WO2023143183A1 - Utilisation d'ergothionéine pour prolonger la durée de vie ou améliorer la santé - Google Patents

Utilisation d'ergothionéine pour prolonger la durée de vie ou améliorer la santé Download PDF

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WO2023143183A1
WO2023143183A1 PCT/CN2023/072351 CN2023072351W WO2023143183A1 WO 2023143183 A1 WO2023143183 A1 WO 2023143183A1 CN 2023072351 W CN2023072351 W CN 2023072351W WO 2023143183 A1 WO2023143183 A1 WO 2023143183A1
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composition
ergothioneine
mammal
regulation
lifespan
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PCT/CN2023/072351
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English (en)
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Kylin LIAO
Shawn Wells
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Nanjing Nutrabuilding Bio-Tech Co., Ltd.
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Publication of WO2023143183A1 publication Critical patent/WO2023143183A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine

Definitions

  • Ergothioneine is a naturally occurring amino acid and is a thiourea derivative of histidine, containing a sulfur atom on the imidazole ring. This compound occurs in relatively few organisms, notably Actinobacteria, Cyanobacteria, and certain fungi. In humans, ergothioneine is acquired exclusively through the diet and accumulates in erythrocytes, bone marrow, liver, kidney, seminal fluid, and eyes.
  • Resveratrol is a stilbenoid, a type of natural phenol. Sources of resveratrol in food include the skin of grapes, blueberries, raspberries, mulberries, and peanuts. Resveratrol is thought to act like antioxidants, protecting the body against damage that can put you at higher risk for things like cancer and heart disease. There is extensive evidence in experimental models (e.g., yeast, fruit flies, fish, and rodents) that resveratrol extends lifespan (K. Pallau et al., Curr. Med. Chem., 2016; 23 (41) : 4639-4680; T. Liu et al., Rejuvenation Res., 2015; 18 (3) : 225-33) . In addition, there is evidence that resveratrol improves risk factors for chronic diseases that are consistent with increased lifespan in humans.
  • C. elegans is a transparent worm about 1 mm in length that lives in temperate soil environments. It has been extensively used as a model organism because it has all the physiological properties of an animal, the ability to replicate human diseases, and a fast life cycle.
  • a model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms. Model organisms are widely used to research human disease when human experimentation would be unfeasible or unethical. This strategy is made possible by the common descent of all living organisms, and the conservation of metabolic and developmental pathways and genetic material over the course of evolution.
  • ETT transporter
  • OCTN1 gene symbol SLC22A4
  • the present invention provides a method for extending lifespan or improving healthspan of a mammal.
  • the method includes administration to the mammal of a composition including an effective amount of ergothioneine, or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof, where the mammal is wild type mammal.
  • the mammal does not over express ⁇ -amyloid peptide.
  • the mammal is a human, horse, cattle or other ruminants, pig, or a pet.
  • the composition is prepared in a form of food, drink, nutritional composition, or pharmaceutical composition.
  • the composition is in a form of solution, liquid suspension, parenteral solution, injectable solution, tablet, pill, granule, powder, film, (micro) capsule, aerosol, tonic, syrup, beverage, or dietary supplement.
  • the administration is at least once a day or more times a day.
  • the administration is through various routes selected from oral, intravenous injectable, intramuscular injectable, intraperitoneal, intranasal, rectal, or sublingual route.
  • the administration of the composition is a daily dose of ergothioneine in the range of 2-2000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg, or 5-50 mg.
  • the daily dose is administered in divided doses or a single dose.
  • ergothioneine is a salt, acid, or derivative.
  • the composition extending lifespan or improving healthspan is through regulation of insulin signaling, autophagy, reactive oxygen species, stress response genes, and/or protein translation.
  • regulation of insulin signaling includes downregulating expression of one or more genes including dsf-2, dsf-16, daf-18, rsks-1, mtl-1, sod-2, and Nrf2.
  • regulation of autophagy includes downregulating cpr-1 gene expression.
  • regulation of reactive oxygen species includes downregulating sod-2 gene expression.
  • regulation of protein translation includes downregulating ife-2 gene expression.
  • regulation of stress response genes includes downregulating expression of one or more genes including skn-1/Nrf2, mtl-1, or sod-2.
  • the present invention provides a composition including an effective amount of ergothioneine, or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof, for extending lifespan or improving healthspan in a mammal, where the mammal is wild type mammal.
  • the mammal does not over express ⁇ -amyloid peptide.
  • the mammal is a human, horse, cattle or other ruminants, pig, or a pet.
  • the composition is in a form of food, drink, nutritional composition, or pharmaceutical composition.
  • the composition is in a form of solution, liquid suspension, parenteral solution, injectable solution, tablet, pill, granule, powder, film, (micro) capsule, aerosol, tonic, syrup, beverage, or dietary supplement.
  • ergothioneine is a salt, acid, or derivative.
  • ergothioneine is administrated at a daily dose of 2-2000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg, or 5-50 mg.
  • the composition extending lifespan or improving healthspan is through regulation of insulin signaling, autophagy, reactive oxygen species, stress response genes, and/or protein translation.
  • regulation of insulin signaling includes downregulating expression of one or more genes including dsf-2, dsf-16, daf-18, rsks-1, mtl-1, sod-2, and Nrf2.
  • regulation of autophagy includes downregulating cpr-1 gene expression.
  • regulation of reactive oxygen species includes downregulating sod-2 gene expression.
  • regulation of protein translation includes downregulating ife-2 gene expression.
  • regulation of stress response genes includes downregulating expression of one or more genes including skn-1/Nrf2, mtl-1, or sod-2.
  • the present invention provides use of the composition disclosed herein, for extending lifespan or improving healthspan in a mammal, where the mammal is wild type mammal.
  • the mammal does not over express ⁇ -amyloid peptide.
  • ergothioneine is administrated at a daily dose of 2-2000 mg, 2-500 mg, 2-200 mg, 2-150 mg, 5-100 mg, or 5-50 mg.
  • the daily dose is administered in divided doses or a single dose.
  • the administration is at least once a day or more times a day.
  • the administration is through various routes selected from oral, intravenous injectable, intramuscular injectable, intraperitoneal, intranasal, rectal, or sublingual route.
  • composition disclosed herein extending lifespan or improving healthspan is through regulation of insulin signaling, autophagy, reactive oxygen species, and/or protein translation.
  • regulation of insulin signaling includes downregulating expression of one or more genes including dsf-2, dsf-16, daf-18, rsks-1, mtl-1, sod-2, and Nrf2.
  • regulation of autophagy includes downregulating cpr-1 gene expression.
  • regulation of reactive oxygen species includes downregulating sod-2 gene expression.
  • regulation of protein translation includes downregulating ife-2 gene expression.
  • regulation of stress response genes includes downregulating expression of one or more genes including skn-1/Nrf2, mtl-1, or sod-2.
  • the present invention provides a method for preparing a composition disclosed herein.
  • Fig. 1 shows the results of growth and toxicity assays. Each panel is one representative image from three replicates used for measuring worm size and growth with ergothioneine exposure.
  • Fig. 2 shows growth and toxicity assay plots of worm size data obtained from high resolution imaging and automated detection and tracking software. Left panel shows the results of worms at L4 stage; right panel shows the results of worms at Day 1 adult stage.
  • Fig. 3 shows the results of acute toxicity assay where L4 stage C. elegans worms were treated with ergothioneine at concentrations from 640 nM to 10 mM.
  • Fig. 4A shows the Kaplan-Meier estimate of the survival function
  • Fig. 4B shows the Nelson-Aalen estimate of hazard rates from lifespan assay.
  • Figs. 5A-5B show the worm activity (aggregate motility) analysis over duration of lifespan.
  • Fig. 5A is the Spatial Distribution, where “ (1-normalized mutual information) ” measures changes in the spatial distribution of active worms between time points.
  • Fig. 5B is the Centroid Distance, where the “normalized minimum centroid distance” measures the changes in the location of active worms as a group between time points.
  • Figs. 6A-6C are showing morphology analysis over duration of lifespan.
  • Fig. 6A shows the average length of worms, which was measured along a central spline fitted to the worm outline.
  • Fig. 6B shows the average width of worms, which was measured at the widest point orthogonal to the central spline.
  • Fig. 6C shows the average area of worms, which is the total pixel area of the worm outline converted to ⁇ m 2 .
  • Fig. 7 shows the average circularity of worm, which measures how close the worm’s shape and posture is to a perfect circle, with a perfect circle having circularity of 1.
  • Fig. 8 is a multidimension scaling plot (MDS) that shows multidimensional scaling to embed expression of all included genes into a 2-dimensional plot.
  • Figs. 9A-9D are the volcano plots for all three comparison groups. Red dots indicate differential gene expression that exceeds defined significance and fold-change thresholds.
  • Fig. 9A Ergothioneine Day 3 vs. Control Day 3.
  • Fig. 9B Ergothioneine Day 10 vs. Control Day 10.
  • Fig. 9C Ergothioneine Day 10 vs. Ergothioneine Day 3.
  • Fig. 9D Control Day 10 vs. Control Day 3.
  • Fig. 10 are heatmaps showing the top thirty differentially expressed genes for each condition. Left panel is from young day 3 worms and right panel is from aged day 10 worms.
  • Fig. 11 is diagram showing a summary of pathway mapping to recognized aging-related pathways for Ergothioneine treatment.
  • the term “or” is meant to include both “and” and “or. ” In other words, the term “or” may also be replaced with “and/or. ”
  • the term “include” or “include” and their conjugations refer to a situation where said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb ‘to consist essentially of’ and ‘to consist of’ .
  • the present invention provides a method for extending lifespan or improving healthspan in a mammal.
  • the method includes administration to the mammal of a composition including an effective amount of ergothioneine, or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof, where the mammal is wild type mammal, and where the mammal does not over express ⁇ -amyloid peptide.
  • the present invention provides a composition including an effective amount of ergothioneine, for extending lifespan or improving healthspan in a mammal, where the mammal is wild type mammal, and where the mammal does not over express ⁇ -amyloid peptide.
  • the present invention provides use of the composition disclosed herein, for extending lifespan or improving healthspan in a mammal.
  • the present invention provides a method for preparing a composition disclosed herein.
  • lifespan refers to the length of time for which a mammal lives. “Extending lifespan” refers to the increase of the length of time for which a mammal lives. An example of lifespan extending can be seen at Example 2 below.
  • healthspan refers to the length of time for which a mammal is generally in good health. Certain features of a mammal can be used as a proxy for its health. For example, moving activity and morphology can serve as a proxy for C. elegans’ health. See Example 3 below. “Improving healthspan” refers to the increase of the length of time for which a mammal is generally in good health.
  • Ergothioneine is a naturally occurring amino acid and is a thiourea derivative of histidine, containing a sulfur atom on the imidazole ring. This compound occurs in relatively few organisms, notably Actinobacteria, Cyanobacteria, and certain fungi. In humans, ergothioneine is acquired exclusively through the diet and accumulates in erythrocytes, bone marrow, liver, kidney, seminal fluid, and eyes. In vitro, ergothioneine requires a specific transporter, ETT, also known as OCTN1 (gene symbol SLC22A4) , to enter cells.
  • ETT also known as OCTN1 (gene symbol SLC22A4)
  • Wild type is the phenotype of the typical form of a species as it occurs in nature.
  • the wild type was conceptualized as a product of the standard "normal” allele at a locus, in contrast to that produced by a non-standard, "mutant” allele.
  • Wild type and mutant are the two types of individuals in the same population with different phenotypes.
  • the main difference between wild type and mutant is that wild type is that wild type refers to individuals with normal phenotype possessed by the majority of the natural population whereas mutant refers to individuals with a phenotype that varies from the normal population.
  • Mutant type arises due to the crossing over of genes with different species of the same genus. Then, the mutant is selected by a long process of natural selection. Although a single mutant type can be identified in a population, several types of mutants can occur.
  • the term "administration” refers to the process of delivering a disclosed combination, composition or kit to a subject.
  • the combination, compositions or kits can be administered in a variety of ways, including orally, intragastrically, and parenterally (e.g., intravenous and intraarterial as well as other suitable parenteral routes) , and the like.
  • the term “pharmaceutically acceptable” means pharmaceutically, physiologically, alimentarily, or nutritionally acceptable, and refers to those compositions or combinations of agents, materials, or compositions, and/or their dosage forms, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term “effective amount” refers to an amount that is required to achieve the effect as taught herein. It will be understood, however, that the total daily usage of ergothioneine may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of ergothioneine employed; the duration of the treatment; drugs used in combination or coincidental with ergothioneine; and like factors well known in the medical arts.
  • a “therapeutically effective amount” is the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect for extending lifespan or improving healthspan in a mammal.
  • the term effective amount may include or be synonymous with a pharmaceutically effective amount or a therapeutically effective amount.
  • an amount may be considered therapeutically “effective” even if the condition is not totally eradicated or prevented, but it or its symptoms and/or effects are improved or alleviated partially in the subject.
  • Various indicators for determining the effectiveness of a method for extending lifespan or improving healthspan in a mammal are known to those skilled in the art.
  • the composition includes from about 5%to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%by weight of ergothioneine, and preferably from about 30%to about 90%by weight of ergothioneine, based upon the total weight of the composition taken as 100%by weight.
  • ingredients may be included in the claimed composition, such as other active agents, preservatives, buffering agents, salts, a pharmaceutically acceptable carrier, or other pharmaceutically acceptable ingredients.
  • a “carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • EtOH Ethanol
  • PEG400 is a commonly utilized carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject.
  • mammal or “subject” may be used interchangeably to refer to any animal to which the presently disclosed methods and compositions may be applied or administered.
  • the animal may have an illness or other disease, but the animal does not need to be sick to benefit from the presently disclosed methods and compositions.
  • “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.
  • the mammal is a human, cattle, or a pet.
  • “Pet” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, fish, reptiles, and sheep.
  • the composition is prepared in a form of food, drink, nutritional composition, or pharmaceutical composition.
  • composition refers to a mixture of ergothioneine with other chemical components, such as diluents or carriers.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.
  • Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
  • a pharmaceutical composition is suitable for human and/or veterinary applications.
  • compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art.
  • a “diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable.
  • a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation.
  • a common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.
  • an “excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition.
  • a “diluent” is a type of excipient.
  • compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
  • compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example include metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • Compositions that can include ergothioneine formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the composition is in a form of solution, liquid suspension, parenteral solution, injectable solution, tablet, pill, granule, powder, film, (micro) capsule, aerosol, tonic, syrup, beverage, or dietary supplement.
  • parenteral solution refers to a solution that can be administered elsewhere in the body than the mouth and alimentary canal. It is not delivered via the intestinal tract.
  • parenteral solution can be delivered intravenously.
  • a “tonic” refers to a medicinal substance taken to give a feeling of vigor or well-being.
  • a “syrup” refers to a thick sticky liquid derived from a sugar-rich plant, for example, sugar cane, corn, and maple.
  • the administration is at least once a day or more times a day.
  • compositions include, but not limited to, oral, rectal, topical, aerosol, injection and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections.
  • a targeted drug delivery system for example, in a liposome coated with a tissue specific antibody. The liposomes will be targeted to and taken up selectively by the organ.
  • the administration is through various routes selected from oral, intravenous injectable, intramuscular injectable, intraperitoneal, intranasal, rectal, or sublingual route.
  • “Intraperitoneal” as used here means within or administered through the peritoneum.
  • the peritoneum is a thin, transparent membrane that lines the walls of the abdominal (peritoneal) cavity and contains/encloses the abdominal organs such as the stomach and intestines.
  • sublingual refers to situated or applied under the tongue.
  • the administration of the composition is by oral with a daily dose of ergothioneine in the range of 2-2000 mg. In some embodiments, the administration of the composition is by oral with a daily dose of ergothioneine in the range of 5-500 mg. In some embodiments, the administration of the composition is by oral with a daily dose of ergothioneine in the range of 5-25 mg.
  • the dosage may range broadly, depending upon the desired effects and the therapeutic indication.
  • the daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.01 mg and 3000 mg of ergothioneine, preferably between 1 mg and 700 mg, e.g., 5 to 200 mg, or between about 0.1 mg and about 1,000 mg of ergothioneine per kg of body weight of the subject.
  • the dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the subject.
  • the compounds are administered for a period of continuous therapy, for example for a week or more, or for months or years.
  • ergothioneine, or a pharmaceutically acceptable salt thereof can be administered less frequently compared to the frequency of administration of an agent within the standard of care. In some embodiments, ergothioneine, or a pharmaceutically acceptable salt thereof, can be administered one time per day. In some embodiments, the total time of the treatment regime with ergothioneine, or a pharmaceutically acceptable salt thereof, can be less compared to the total time of the treatment regime with the standard of care.
  • human dosages for ergothioneine have been established for at least some condition, those same dosages may be used, or dosages that are between about 0.1%and 500%, more preferably between about 25%and 250%of the established human dosage.
  • a suitable human dosage can be inferred from ED 50 or ID 50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
  • ED 50 refers to the dose that produces the desired effect in 50%of the population, or median effective dose.
  • dosages may be calculated as the free base.
  • dosages may be calculated as the free base.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to maintain the modulating effects, or minimal effective concentration (MEC) . Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen that maintains plasma levels above the MEC for 10-90%of the time, preferably between 30-90%and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
  • MEC minimal effective concentration
  • the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) .
  • the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • the daily dose is administered in divided doses or a single dose.
  • ergothioneine is referred to its corresponding salts, acids, or derivatives.
  • the composition including an effective amount of ergothioneine, or a pharmaceutically acceptable salt, acid, ester, analog or derivative thereof extending lifespan or improving healthspan is through regulation of insulin signaling, autophagy, reactive oxygen species, and/or protein translation.
  • regulation of insulin signaling includes downregulating expression of one or more genes including dsf-2, dsf-16, daf-18, rsks-1, mtl-1, sod-2, and Nrf2.
  • regulation of autophagy includes downregulating cpr-1 gene expression.
  • regulation of reactive oxygen species includes downregulating sod-2 gene expression.
  • regulation of protein translation includes downregulating ife-2 gene expression.
  • the term “comprising” is to be interpreted synonymously with the phrases “having at least” or “including at least. ”
  • the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
  • the term “comprising” means that the composition, composition or device includes at least the recited features or components, but may also include additional features or components.
  • a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and/or’ unless expressly stated otherwise.
  • a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should be read as “and/or” unless expressly stated otherwise.
  • C. elegans strain used wild type, and not over express ⁇ -amyloid peptide
  • N2 (Bristol) wild type strain has been used in this application.
  • worms were fed on a lawn of UV-killed bacteria (UV-i bacteria) .
  • UV-i bacteria UV-killed bacteria
  • An overnight culture of E. coli strain WP-2 was pelleted, washed, filtered, and irradiated.
  • a suspension of UV-i bacteria was spotted on NGM agar containing Streptomycin to inhibit the growth of contaminating bacteria.
  • the quantity and distribution of food bacteria were calibrated to ensure adequate access to food for the duration of assay while maintaining visibility of the worms.
  • the ALM used herein is based on the Caenorhabditis elegans lifespan machine published by Stroustrup et. al (Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. Nat Methods 10, 665–670 (2013) ) , with proprietary modifications to improve temperature stability and image acquisition.
  • the scanner unit consists of a modified EPSON V850 and images are processed and analyzed using the ALM software (see id. ) .
  • the machine time-of-death calls are trained and validated using the “storyboarding” feature of the ALM software.
  • the lifespan assay was designed and performed according to published and validated methods (Amrit, F.R.G., Ratnappan, R., Keith, S.A. &Ghazi, A. The C. elegans lifespan assay toolkit. Methods 68, 465–475 (2014) ) , using a modified version of the Automated Lifespan Machine (Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. Nat Methods 10, 665–670 (2013) ) . Worms were age synchronized by bleaching and eggs were plated directly onto NGM agar seeded with UV-i bacteria (Day 1, hatch) .
  • worms On Day 3, when the worms had reached the late L4 stage, they were transferred to dishes containing 100 ⁇ M 5-Fluorodeoxyuridine (FUdR) to suppress progeny. On day 5, the worms were washed off the dishes, and 30-50 worms were plated on each scanner dish. Plates were immobilized inverted on the bed of an Automated Lifespan Machine which scanned two images per hour of the plates continuously for the next 40 days.
  • FdR 5-Fluorodeoxyuridine
  • Time of death calls exported from the ALM software was analyzed and plotted using the Lifelines software package developed by Cam Davidson-Pilon et. al (Lifelines Version: v0.25.9) . Additional analysis performed using the OASIS2 analysis software (Seong Kyu Han et. al. OASIS 2: online application for survival analysis 2 with features for the analysis of maximal lifespan and healthspan in aging research. Oncotarget. 7: 56147-56152. (2016) ) .
  • Worm movement was tracked from the images acquired by the ALM during the lifespan assay. Worm size and movement features were extracted and analyzed using custom software.
  • RNA samples were submitted to Novogene Co. Ltd and subjected to more stringent QC, being tested on a Qubit for concentration and run on an agarose gel and on the Agilent 2100 to assess RNA quality and integrity. All samples had an RNA Integrity Number (RIN) of 8.8 or higher (range is 0-10, with 10 being “perfect” ) .
  • RIN RNA Integrity Number
  • RNA is then enriched for poly-mRNA using oligo (dT) paramagnetic beads.
  • DNA libraries were then constructed from this input mRNA using the NEB Next UltraTM II RNA Library Prep Kit. This creates a ready-to-sequence dsDNA library that retains the strand-specific information in the original mRNA. These libraries were then further tested by the Qubit for concentration and the Agilent 2100 for library size distribution and quality. In order to properly pool the libraries and load them onto sequencing lanes to ensure the correct number of reads per sample, an even more precise quantification of the library was done via qPCR, and the samples were loaded onto the NovaSeq 6000 platform for a paired-end sequencing run of 150 bp for each end (PE150) . The loading concentrations were designed to obtain at least 6.0 Gb (which is the number of billion bases of raw data, determined by the number of reads multiplied by the length of each read) .
  • Sequencing run data quality control was performed both by Novogene and again by inventors of this application.
  • This example describes the tests of various dosages of ergothioneine and their toxicity on C. elegans (wild type, and not over express ⁇ -amyloid peptide) .
  • the ideal dose of a lifespan-extending composition will balance providing a high enough dose to be effective with low enough doses not to cause toxic or aversive reactions to the treating animals.
  • C. elegans was used in this study because it has advantages including having all the physiological properties of an animal, the ability to replicate human diseases, and a fast life cycle.
  • the body of C. elegans is encased in a selectively permeable cuticle that only permits some compounds to be absorbed efficiently through the skin, so the most reliable mechanism for delivering compounds to the worms is through ingestion. Water-soluble compounds permeate the media and food and are readily taken up by the worms.
  • C. elegans is usually grown in the laboratory using E. coli as a food source.
  • Nematode Growth Medium (NGM) plates have been prepared by aseptically pouring NGM agar into petri plates. Ergothioneine was dissolved in water to make a working solution of 0.5 M and then mixed with E. coli liquid culture before seeding on NGM agar plates. The seeding spots were dried slowly, allowing ergothioneine to diffuse into the food bacteria and the agar for at least 24 hours before worms are introduced.
  • the indicated ergothioneine dosages (0, 640 nM, 3.2 ⁇ M, 16 ⁇ M, 80 ⁇ M, 400 ⁇ M, 2 mM, and 10 mM) are based on the total volume of the plates.
  • High-resolution imaging and automated detection were used to precisely measure the growth rate of C. elegans worms from hatching to the first day of adulthood (total of 4 days) .
  • the C. elegans growth and development assay is highly sensitive and widely used in toxicology studies. Performing this test over a range of doses helps to identify a set of doses that have a physiological impact and exclude dose ranges that are likely too toxic to benefit lifespan.
  • Fig. 1 shows the results of growth and toxicity assays. Each panel is one representative image from three replicates used for measuring worm size and growth with ergothioneine exposure. Concentrations of ergothioneine are shown above the top row. Images were acquired using WormLab imager and measured using automated detection and measurement. As shown in Fig. 1, 16 ⁇ M and 400 ⁇ M ergothioneine treatment resulted in comparable number and size of C. elegans for L4 and day 1 adult. On the other hand, 10 mM resulted in less worms at both L4 and day 1 adult stages.
  • Fig. 2 shows growth and toxicity assay plots of worm size data obtained from high resolution imaging and automated detection and tracking software.
  • Line mean area
  • box 25th to 75th percentile
  • whiskers 10th to 90th percentile. Circles indicate individuals outside the 10th-90th percentile.
  • the growth rate was largely unaffected at concentrations up to 10 mM at L4 stage (left panel) .
  • the growth rate was largely unaffected at concentrations up to 2mM, and at the 10mM concentration, the young adults were smaller than normal (right panel) .
  • the acute toxicity assay tests toxicity in adult worms by simulating the actual conditions of the lifespan assay. This accomplishes two things. First, in the early stages, it determines doses that are acutely toxic to adult worms. Second, over time worm deaths are scored to rule out doses that will likely have a negative impact on lifespan despite lacking immediate toxicity. Worms are plated on the exact media and sealed plates that will be used in the lifespan assay and then incubated at 25 °C to provide a pilot lifespan that helps catch any other dosing and delivery pitfalls early on. Adult worms were monitored in the days immediately following exposure for early lethality or any other obvious defects.
  • Worms were treated with either vehicle control or ergothioneine at doses spanning a range from 650 nM to 10 mM on media identical to what would be used in the lifespan experiment.
  • Fig. 3 shows the results of acute toxicity assay. Large L4 stage worms were plated on solid media identical to that of the actual lifespan assay. Worms were examined for immediate toxic effects and then scored intermittently for survival until the start of lifespan experiment in the next step (see example 2 below) . Of all the conditions, as shown in Fig. 3, only the 10 mM dose showed an early lethality at day 10 post treatment, and 2mM was on the upper end of the remaining doses that did not cause early lethality.
  • the dose range of ergothioneine for the lifespan experiment can be at least up to 2mM.
  • 5 mM ergothioneine has been used before for lifespan extending experiment (Cheah, Irwin K., et. al., Inhibition of Amyloid-Induced Toxicity by Ergothioneine in a Transgenic Caenorhabditis Elegans Model, FEBS Letters 2019, 593 (16) : 2139–50) .
  • a maximum dose of 5mM was tested along with two lower concentrations of 0.5 mM and 0.1 mM in the following examples.
  • This example describes assessment and measurement of how ergothioneine mitigates lifespan and healthspan decline.
  • the lifespan assay was initiated by expanding all replicate groups to more than 1000 worms, then synchronizing by bleaching and allowing larval worms to hatch and arrest.
  • C. elegans wild type, and not over express ⁇ -amyloid peptide
  • synchronized worms were only exposed to dead food bacteria.
  • worms were transferred to media containing 5-Fluorodeoxyuridine (FUdR) within 54-60 hours post-plating. Worms were inspected 24 and 48 hours after this transfer to confirm infertility. Finally, the worms were inspected for general health and morphology before transferring to scanner plates. The scanner plates were incubated for an additional 2 days and inspected again before loading onto scanners.
  • the analysis of lifespan data is grounded in the study of two mathematical functions: the survival curve and the hazard function.
  • the survival curve describes the fraction of a tested population that remains alive over time.
  • the hazard function is related to the survival curve and provides an intuitive measure of the risk of death; this function describes the probability that a typical individual who is currently alive will soon die, providing a clear visualization of the way a treatment may change patterns in mortality.
  • lifespan data was collected using an Automated Lifespan Machine (ALM) (Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. Nat Methods 10, 665–670 (2013) ) .
  • ALM Automated Lifespan Machine
  • Table 1 Lifespan assay summary of Ergo (ergothioneine) and resveratrol. Lifespan is counted with day 0 set at day 1 of adulthood. No death times are recorded until worms are placed on the scanner on day 8 so earlier deaths are excluded from calculations of mean and median. Median lifespan is equal to the time at which 50%of the worms have died. Mean lifespan is calculated from the area under the survival curve. Maximum lifespan is equal to 95th percentile of lifespans in each group. C.I.: Confidence Interval. See Figs. 4A-4B and Table 1 for statistical analysis.
  • Lifespan data are represented as the percent of worms surviving over time and the profile of this curve can be modeled by the Kaplan-Meier Estimate of Survival function (Figs. 4A-4B) .
  • Fig. 4A is the Kaplan-Meier estimate of the survival function;
  • Fig. 4B is the Nelson-Aalen estimate of hazard rates from lifespan assay.
  • the survival curves can be compared by several statistics including mean, median, and maximum lifespan (see Table 1) , as well as the age of worms at a specific percent survival (see Table 2) .
  • Age in days at percent mortality is the age in days at which the given percentage of worms are dead.
  • Age at 50%mortality is equal to the median.
  • survival curves are holistically compared using a log-rank test (see Table 3) .
  • Table 3 Pairwise statistical analysis of survival curves.
  • the Mantel-Cox log-rank test is a non-parametric test that compares two survival functions across the duration of the lifespan.
  • the Wilcoxon-Breslow-Gehan test weights each death time by the total number of subjects at risk, thus assigning more weight to earlier death times.
  • P-value is corrected for multiple comparisons (Bonferroni correction) . Numbers and asterisks represent P-value and significance, respectively.
  • Tables 1-3 detail the raw measurements, calculations, and statistical analysis related to the survival curve in Figs. 4A-4B.
  • the lifespan assay detected a significant positive increase in lifespan in the highest Ergothioneine treatment group as well as the Resveratrol positive control.
  • a minimum of 184 and a maximum of 253 worm lifespans per condition was recorded (Table 1) surpassing the requirement of 150 required to eliminate subsampling errors and detect lifespan differences of 10%or more (Gruber, Jan, Li Fang Ng, Suresh Kumar Poovathingal, and Barry Halliwell. 2009. “Deceptively Simple but Simply Deceptive--Caenorhabditis Elegans Lifespan Studies: Considerations for Aging and Antioxidant Effects. ” FEBS Letters 583 (21) : 3377–87) .
  • Fig. 4A is the Kaplan-Meier estimate of the survival function, which shows that treatment of worms with Ergothioneine produced a dose-dependent increase in lifespan, with the highest dose having a significant effect comparable to Resveratrol (Fig. 4A) .
  • factors that act upon the rate of aging typically produce a survival curve that remains parallel to the control but right-or left-shifted. This is known as “temporal scaling” of lifespan.
  • the Ergothioneine and Resveratrol treatments follow this paradigm running mostly parallel but right-shifted relative to the control group.
  • the hazard rate function is the instantaneous risk of death at a given time and can help indicate whether different survival curves are determined by similar or different risk factors.
  • Fig. 4B is the Nelson-Aalen estimate of hazard rates from lifespan assay. In this case, the roughly parallel hazard rate functions indicate that all treatment groups are responding to similar aging hazards, albeit aging at different rates (Fig. 4B) .
  • Survival curves can be compared descriptively with statistics such as mean, median, and maximum lifespan (Table 1) .
  • Treatment with 5.0mM Ergotheionine increased each of these statistics as did Resveratrol.
  • the vehicle-treated control group had a median lifespan of 23.6 days
  • the Ergothioneine and Resveratrol treated groups had median survival of 25.9 and 27.3 days, respectively (see Table 1) .
  • the “maximum lifespan” is typically the 95th percentile of lifespans recorded.
  • Treatment with 5.0mM Ergothioneine increased the maximum lifespan from the control of 32.4 days to 34.6 days; the Resveratrol group had a maximum lifespan of 37.3 days (see Table 1) .
  • Survival curves also are commonly compared statistically using a log-rank test, which tests the hypothesis that the two curves are sampled from the same population. Treatment with 5.0mM Ergothioneine produced a statistically significant shift in survival when the curves were analyzed using two contrasting log-rank tests.
  • a standard Mantel-Cox log-rank test compares the curves globally over the course of the lifespan assigning equal weight to each timepoint, whereas the Wilcoxon-Breslow-Gehan weights each death by the number of subjects at risk, assigning greater weight to earlier deaths. Pairwise statistical analysis of survival curves is summarized in Table 3. Each of these tests indicated highly significant differences between 5.0mM Ergothioneine treatment and controls (Table 3) . These same tests indicated that 5.0mM Ergothioneine treatment increases lifespan similarly to, but slightly less than the Resveratrol positive control.
  • Ergothioneine and Resveratrol treatments increased lifespan while maintaining the overall shape of the survival curve. Additional analyses are useful when the curves have a more complex shape or cross. Table 2 shows the age in days for multiple levels of mortality. A Fisher’s exact test can be also applied to specific time or mortality points of interest.
  • Table 4 Fisher’s Exact Test for survival differences at key time points. Statistical analysis corresponding to the age at specific percent mortality shown in Table 2. Age at 50%mortality is equal to the median. These analyses are useful for examining early or late life-specific effects or when the survival curves are not parallel.
  • This example describes healthspan, movement, and morphology of Ergothioneine-treated C. elegans (wild type, and not over express ⁇ -amyloid peptide) worms.
  • Worm Activity serves as a proxy for animal health. Changes in spatial distribution of the worms between time points is used to derive aggregate movement for the population over time. Worm Activity is described by two complementary measurements of aggregate movement, Centroid Distance and Spatial Distribution:
  • Centroid Distance calculates a geometric center for each individual worm, and then measures the minimum collective distance that a group of worms moved between time points. It achieves measurement of the changes in spatial distribution between time-points.
  • Spatial Distribution uses changes in worm contours between time points to measure the changes in the spatial distribution of the worms’ bodies.
  • the normalized distribution of these distances in the population provides a measure of how much the group of worms altered their positions and posture on the plate between two time-points.
  • Worm morphology is measured from the worm contours detected in the images. In the process of aging, worms become shorter and stouter over time and their shape is an indicator of their overall health and biological age. The Length is calculated from the central spline fitted to the worm contour and Width is measured from each worm’s widest point. The worms’ posture also changes with age as they lose the ability to maintain an elongated position. Average Circularity measures how close the shape and posture come to being enclosed by a circle.
  • Each of these measures are obtained by averaging data for all active worms detected on a plate, then averaging across different replicate plates of the same condition. All measurements are based on worms that are still alive and moving at the time of quantification. All measures start when worms are placed on the scanner at day 3 of adulthood.
  • Figs. 5A-5B show the worm activity (aggregate motility) analysis over duration of lifespan.
  • Fig. 5A is the Spatial Distribution, where “ (1-normalized mutual information) ” measures changes in the spatial distribution of active worms between time points.
  • Fig. 5B is the Centroid Distance, where the “normalized minimum centroid distance” measures the changes in the location of active worms as a group between time points. Both the Centroid Distance and the Spatial Distribution are concordant with the results and relative ranking of strains in the lifespan analysis. Both the 5mM Ergothioneine and the Resveratrol groups showed more vigorous movement over the duration of lifespan (Figs. 5A-5B) .
  • the plots are normalized by the number of worms still alive and moving. The Resveratrol plot does not reach zero due to a small number of worms still moving at the end of the experiment.
  • Figs. 6A-6C are showing morphology analysis over duration of lifespan.
  • Fig. 6A shows the average length of worms, which was measured along a central spline fitted to the worm outline.
  • Fig. 6B shows the average width of worms, which was measured at the widest point orthogonal to the central spline.
  • Fig. 6C shows the average area of worms, which is the total pixel area of the worm outline converted to ⁇ m 2 .
  • the overall length of worms was consistent with expected worm size and no differences were detected between the three treatments over the course of the lifespan.
  • the Resveratrol treated worms were overall slightly smaller earlier in the assay but remained larger in the late life stages.
  • the Average Circularity Assay indicates worm heath by describing how closely the shape and posture of the worms is enclosed within a circle. Healthy, active worms maintain an elongated, albeit sinusoidal posture. As unhealthy and aged worms lose muscle function, they increasingly adopt a curled, bunched, or folded state in addition to a stout and wrinkled morphology. Hence, the shape of unhealthy worms is more readily enclosed by a perfect circle--their circularity is closer to 1.
  • Fig. 7 shows the average circularity of worm, which measures how close the worm’s shape and posture is to a perfect circle, with a perfect circle having circularity of 1.
  • This example describes the gene expression pattern in Ergothioneine-treated C. elegans (wild type, and not over express ⁇ -amyloid peptide) worms.
  • RNA-Seq mRNA sequencing
  • DEG differentially expressed genes
  • Multidimensional scaling makes it possible to see strong patterns in large, complex data sets by reducing the data to two or three dimensions.
  • the samples form clustered based on their overall similarity to one another.
  • the distance between samples is calculated based on the Biological Coefficient of Variation (BCV) . Volcano plots for each comparison were used to show log fold change of gene expression against its p-value of the specified comparison.
  • BCV Biological Coefficient of Variation
  • Reading Volcano Plots (log2 fold change, LogFC) : Positive values mean that the gene is more expressed in Treatment, and negative values mean the gene is more expressed in Control. The farther the dot is from the origin point on the x-axis, the greater the expression fold-change. Genes represented in green show a larger than 2-fold change in expression but are not deemed statistically significant due to replicate variability or low read count. Genes represented by red dots show greater than 2-fold change and expression that is statistically significant. The higher on the y-axis, the greater the calculated level of significance.
  • Reading Gene Ontology Enrichment (Pathway) Analysis Tables: Functional characterization of gene lists using Gene Ontology (GO) enrichment analysis is a common approach in transcriptomic analysis. Once the table of differentially expressed genes has been created, the annotation of those genes by biological process (BP) , molecular function (MF) , or cellular compartment (CC) , is cataloged and a comparison is made between the likelihood of seeing genes in that category (ontology) being enriched in the list of differentially-expressed genes when compared to a random selection of genes. This allows patterns due to the interactions of multiple genes to emerge.
  • BP biological process
  • MF molecular function
  • CC cellular compartment
  • Fig. 8 is a multidimension scaling plot (MDS) and shows multidimensional scaling to embed expression of all included genes into a 2-dimensional plot.
  • BCV Biological coefficient of variance.
  • Dimensional reduction of the data to a 2-dimensional plot shows the replicates for each condition in six distinct, non-overlapping clusters. This indicates that each condition holistically exhibited a distinct and reproducible expression profile (Fig. 8) .
  • the relative positions of each condition on the plot also agrees with the expected results. All of the Day 3 treatments (black, green) are horizontally separated from all of the Day 10 treatments (blue, red) but vertically aligned with each other, as are the Day 10 treatments (Fig. 8) .
  • X-axis components therefore, represent differences between young and aged worms regardless of treatment.
  • the Day 3 replicates were more vertically spread whereas there was little if any separation between the Day 10 replicates, indicating a stronger impact of treatment at the Day 3 time point.
  • the lack of separated clusters between the treated and untreated samples indicates that differences are more likely to arise in individual key genes rather than global changes in gene expression.
  • Figs. 9A-9D are the volcano plots for all three comparison groups. Red dots indicate differential gene expression that exceeds defined significance and fold-change thresholds.
  • Fig. 9A Ergothioneine Day 3 vs. Control Day 3.
  • Fig. 9B Ergothioneine Day 10 vs. Control Day 10.
  • Fig. 9C Ergothioneine Day 10 vs. Ergothioneine Day 3.
  • Fig. 9D Control Day 10 vs. Control Day 3.
  • the volcano plots in Figs. 9A-9D show the distribution of genes for each condition with a P-value 2.0. Ergothioneine treatment produced only 3 genes differentially expressed above threshold at Day 3: cpr-1, mtl-1, argk-1. However, each of these genes has been previously implicated in worm longevity and will be discussed below. Only 1 gene was differentially expressed above threshold at Day 10. This gene F55G11.4 is uncharacterized in C. elegans and does not have a clear ortholog in humans or other animal models.
  • Fig. 10 are heatmaps showing the top thirty differentially expressed genes for each condition.
  • Left panel is from young day 3 worms and right panel is from aged day 10 worms. In both panels, the left two columns are controls and the right three columns are samples treated with Ergothioneine at 5mM.
  • Heatmap columns represent individual biological replicates. Colormap indicates log2 (expression in CPM) .
  • Fig. 10 shows that the response was consistent within and between groups. Although most do not surpass the stringent significance cutoff shown in the volcano plots (see Figs.
  • Table 6 shows the top 20 differentially expressed genes under Ergothioneine treatment at Day 3 ranked by P-value.
  • Table 7 shows the top 20 differentially expressed genes under Ergothioneine treatment at Day 10.
  • Table 7 Ergothioneine 5mM vs. Vehicle Control: Top differentially expressed genes at Day 10. Gene function annotations collected from WormBase are shown.
  • Table 8 Top over-represented GO terms Ergothioneine 5mM vs control day 3.
  • GO ID Unique GO ID#cataloged at geneontology. org.
  • Ont Ontology class biological process (BP) , molecular function (MF) , cellular compartment (CC) .
  • N Total number of Genes classified in that GO term. Up/Down: The number of genes in that GO term (out of N) that are up or down-regulated.
  • P-value Significance of gene enrichment (up) or depletion (down) in the set of differentially expressed genes vs. the null set.
  • Table 9 Top under-represented GO terms Ergothioneine 5mM vs control day 3. GO terms under-represented in Ergothioneine-treated worms at day 3. GO ID: Unique GO ID#cataloged at geneontology. org. Ont: Ontology class biological process (BP) , molecular function (MF) , cellular compartment (CC) . N: Total number of Genes classified in that GO term. Up/Down: The number of genes in that GO term (out of N) that are up or down-regulated. Range is shown for all conditions. P-value: Significance of gene enrichment (up) or depletion (down) in the set of differentially expressed genes vs. the null set.
  • the GO enrichment tables are shown here for illustration and to highlight ontology represented among the DEG. Multiple processes are likely represented by the same single gene. As GO enrichment analysis would not be strongly informative for this specific data set, analysis will focus on the individual genes and the longevity pathways represented in the Mechanism of Action report below.
  • This example describes identification of cellular pathways that are most likely modulated by treatment with Ergothioneine.
  • the genes differentially expressed after Ergothioneine treatment were first mapped to core established longevity pathways from the literature. Then the mapping was expanded to intersecting and supporting pathways. This placed the transcriptomic data within the context of well-characterized biological pathways, particularly several related to longevity.
  • DEGs Differentially expressed genes
  • Fig. 11 is a summary of pathway mapping to recognized aging-related pathways for Ergothioneine treatment. Colored score increments indicate degree of up- (red) or down- (blue) regulation weighted by the log of the P value such that each increment approximates one inverse log of P value. Uncolored objects indicate components that were not detected in the data. Top red indicates P value ⁇ 0.0001 and so on. Solid lines indicate direct pathway connections and dashed lines indicate indirect or multi-step connections. Where lists of differentially expressed genes have been condensed into a module, the number of genes is indicated.
  • Insulin signaling (daf-2, daf-18, rsks-1)
  • the insulin/insulin-like growth factor-1 signaling (IIS) pathway was the first pathway implicated in genetic regulation of lifespan and aging.
  • the IIS signaling pathway regulates longevity through three key components: the worm insulin receptor DAF-2, the kinase AGE-1, and the transcription factor DAF-16.
  • Ergothioneine treatment modulated the expression of several genes linked to this pathway.
  • the discovery that loss of function of the worm insulin receptor DAF-2 could more than double lifespan in C. elegans was a landmark finding that helped launch the field of aging research. In response to Ergothioneine treatment, daf-2 and three other key pathway components were slightly downregulated.
  • Both daf-18 which encodes the ortholog of human Phosphatase and Tensin (PTEN)
  • rsks-1 which encodes Ribosomal Protein S6 Kinase, regulate the activity of the Phosphatidylinositol 3-Kinase, AGE-1, which transduces the insulin response signal.
  • the transcriptional output of this pathway is carried out by the transcription factor DAF-16, which controls expression of a large number of genes (Murphy, Coleen T., Steven A. McCarroll, Cornelia I. Bargmann, Andrew Fraser, Ravi S. Kamath, Julie Ahringer, Hao Li, and Cynthia Kenyon. 2003.
  • Autophagy is a cellular process that catabolizes cellular components to maintain energy homeostasis and protect against stress. Activation of autophagy is associated with increased longevity (Hansen, Malene, David C. Rubinsztein, and David W. Walker. 2018. “Autophagy as a Promoter of Longevity: Insights from Model Organisms. ” Nature Reviews. Molecular Cell Biology 19 (9) : 579–93) .
  • the gene cpr-1 which encodes a worm ortholog of Cathepsin B, was significantly downregulated in Ergothioneine-treated worms. Cathepsins control proteolytic degradation within the lysosome.
  • mTOR is a key nutrient sensor and master regulator of growth and energy metabolism in animals. Signaling through mTOR involves two distinct protein complexes, mTORC1 and mTORC2 that regulate different physiological processes. Although TOR signaling pathways share many components and interact with the IIS pathway, in this case no evidence indicated substantial direct modulation of the TOR pathway.
  • Mitochondria provide essential energy for the cell.
  • This organelle is also a major source of reactive oxygen species (ROS) that causes oxidative stress and damage to proteins.
  • ROS reactive oxygen species
  • Disruptions in mitochondria function, particularly in electron transport exacerbates overproduction of ROS.
  • Conditions that promote mitochondrial maintenance and/or turnover have been linked to extended lifespan and improved health. All animals treated with Ergo showed a slightly decreased expression of superoxide dismutase (SOD-2) , a key antioxidant enzyme that breaks down reactive oxygen species. Modulation of several genes involved in oxidative phosphorylation by Ergo treatment can also be an indicator of mitochondrial health and maintenance.
  • SKN-1 is an ortholog of human Nuclear Respiratory Factor (Nrf2) that works in conjunction with DAF-16 to activate transcriptional responses to xenobiotic and oxidative stress.
  • Nrf2 nuclear Respiratory Factor
  • eukaryotic initiation factor 4E (eIF4E) is encoded by the C. elegans gene ife-2. Although there is a connection between TOR signaling and eIF4E activity, knockdown of ife-2 in C. elegans can extend lifespan independently of both IIS and TOR signaling, suggesting a possible distinct pathway of life extension.

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Abstract

La présente invention concerne des méthodes permettant de prolonger la durée de vie ou améliorer la santé d'un mammifère, qui comprend l'administration au mammifère d'une composition contenant une dose efficace d'ergothionéine, ou d'un sel, acide, ester, analogue ou dérivé pharmaceutiquement acceptable de celle-ci.
PCT/CN2023/072351 2022-01-27 2023-01-16 Utilisation d'ergothionéine pour prolonger la durée de vie ou améliorer la santé WO2023143183A1 (fr)

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