described in PCT/US2015/027977 and incorporated by reference.

Example 2: determination of telomere length

To determine the effect on telomere length variation, several methods may be used, some of which are described in US8084203 and incorporated by reference. For example, for telomere length analysis, samples from the tail and back skin, small intestine, cornea, testis, and brain of mice were harvested and fixed overnight in neutral buffered formalin at 4 ℃, dehydrated by fractionation alcohol and xylene, and embedded in paraffin. Prior to embedding, the dissected skin is cut parallel to the spine to obtain longitudinal hair follicle sections. The intestinal tract was rinsed with PBS and rolled into compact circles using longitudinally oriented jejunal sections for analysis. For corneal and testicular analysis, the whole eye and testis were cut in half prior to dehydration. Finally, the brain was coronally dissected to harvest the coracoid hippocampus. In all cases 5"M sections were used for qflish and immunostaining analysis. As an example of using Flow-FISH telomere length measurements in K15-EGFP mice, freshly isolated keratinocyte suspensions (EGFP+ and EGFP-) from K15-EGFP mice were fixed in methanol/acetic acid (3:1), permeabilized with 100% methanol and washed in PBS. Cells were blocked in BSA 10% PBS for 15 min at room temperature and incubated with BD Living Colors AV monoclonal antibody JL-8 (Bidi corporation of san Jose, calif.) for 30 min at 1:250 dilution at room temperature. After washing twice in Tween20-PBS, cells were blocked in BSA 10% PBS for 15 min at room temperature and incubated with goat anti-mouse antibodies (Molecular Probes) conjugated with Alexa 647, england (Invitrogen)) at 1:500 dilution for 30 min at room temperature. After washing twice in Tween20-PBS, the cells were fixed in formaldehyde 0.5% PBS for 5 minutes and washed twice in PBS. Telomeric flow-FISH was then performed using FITC-labeled PNA-tel probes and propidium iodide (PI, sigma) as described (Rufer et al, 1998) to counterstain DNA and analyzed in a FACScanto cell counter (BD Biosciences). Cells of sufficient size and complexity as determined by the forward and side scatter channels were gated at the G0/G1 phase using PI signals acquired in the FL2 channel. Their markers for Alexa 467 were obtained in the FL4 channel and used to identify GFP positive and negative cell populations. Telomeric fluorescence as FITC signal was obtained for both cell populations in FL 1. To compensate for the contribution of cell autofluorescence, the fluorescence value of the negative control cells (i.e., cells hybridized in the absence of FITC PNA-tel probe) was subtracted from each sample. L5178Y-R and L5178Y-S cell lines (Michaex (McIlracth) et al, 2001) of known telomere lengths of 10.2 and 79.7kb, respectively, were treated in parallel and used to convert fluorescent values to kb. Negative controls and collection settings for each fluorescent dye were established with either unstained or single stained cell populations.

Example 3: influence of ergothioneine on telomere length and shortening rate

Materials and methods

Cell cultures and reagents

Establishment of human neonatal dermal fibroblast under standard culture conditionPCS-201-010 ^TM ) Is a primary culture of (a) a plant. Cells at 3X10 ³ Individual cells/cm ² Inoculated and grown and maintained in fibroblast medium (Innoprot). />

More than 98% of ergothioneine is produced by california in blue by a proprietary fermentation process using a modified e.coli K12 strain that is non-pathogenic and non-toxigenic and contains a gene encoding a protein that converts histidine to ergothioneine.

MTT assay

The MTT assay is a colorimetric assay that uses a yellow tetrazolium salt (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide, or MTT) to measure the metabolic activity of cells as cellsRepresentative of viability. MTT reduction rate is an indicator of mitochondrial functional integrity and thus of cell viability [17,18 ]]. To determine the cytotoxicity of ergothioneine on primary neonatal fibroblasts, the cells were grown at 0.35×10 ⁴ Individual cells/plate and 0.5X10 ⁴ Individual cells/plates were seeded in 96-well plates (ken (Nunc)), treated for 72 hours and 1 week. 24 hours from seeding, cells were washed once with Phosphate Buffered Saline (PBS) and treated with the corresponding compounds in cell culture medium. Ergothioneine was dissolved in Dimethylsulfoxide (DMSO). Eight serial dilutions (1 mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.0313mg/ml, 0.0156mg/ml, 0.00078 mg/ml) were performed to evaluate the compounds. With and without 10 mu M H for each condition ₂ O ₂ In triplicate. For positive and negative controls, 8mM Methyl Methanesulfonate (MMS) and 100% DMSO, respectively, were used. After addition of the compounds, the plates were incubated for 72 hours and one week. The medium containing the compound was changed every two days. After the treatment period, the cells were washed twice with PBS and the medium was replaced with 0.5mg/ml MTT reagent in Dulbeck's Modified Eagle Medium (DMEM) without phenol red. The plates were gently shaken and incubated for 4 hours. After incubation, the medium was removed and replaced with DMSO. The plate was gently shaken to dissolve the formazan crystals. Absorbance was measured at 570nm wavelength using an Envision multiple plate reader (multiplex reader).

The percent cell death was calculated using the following equation:

cell death (%) = (OD _{Sample of} –OD _PC /OD _NC –OD _PC )x 100

Wherein OD _NC Is the optical density of the negative control, and OD _PC Is the optical density of the positive control.

Proliferation assay

A primary culture of neonatal human fibroblasts is established. Cells at 5X10 ³ Individual cells/cm ² Inoculated in fibroblast medium (Inonoprate). The medium was refreshed every 2 to 3 days and the cells were passaged at sub-confluence (70-80%) every 7 days. As previously described, in the standard and oxidized (10. Mu. M H) ₂ O ₂ ) Cells were expanded during eight cycles under cell culture conditions. Different concentrations of ergothioneine (0.04 mg/ml, 0.3mg/ml, 0.1mg/ml, and 1.0 mg/ml) or vehicle control were added to the treated or control cells, respectively. Treatment dilutions were freshly prepared with fresh medium at each passage. By using Countess ^TM Cell counter (invitrogen) counts the number of cells per passage to monitor cell growth for each condition. Population Doublings (PD) were calculated using the following: pd= 3.322 (Log (Cf) -Log (Ci)) +x (where Cf: final concentration; ci: initial concentration; X: PD of last passage). One PD corresponds to one round of cell replication.

Each treatment was performed in triplicate and tested in different plates. Each plate contained a control sample. Frozen vials were obtained at different time points for analysis of relative telomerase activity and telomere length.

Relative telomerase activity

Quantitative Polymerase Chain Reaction (PCR) -based telomere repeat amplification protocol (Q-TRAP) is a sensitive and accurate PCR-based assay that enables measurement of telomerase activity. Cell pellets from neonatal fibroblasts were lysed using CHAPS (3-cholesteryl propyl dimethyl ammonium 1-propanesulfonate) lysis buffer for protein extraction [19]. The samples were stored at 4 ℃ and used within 24 hours. Protein quantification was performed for each sample using a Biorad protein assay (Bio-Rad kit 50000002). A minimum protein concentration of 0.3mg/ml is required for sample analysis. The telomerase protein extract was then incubated at 27 ℃ for 30 minutes to allow the telomerase in the protein extract to extend the TS primer by adding TTAGGG repeats. After the enzymatic reaction, the telomerase extension product was amplified and quantified by real-time qPCR using SYBR Green, a Green fluorescent cyanine dye [19-21]. PCR was started at 95℃for 10 minutes, followed by 40 cycles of amplification (95℃for 15 seconds, 60℃for 60 seconds) and a melting curve stage step. The reaction was monitored and analyzed using quantsudio 5 (applied biosystems (Applied Biosystems)). Telomerase activity in a cell line or sample is calculated from the cycle threshold (Ct). All samples were run in triplicate.

Telomere is preparedEnzyme-positive standard dilution series and telomere protein concentration (r ² >0.9 Plotted as a standard curve of Ct values. The standard curve was generated by plotting the cycle threshold (Ct value) of HeLa cell line standards against the logarithm of 1000, 333, 111, 37.03, 12.34, 4.11, 1.37 and 0.45ng protein (whole cell extract).

Using telomere analysis techniquesMeasurement of telomere Length

Proprietary telomere analysis techniques using Life Length for all telomere Length measurementsIs carried out. />Telomere length was measured using High Throughput (HT) Q-FISH (quantitative fluorescence in situ hybridization) techniques as described previously [22 ]]. This method is based on a quantitative fluorescent in situ hybridization method, which is modified to measure individual chromosomes of the interphase cells. Briefly, the telomeres were hybridized with a fluorescent peptide nucleic acid Probe (PNA) (sequence: alexa488-OO-CCCTAACCCTAACCCTAA, available from panacon (Panagene)) that recognizes three telomere repeats. Images of nuclei and telomeres were captured by the high content screening system. The intensity of fluorescent signal from telomere PNA probes hybridized to a given telomere is proportional to the length of the telomere. Fluorescence intensity was converted to base pairs (bp) by using a standard regression curve generated with control cell lines of known telomere length. / >Not only is the telomere length measured in absolute base pair units, but also a distribution assessment, 50 th and 20 th percentiles of telomere length, and percent of short telomeres are provided, allowing a more comprehensive analysis of each sample. All samples were run in duplicate.

Statistical analysis

The results obtained for each treatment and each time point were compared with the appropriate control group using student's t-test (independent sample t-test) [23].

Results

Cell viability

All ergothioneine concentrations tested (0.04 mg/ml, 0.1mg/ml, 0.3mg/ml and 1.0 mg/ml) produced cell death values <20%, indicating no significant toxic effects. Morphological analysis assessment by optical microscopy visualization showed that ergothioneine concentrations as high as 1.0mg/ml did not have any detrimental effect.

Proliferation assay

Under standard culture conditions, the difference in proliferation potency between control and treated fibroblasts was not confirmed (fig. 3A). Cells treated with either 1.0mg/ml or 0.3mg/ml ergothioneine showed an increase in proliferation capacity after week 4 compared to the control group under oxidative culture conditions (FIG. 3B).

Telomerase activity

Telomerase activity of human fibroblasts was measured over a 72 hour period (fig. 4). At 6 hours, the telomerase activity of all dose levels of ergothioneine-treated cells was significantly lower than control cells. At 24 hours, the telomerase activity of ergothioneine-treated cells was 50% -90% higher at all dose levels compared to control cells (p <0.05 for all dose levels). At 48 hours, telomerase activity was still elevated compared to the control, although this effect was only statistically significant for 0.04mg/ml and 1.0mg/ml ergothioneine. At 72 hours, there was no significant difference in telomerase activity between the treated group and the control.

Telomere length

In standard and oxidation (10. Mu. M H) ₂ O ₂ ) Telomere length was assessed over a period of 8 weeks under cell culture conditions. At week 4, cells treated with 0.3mg/ml or 1.0mg/ml ergothioneine under standard conditions had longer median telomere length and telomere length at 20 th percentile compared to controls (figures 5A and 5B). The median telomere length of the control cells is 9378+ -107 base pairs (bp), the median telomere length of 0.3mg/ml ergothioneine is 9637+ -340 bp, and the median telomere length of 1.0mg/ml ergothioneine is 9745+ -206And (5) bp. Cells treated with 0.3mg/ml or 1.0mg/ml ergothioneine also had significantly reduced short telomeres @ 4 weeks compared to the control<3kbp or<3000 bp) percent (fig. 5C). Control short telomeres [ ]<3 kbp) was 8.1.+ -. 0.6%, the short telomere ratio of 0.3mg/ml ergothioneine was 7.3.+ -. 1.1%, and the short telomere ratio of 1.0mg/ml ergothioneine was 6.8.+ -. 1.0%. The telomere shortening rate was assessed and normalized by the number of population doublings (cell replications) to assess the specific effects of ergothioneine. At week 4, the rate of telomere shortening was significantly reduced for cells treated with 0.1mg/ml or 0.3mg/ml ergothioneine compared to the control (fig. 5D), and there was a trend to reduce the rate of telomere shortening for cells treated with 1.0mg/ml ergothioneine (p=0.09). At week 8, all protection previously seen at week 4 was no longer present (fig. 5).

Under oxidative conditions, ergothioneine appears to have greater protection. Cells treated with 0.1mg/ml or 1.0mg/ml of ergothioneine had a higher median telomere length compared to the control at week 4 (p <0.05, fig. 6A), and treatment with all concentrations of ergothioneine significantly increased median telomere length compared to the control at week 8 (fig. 6A). At week 8, under oxidative conditions, the median telomere length of 0.04mg/ml ergothioneine was 9015+ -112 bp, the median telomere length of 0.1mg/ml ergothioneine was 8794+ -212 bp, the median telomere length of 0.3mg/ml ergothioneine was 9068+ -24 bp, the median telomere length of 1.0mg/ml ergothioneine was 8997+ -171 bp, in contrast to the control median telomere length of 8725+ -94 bp (p <0.05 for all cases). At week 4, the results for the telomere length of the 20 th percentile were similar to the longer telomere length (p < 0.05) obtained with 0.1mg/ml, 0.3mg/ml, or 1.0mg/ml ergothioneine treatment. These effects continued until week 8, at which time all concentrations of ergothioneine produced a significant increase in 20 th percentile telomere length compared to the control (fig. 6B). All concentrations of ergothioneine reduced the percentage of short telomeres (< 3 kbp) at both weeks 4 and 8 compared to the control (figure 6C). At week 4, all concentrations of ergothioneine reduced the telomere shortening rate by 27% -52%, and this was statistically significant for 0.04mg/ml, 0.1mg/ml, and 1.0mg/ml (fig. 6D). At week 8, the rate of telomere shortening for the ergothioneine treated group appeared to be low, but these effects did not reach statistical significance.

Discussion of the invention

The results indicate that ergothioneine can protect telomeres, especially under oxidizing conditions. The tests were performed under both standard and oxidizing conditions. During 8 weeks under oxidative conditions, ergothioneine treatment resulted in longer median telomere length, longer 20 th percentile telomere length, lower percentage of short telomeres (< 3 kbp), and concomitant reduction in telomere shortening rate. Because the rate of telomere shortening was normalized using population doublings, the observed decrease in rate of telomere shortening suggests that these decreases are due to differences in ergothioneine treatment and not the number of cell divisions. These benefits are further supported by the observation that transient increases in the relative telomerase activity of ergothioneine treatment. These effects appear not to be dose dependent.

One of the problems with the use of cultured cells for operation is that they are known to have limited ability to divide, after which they become senescent. Previous work showed that cell proliferation in culture was initially strong, but decreased over time, and that after about 50 replication cycles the cells became senescent, a number known as the hfreck limit [24]. During the course of 8 weeks, the cells in our experiments underwent 13-16 replication cycles, which were well below the hfly limit. If the telomeres become extremely short, the cells may also become senescent, although what length is considered extremely short and there is no standard threshold. In our study, the median telomere length is in the range of about 8.5kbp to 9.8kbp, well above what is considered short, and only 7% -11% of the telomeres are less than 3kbp. Both proliferation data and telomere length data indicate that the cells used in this study may not be senescent.

The ergothioneine concentrations (17. Mu.M to 4.4 mM) used in this study were within the physiological range observed in tissues and blood. Previous studies reported high concentrations of ergothioneine in erythrocytes, bone marrow, liver, kidney, semen, and lens and cornea of the eye of 100 μm to 2mM [25]. In humans, supplementation with ergothioneine for 7 days resulted in plasma concentrations of approximately 3. Mu.M, and whole blood concentrations of 30. Mu.M-40. Mu.M [26]. Thus, in our study, physiological concentrations of ergothioneine show beneficial effects by reducing the rate of telomere shortening and preserving telomere length under oxidative stress conditions. Overall, our data support the potential role of ergothioneine in healthy aging.

Example 4: treatment of cognitive related disorders, diseases or other conditions using ergothioneine

In 16 weeks of the study, an effective amount of a pharmaceutical composition comprising ergothioneine was administered to elderly patients suffering from a cognition related disorder or disease. The primary outcome variable measured was the change in CNS-VS test combination composite memory criteria score from baseline to week 16. The secondary outcome variable comprises one or more of the following: changes in CNS-VS integrated memory criteria scores from baseline to week 4 and week 8; changes from baseline to week 4, week 8 and week 16 in the following CNS-VS fields: neuro-cognitive index, linguistic memory, visual memory, mental motor speed, reaction time, complex attention, cognitive flexibility, processing speed, executive function, simple attention, and motor speed; LSEQ changes from baseline to week 4, week 8 and week 16; POMS questionnaire varied from baseline to week 4, week 8, and week 16; changes in hs-CRP from baseline to week 4, week 8 and week 16; changes in ergothioneine levels from baseline to week 4, week 8, and week 16; telomere length varies from baseline to week 16. Plasma and/or serum samples were archived at 1b-4 access for analysis of non-genetic markers of inflammation.

Ergothioneine is effective in maintaining telomere length, improving cell viability, and thus improving symptoms associated with cognitive related disorders or diseases.

Example 5: treatment of oxidative stress related viral infections using ergothioneine

The objective of this study was to determine whether ergothioneine is safe and effective for treating patients with oxidative stress-related viral infections (e.g., HCV infection, HIV infection, etc.). In some embodiments, the viral infection is a respiratory viral infection (e.g., an infection caused by influenza virus, influenza (IV), human Respiratory Syncytial Virus (HRSV), human Rhinovirus (HRV), human Metapneumovirus (HMPV), parainfluenza virus, and adenovirus and coronavirus (CoV) (e.g., SARS-CoV, MERS, and SARS-CoV-2). In some embodiments, the viral infection is COVID-19 caused by SARS-CoV-2. Without wishing to be bound by theory, it is believed that ergothioneine will improve symptoms by exerting a protective effect on telomeres under oxidative conditions. For example, ergothioneine is believed to treat or prevent inflammation and/or cytokine storm, protect against acute respiratory distress syndrome, alleviate oxidative stress, lung injury, and other long-term conditions, and the like.

Example 6: treatment of covd-19 using ergothioneine

The purpose of this study was to determine if ergothioneine treatment of patients with covd-19 was safe and effective. An effective amount of a pharmaceutical composition comprising ergothioneine is administered to a patient having covd-19 to treat a disease. Ergothioneine can protect telomeres, and especially improve cell viability under oxidative conditions prevalent in patients with covd-19, thereby improving symptoms of covd-19.

Reference to the literature

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Equivalents and scope

In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or apparent from the context. Unless indicated to the contrary or apparent from the context, a claim or description that includes an "or" between one or more members of a group is deemed to satisfy the existence, employment, or association of one, more than one, or all of the group members in a given product or process. The present invention includes embodiments in which exactly one member of the group exists, is used in, or otherwise related to a given product or method. The present invention encompasses embodiments in which more than one or all of the group members are present, employed in, or otherwise related to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are presented in a manifest form (e.g., in Markush group (Markush group)), each subset of the elements is also disclosed, and any elements can be removed from the group. It should be understood that where the invention or aspects of the invention are referred to as comprising particular elements and/or features, in general, certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements and/or features. For simplicity, those embodiments are not specifically set forth herein as such. It should also be noted that the terms "comprising" and "including" are intended to be open and allow for the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges may assume any particular value or subrange within the ranges described in the different embodiments of the invention to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

The present application is directed to various issued patents, published patent applications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and this specification, the present specification shall control. In addition, any particular embodiment of the invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention may be excluded from any claim for any reason, whether or not related to the presence of prior art.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments of the invention described herein is not intended to be limited by the foregoing description, but rather is set forth in the following claims. It will be apparent to those skilled in the art that various changes and modifications can be made to this description without departing from the spirit or scope of the invention as defined in the following claims.

Claims (44)

1. A method of improving cell viability or maintaining telomere length comprising contacting or incubating a cell, such as a test cell population, with a composition comprising ergothioneine (e.g., a natural form of purified ergothioneine), optionally wherein the ergothioneine has a purity of at least or about 98%.

2. The method of claim 1, wherein the ergothioneine comprises L-ergothioneine.

3. The method of any one of claims 1-2, wherein the cell viability is improved by maintaining the average telomere length of the cells, such as the test cell population.

4. A method according to any one of claims 1 to 3, wherein the telomere length is determined by single cell telomere mapping techniques.

5. The method of claim 4, wherein the single cell telomere mapping technique is based on a fluorescent in situ hybridization assay.

6. The method of any one of the preceding claims, wherein the composition is in the form of a free-flowing powder.

7. The method of any one of the preceding claims, wherein the average telomere length of the cells, such as the test cell population, is maintained when compared to the average telomere length of a control cell, such as a control cell population, without contact or incubation with the ergothioneine.

8. The method of any one of the preceding claims, wherein the cell, such as the test cell or population of test cells, is a tissue sample selected from the group consisting of skin, small intestine, testis, cornea, blood, bone marrow, brain tissue, and tumor tissue.

9. The method of any one of the preceding claims, wherein the control cell is a cell of an immortalized cell line selected from the group consisting of Hela 2, hela, MCF7, hela S3, 293T, L5178Y-S, MEFs BL 6G 3 tec-/-, MEFs BL6 wild type, hela 1211, MEFs 129Sv/BL6 wild type, and L5178Y-R.

10. The method of any one of the preceding claims, wherein the amount of ergothioneine contacted with or incubated with the cells, such as cells in the test cell population, is about 0.04mg/ml-1.0mg/ml.

11. The method of any one of the preceding claims, wherein the amount of ergothioneine contacted with or incubated with the cells, e.g., cells in the test cell population, is about 0.04mg/ml, about 0.1mg/ml, about 0.3mg/ml, or about 1mg/ml.

12. A method of treating or preventing a telomere-related disorder, disease, or other condition, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising ergothioneine (e.g., a purified natural form of ergothioneine), optionally wherein the ergothioneine has a purity of at least or about 98%.

13. The method of claim 12, wherein the ergothioneine comprises L-ergothioneine.

14. The method of claim 12 or 13, wherein the composition is in the form of a free-flowing powder.

15. The method of any one of claims 12-14, wherein the treating or preventing the telomere related disorder, disease, or other condition comprises maintaining the average telomere length of cells in a target environment in the subject in need thereof.

16. The method of any one of claims 12 to 15, wherein the telomere related disorder, disease or other condition is associated with premature aging.

17. The method of any one of claims 12 to 15, wherein the telomere related disorder, disease or other condition is associated with chronic inflammation.

18. The method of any one of claims 12 to 15, wherein the telomere related disorder, disease or other condition is caused by or associated with oxidative stress.

19. A method of treating or preventing a cognition-related disorder, disease, or other condition, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising ergothioneine (e.g., a purified natural form of ergothioneine), optionally, wherein the ergothioneine has a purity of at least or about 98%.

20. The method of claim 19, wherein the ergothioneine comprises L-ergothioneine.

21. The method of claim 19 or 20, wherein the composition is in the form of a free-flowing powder.

22. The method according to any one of claims 19-21, wherein the treating or preventing the cognition-related disorder, disease, or other condition comprises maintaining an average telomere length of cells in a target environment in the subject in need thereof.

23. The method according to any one of claims 19 to 22, wherein the cognition-related disorder, disease or other condition is associated with premature aging.

24. The method according to any one of claims 19 to 22, wherein the cognition-related disorder, disease or other condition is associated with chronic inflammation.

25. The method according to any one of claims 19 to 22, wherein the cognition-related disorder, disease or other condition is alzheimer's disease, frontotemporal dementia, schizophrenia, huntington's disease, lewy body disease, vascular dementia, traumatic Brain Injury (TBI), parkinson's disease, prion disease, dementia, amnesia or delirium.

26. A method of treating or preventing an oxidative stress-related disorder, disease, or other condition, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising ergothioneine (e.g., a natural form of purified ergothioneine), optionally wherein the ergothioneine has a purity of at least or about 98%.

27. The method of claim 26, wherein the ergothioneine comprises L-ergothioneine.

28. The method of claim 26 or 27, wherein the composition is in the form of a free-flowing powder.

29. The method of any one of claims 26-28, wherein the treating or preventing the oxidative stress-related disorder, disease, or other condition comprises maintaining an average telomere length of cells in a target environment in the subject in need thereof.

30. The method of any one of claims 26-29, wherein the oxidative stress-related disorder, disease, or other condition is associated with telomere shortening.

31. The method of any one of claims 26-30, wherein the oxidative stress-related disorder, disease, or other condition is associated with premature aging.

32. The method of any one of claims 26-30, wherein the oxidative stress-related disorder, disease, or other condition is associated with chronic inflammation.

33. The method of any one of claims 26-30, wherein the oxidative stress-related disorder, disease, or other condition is a viral infection, cardiovascular disease, diabetes, a diabetes-related complication, cancer, gout, or arthritis.

34. The method of claim 33, wherein the oxidative stress-related disorder, disease, or other condition is a respiratory viral infection.

35. The method of claim 33 or 34, wherein the viral infection is covd-19.

36. The method of any one of claims 12-35, wherein the ergothioneine is administered at a dose of about 5mg to about 25mg per day.

37. The method of claim 36, wherein the ergothioneine is administered at a dose of about 5mg per day or about 25mg per day.

38. A composition comprising ergothioneine (e.g., a natural form of purified L-ergothioneine) and optionally one or more pharmaceutically acceptable carriers, and further optionally, wherein the ergothioneine has a purity of at least or about 98%.

39. The composition of claim 38, wherein the ergothioneine comprises L-ergothioneine.

40. The composition of claim 38 or 39, wherein the composition is in the form of a free-flowing powder.

41. A composition according to any one of claims 38 to 40, wherein the amount of ergothioneine is about 0.04mg/ml-1.0mg/ml.

42. The composition according to claim 41, wherein the amount of ergothioneine is about 0.04mg/ml to 0.1mg/ml, about 0.1mg/ml to 0.5mg/ml, or about 0.5mg/ml to 1.0mg/ml.

43. The composition of claim 41, wherein the amount of ergothioneine is about 0.04mg/ml, about 0.1mg/ml, about 0.3mg/ml, or about 1mg/ml.

44. A pharmaceutical formulation comprising a therapeutically effective amount of the composition of any one of claims 38 to 43 and one or more pharmaceutically acceptable carriers.