Urolithin a as immune enhancer

Abstract

To provide compounds for modulating stem cell function.SOLUTION: The present invention provides compounds of formula (I) or salts thereof for use in methods for modulating stem cell function (where A, B, C, D, W, X, Y, and Z are independently selected from H and OH).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to compounds for use in increasing an immune response to an antigen and / or enhancing, modulating, or enhancing an immune response to an antigen. In particular, the use of urolithins. The present invention also relates to immune enhancing agents comprising urolithins, methods of using such immune enhancing agents, and processes for the preparation of such immune enhancing agents. The present invention also relates to the use of urolithins in methods for modulating stem cell function, such as increasing stem cell numbers, promoting stem cell renewal, and promoting stem cell differentiation. [Background technology]

[0002] Viruses cause a wide range of human diseases, ranging from acute self-resolving conditions to acute, fatal diseases such as rabies, measles, diarrheal diseases, hepatitis, polio, herpes labialis, HIV, Ebola, Zika and influenza. These microscopic particles spread easily, typically by person-to-person contact or contact with contaminated surfaces. Once inside the body, the virus enters cells and reproduces quickly. Effective antiviral drugs exist for only a small proportion of the many human viral diseases. Treatment of viral illnesses often involves alleviating symptoms until the body's immune system clears the infection.

[0003] Influenza places a heavy burden on health services, causing significant morbidity and mortality in the elderly, very young children, and people with chronic diseases. The World Health Organization estimates that seasonal influenza causes 250,000 to 500,000 deaths worldwide each year. Non-influenza-related viral respiratory tract infections are also common human diseases and similarly pose a significant health burden on health services [Non-Patent Document 1].

[0004] Bacterial diseases also pose a significant health burden. One of the bacterial diseases with the highest disease burden is tuberculosis, caused by the bacterium Mycobacterium tuberculosis, which causes approximately 2 million deaths per year, mainly in sub-Saharan Africa. Pathogenic bacteria are responsible for other globally important diseases such as pneumonia, which can be caused by bacteria such as Streptococcus and Pseudomonas, and food-borne diseases, which can be caused by bacteria such as Shigella, Campylobacter, and Salmonella. Pathogenic bacteria also cause infectious diseases such as tetanus, typhoid, diphtheria, syphilis, and leprosy. Pathogenic bacteria are also responsible for high infant mortality rates in developing countries.

[0005] Vaccination with inactivated or attenuated forms of the causative pathogens builds cellular (T cell-cytokines and memory) and humoral (B cell-antibody) immune responses in the host to offset the harmful burden of exposure to the pathogen. A representative example of a global vaccination strategy relates to vaccination with influenza vaccines. Every year, people may be exposed to various strains of influenza virus, which can lead to the development of upper respiratory tract infections (influenza) and even life-threatening pneumonia in advanced cases, and every year, there is an opportunity to be immunized with influenza vaccines against the most prevalent strains of that year. Studies have shown that elderly people aged 65 years and older, whose immune function and health status have declined due to aging, are the most sensitive to exposure to influenza strains, with an incidence rate of over 30% in the elderly population in the United States alone, compared to less than 10% in the healthy adult population (18-49 years old) with a strong immune system (Non-Patent Document 2).

[0006] Influenza vaccination is effective, but the seasonal effectiveness rate depends on the type of vaccine and adjuvant used, the age of the vaccinee, and the virulence of the dominant strain of that season, so it cannot be said to be effective. On average, the effectiveness rate of the entire US population varies between 10% and 60%, and although elderly people benefit more from vaccination, they have a lower effectiveness rate than average due to their poor immune function status (Non-Patent Document 3 and Non-Patent Document 4).

[0007] Aging is associated with immunosenescence and age-related inflammation (Non-Patent Document 5; Non-Patent Document 6). With age-related declines in immune function and health, the risk of being affected by infectious pathogens and diseases increases, leading to higher mortality and morbidity in the elderly. The elderly population is expected to account for more than 25% of the world's population by 2030 (Non-Patent Document 7), and maintaining immune function or preventing its decline is key to maintaining a good quality of life for this population. Furthermore, declining immune function leads to reduced efficiency in responding to vaccinations in populations at highest risk of developing diseases such as influenza.

[0008] Aging is believed to be due in part to the aging of the self-renewing stem cells of an organism and the decrease in stem cell number. This stem cell aging is believed to be due to intrinsic events such as DNA damage and extrinsic events such as changes in stem cell niches or the protected environment that supports stem cells. Various niches have been shown to decline with age, and therefore even in the absence of a significant effect of aging on quiescent stem cell populations, stem cell functionality may be affected due to the decline in signals from local niches that regulate the function of stem cells and their progeny.

[0009] The decline in stem cell function refers to a decline in common molecular processes that underlie the core stem cell properties of self-renewal and generation of differentiated progeny (Non-Patent Document 8). This decline in stem cell function, self-renewal and stem cell numbers is thought to affect age-related conditions such as frailty, atherosclerosis and type 2 diabetes (Non-Patent Document 9). The role of aging in stem cells has been best studied in the hematopoietic system, where stem cells can be isolated nearly homogeneously and their functions can be studied using validated assays.

[0010] The hematopoietic system is composed of all the cellular components found in blood (Figure 1A). Cells of the hematopoietic system require constant renewal, occurring approximately every 10 11 ~10 12It is necessary that new blood cells are produced daily to maintain steady-state cell levels in the average human. The production of these cells occurs in the organs of the hematopoietic system, the spleen and bone marrow, and all the different lineages are derived from multipotent progenitor cells (MPPs). The population of multipotent progenitor cells (MPPs) that maintain the blood lineages are themselves maintained by hematopoietic stem cells (HSCs) (Figure 1B). These cells can be identified by the expression of various proteins at the cell surface. As shown in Figure 1B, HSCs and the various MPP progenitor cell types have different combinations of cell surface proteins, and by using various antibodies against these cell surface proteins, these various cells can be identified and separated. HSCs are stem cells that circulate less frequently and are able to maintain the more regularly cycling MPP population. The infrequent circulation of the HSC population protects these cells from DNA damage and helps to maintain them for the lifespan of the organism. Their depletion and the deterioration of the HSC niche that supports them are thought to be important in aging.

[0011] In bone marrow transplantation, the age of the donor HSCs used to reconstitute the host's blood lineage plays an important role in the success of the transplant: Aged HSCs have been shown to have impaired ability to engraft and reconstitute bone marrow (Non-Patent Document 10).

[0012] Deficient immune responses during aging in general indicate a high need for new immune enhancing agents, especially in immune system deficient populations such as the elderly and children whose immune systems are still developing. The compounds of formula (I), which are the subject of this patent, may serve as such immune enhancing agents.

[0013] Urolithins are natural gut metabolites that are produced by the host's gut microbiota upon exposure to dietary precursors (e.g., ellagitannins) found in large amounts in certain fruits and nuts, such as pomegranates, berries, and walnuts. Urolithin production in the gut shows large human interindividual variability, which is associated with differences in the colonic microbiota

[11] . Several studies have shown health benefits from the consumption of these polyphenol-rich fruits, but the results between these studies are inconsistent, probably due to the large variation in gut microbiota between individuals that results in different metabolite profiles that greatly affect their biological efficacy. This poses another major challenge in designing optimal dietary interventions and limits the interpretation of studies investigating the health aspects of polyphenol-rich fruit consumption. [See, for example, Non-Patent Documents 12 and 13]. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Fendrick et al (2003) Arch.Intern.Med.163(4), 487-94 [Non-Patent Document 2] CDC, 2011~2012 https: / / www.cdc.gov / mmwr / preview / mmwrhtml / mm6122a4.htm [Non-Patent Document 3] N Engl J Med.2007 Oct 4; 357(14):1373-81 [Non-Patent Document 4] Interim Estimates of 2017-18 Seasonal Influenza Vaccine Effectiveness - United States, February 2018.MMWR Morb Mortal Wkly Rep. 2018 Feb 16; 67(6):180-185 [Non-Patent Document 5] Aging Clin Exp Res.2009 Jun;21(3):201-9 [Non-Patent Document 6] Front Immunol.2018 Jan 10;8:1960. doi: 10.3389 / fimmu.2017.01960 [Non-Patent Document 7] United Nations Department of Economic and Social Affairs, Population Division (2015). World Population Ageing 2015 (ST / ESA / SER.A / 390) [Non-Patent Document 8] D. Melton, in Essentials of Stem Cell Biology (Third Edition).(Academic Press, Boston, 2014), pp.7-17 [Non-Patent Document 9] Sharpless, 2007 Nat Rev Mol Cell Biol 2007 Sep; 8(9):703-13 [Non-Patent Document 10] Stem Cells Transl Med.2015 Feb;4(2):186-94 [Non-Patent Document 11] Garcia-Villalba et al (2013) J.Agric.Food Chem., 2013, 61, 8797-8806 [Non-Patent Document 12] Tomas-Barberan FA et al (2014) J Agric Food Chem.62(28):6535-8 [Non-Patent Document 13] Gonzalez-Sarrias et al (2017) Mol Nutr Food Res.61(5). doi: 10.1002 / mnfr.201600830 Summary of the Invention

[0015] According to a first aspect of the present invention there is provided a compound of formula (I) for use in (i) raising an immune response to an antigen and / or (ii) enhancing, modulating or enhancing an immune response to an antigen in a human or animal subject. [ka] (In the formula, A, B, C, D, W, X, Y, and Z are each independently selected from H and OH. or a salt thereof.

[0016] Urolithins, such as Urolithin A, are surprisingly believed to promote immune function and health by improving immune cell and antibody (humoral) responses. Urolithins, such as Urolithin A, maintain optimal functioning of the immune system and may be used prior to vaccination as an additional intervention to substantially enhance the efficacy of current vaccines, particularly in susceptible populations such as the elderly or children.

[0017] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for (i) increasing the immune response to an antigen, and / or (ii) enhancing, modulating or potentiating the immune response to an antigen.

[0018] According to a further aspect of the invention there is provided a method of (i) increasing the immune response to an antigen, and / or (ii) enhancing, modulating or potentiating the immune response to an antigen in a human or animal subject, comprising administering an effective amount of a compound of formula (I).

[0019] According to a further aspect of the invention there is provided a method of (i) increasing the immune response to an antigen, and / or (ii) enhancing, modulating or potentiating the immune response to an antigen in a human or animal subject, comprising administering an effective amount of a compound of formula (I) as an immune enhancer.

[0020] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of improving immune function and / or immune health in a human or animal subject.

[0021] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of improving immune function and / or immune health in a human or animal subject when administered as an immune enhancer.

[0022] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for use in a method of improving immune function and / or immune health in a human or animal subject.

[0023] According to a further aspect of the invention there is provided a method of improving immune function and / or immune health comprising the administration of an effective amount of a compound of formula (I).

[0024] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for boosting the immune system.

[0025] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for boosting the immune system.

[0026] According to a further aspect of the invention there is provided a method of boosting the immune system comprising the administration of an effective amount of a compound of formula (I).

[0027] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for modulating the immune system.

[0028] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for modulating the immune system.

[0029] According to a further aspect of the invention there is provided a method of modulating the immune system comprising administering an effective amount of a compound of formula (I).

[0030] In one embodiment, modulating the immune system comprises enhancing an immune response. In another embodiment, modulating the immune system comprises modulating an inflammatory response. In a further embodiment, modulating the immune response comprises enhancing an immune response and modulating an inflammatory response.

[0031] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of treating an immune deficiency in a human or animal subject.

[0032] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for use in a method of treating an immune deficiency in a human or animal subject.

[0033] According to a further aspect of the invention there is provided a method of improving immune function and / or immune health comprising administering to a human or animal subject an effective amount of a compound of formula (I).

[0034] Age-related inflammation is a chronic progressive increase in proinflammatory states that affect the incidence of aging and age-related diseases. Inflammation is a series of complex response events triggered by the host system facing pathogen infection or various types of tissue damage. In general conditions, the inflammatory response disappears when the proinflammatory factors of infection and tissue damage are eliminated, and then changes to a highly active, well-regulated, and balanced state called resolution of inflammation. However, in some situations, such as during aging, inflammation cannot transition to a steady state of anti-infection and tissue damage repair, and instead inflammation continues and transitions to a non-resolving inflammatory state.

[0035] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for treating age-associated inflammation in a human or animal subject. According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for preventing, reducing or delaying age-associated inflammation in a human or animal subject.

[0036] According to a further aspect of the invention there is provided the use of a compound of formula (I) in a method for treating inflammation associated with aging. According to a further aspect of the invention there is provided a compound of formula (I) in a method for preventing, reducing or delaying inflammation associated with aging.

[0037] According to a further aspect of the present invention, there is provided a method for treating age-associated inflammation, comprising administering to a human or animal subject an effective amount of a compound of formula (I). According to a further aspect of the present invention, there is provided a method for preventing, reducing or delaying age-associated inflammation, comprising administering to a human or animal subject an effective amount of a compound of formula (I).

[0038] Measuring the ratio of interleukin 10 (IL10) to interleukin 12 (IL12) is a useful measure of the inflammatory phenotype of cells, with a higher IL10 / IL12 ratio indicating a more anti-inflammatory phenotype. Thus, according to a further aspect of the present invention, there is provided a method of increasing the IL10 / IL12 ratio using a compound of formula (I).

[0039] The compounds of the invention are also believed to be useful in the treatment of inflammatory disorders such as Behcet's disease. Thus, according to a further aspect of the invention there is provided a compound of formula (I) for use in a method for the treatment of Behcet's disease.

[0040] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of treating immunosenescence in a human or animal subject.

[0041] According to a further aspect of the invention there is provided the use of a compound of formula (I) in a method for treating immunosenescence.

[0042] According to a further aspect of the invention there is provided a method of treating immunosenescence comprising administering to a human or animal subject an effective amount of a compound of formula (I).

[0043] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of preventing, reducing or delaying stem cell senescence in a human or animal subject.

[0044] According to a further aspect of the invention there is provided the use of a compound of formula (I) in a method for preventing, reducing or delaying senescence of stem cells.

[0045] According to a further aspect of the invention there is provided a method of preventing, reducing or delaying stem cell senescence comprising administering to a human or animal subject an effective amount of a compound of formula (I).

[0046] Immunosenescence can result from senescence of hematopoietic stem cells which differentiate into cells of the immune system and thus, according to a further aspect of the invention there is provided a compound of formula (I) for use in a method for treating stem cell aging, in particular hematopoietic stem cell aging, in a human or animal subject.

[0047] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for use in a method of treating stem cell ageing, particularly haematopoietic stem cell ageing, in a human or animal subject.

[0048] According to a further aspect of the invention there is provided a method of treating stem cell ageing, in particular hematopoietic stem cell ageing, comprising administering to a human or animal subject an effective amount of a compound of formula (I).

[0049] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD34 negative hematopoietic stem cells.

[0050] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the numbers of CD34-negative hematopoietic stem cells and CD34-positive, CD48-negative hematopoietic progenitor cells.

[0051] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD34 positive hematopoietic progenitor cells.

[0052] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD34 positive, CD48 negative hematopoietic progenitor cells.

[0053] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD48 positive hematopoietic progenitor cells.

[0054] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD48+, CD150+ hematopoietic progenitor cells.

[0055] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD150-negative hematopoietic progenitor cells.

[0056] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD150-negative, CD135-negative hematopoietic progenitor cells.

[0057] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD135 positive hematopoietic progenitor cells.

[0058] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD34+, CD48+ hematopoietic progenitor cells.

[0059] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD48 positive, CD150 negative hematopoietic progenitor cells.

[0060] Compounds of formula (I) are believed to affect stem cells themselves in addition to hematopoietic stem cells. Thus, according to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method of modulating stem cell function. Modulation of stem cell function may include: (i) promoting stem cell regeneration; (ii) promoting an increase in stem cell numbers; (iii) preventing, reversing, or slowing stem cell aging; (iv) preventing, reversing, or delaying stem cell senescence; and / or (v) Promoting stem cell differentiation.

[0061] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing stem cell numbers, either in vivo, in vitro or ex vivo.

[0062] According to a further aspect of the invention there is provided a compound of formula (I) for use in the ex vivo expansion of stem cells prior to return to the patient for subsequent regenerative treatment.

[0063] According to a further aspect of the invention there is provided cells produced following ex vivo expansion of stem cells.

[0064] Examples of stem cells include pluripotent stem cells, induced pluripotent stem cells (iPS), embryonic stem cells, and adult stem cells. Further examples of stem cells include hematopoietic stem cells, muscle stem cells, skin stem cells, liver stem cells, neural stem cells, melanocyte stem cells, hair follicle stem cells, mesenchymal stem cells, adipose-derived stem cells, limbal stem cells (corneal stem cells), cardiac stem cells, intestinal stem cells, epithelial stem cells, autologous stem cells, and allogeneic stem cells.

[0065] According to a further aspect of the present invention, -How to support stem cell regeneration; -Methods to help optimize stem cell regeneration; -Methods to promote stem cell regeneration; -Methods to stimulate stem cell regeneration; -Methods to support stem cell function; -A method for helping to optimize stem cell function; and / or - There is provided a compound of formula (I) for use in a method for promoting stem cell function.

[0066] According to a further aspect of the present invention, -How to support stem cell regeneration; -Methods to help optimize stem cell regeneration; -Methods to promote stem cell regeneration; - a method for enhancing stem cells; and / or - There is provided a compound of formula (I) for use in a method for increasing stem cell numbers.

[0067] According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for increasing the number of stem cells.In one embodiment, the number of stem cells is increased by at least about 5%, for example, at least about 10%, at least about 15%, at least about 20%, or at least about 25%.

[0068] Stem cells are generally quiescent, and can be arrested in the cell cycle, proliferate to generate new stem cells when necessary, and return to quiescence until they need to proliferate again. During aging, stem cells enter a senescent state and are unable to re-enter the cell cycle. Stem cells may require support to prevent them from entering a senescent state and maintain a vigorous and healthy stem cell population. Thus, according to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for supporting stem cells. According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for preventing and / or inhibiting stem cell aging.

[0069] Changes reported in aged hematopoietic stem cells include a decreased ability to differentiate into lymphocytes, an increased ability to differentiate into myeloid cells, and an increased expression of the cell cycle inhibitor p16 INKaThus, according to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for treating stem cell aging in a human or animal subject, the treatment comprising improving the ability to differentiate into lymphocytes, reducing the ability to differentiate into myeloid cells, increasing the expression of the cell cycle inhibitor p16 INKa and improving the ability to repopulate hematopoietic stem cell progenitors.

[0070] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for use in a method of treating stem cell senescence in a human or animal subject, the treatment comprising improving the capacity to differentiate into lymphocytes, reducing the capacity to differentiate into myeloid cells, inhibiting the expression of the cell cycle inhibitor p16 INKa and improving the ability to repopulate hematopoietic stem cell progenitors.

[0071] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of treating stem cell senescence in a human or animal subject, the treatment comprising improving the capacity to differentiate into lymphoid lineages, reducing the capacity to differentiate into myeloid lineages, inhibiting the cell cycle inhibitor p16 INKa and improving the ability to repopulate hematopoietic stem cell progenitors, the method comprising administering to the human or animal an effective amount of a compound of formula (I).

[0072] The changes observed during immunosenescence also include impaired functioning of the innate immune system, impaired B cell development, thymic involution, reduced T cell output, and reduced T cell diversity.According to a further aspect of the present invention, there is thus provided a compound of formula (I) for use in a method of treating immunosenescence in a human or animal subject, the treatment comprising reversing, reducing or stopping alterations in the innate immune system, reversing, reducing or stopping impaired B cell development, reversing, reducing or stopping thymic involution, reversing, reducing or stopping reduced T cell output, and reversing, reducing or stopping reduced T cell diversity.

[0073] According to a further aspect of the invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for use in a method of treating immunosenescence in a human or animal subject, the treatment comprising reversing, reducing or stopping alterations of the innate immune system, reversing, reducing or stopping impairment of B cell development, reversing, reducing or stopping thymic involution, reversing, reducing or stopping the reduction in T cell output and reversing, reducing or stopping the reduction in T cell diversity.

[0074] According to a further aspect of the invention there is provided a method of treating immunosenescence in a human or animal subject, the treatment comprising reversing, reducing or stopping alterations of the innate immune system, reversing, reducing or stopping impairment of B cell development, reversing, reducing or stopping thymic involution, reversing, reducing or stopping reduction in T cell output and reversing, reducing or stopping reduction in T cell diversity, the method comprising administering to the human or animal subject an effective amount of a compound of formula (I).

[0075] One important role of immune system is to fight against external infection, and vaccination is an important tool for this purpose.Immunocompromised subjects and elderly subjects generally have a reduced immune response, which impairs response to vaccination, reduces the effectiveness of vaccination, and generally makes subjects more susceptible to influenza.According to another aspect of the present invention, there is provided a compound of formula (I) for use in (i) enhancing the immune response to antigen and / or (ii) enhancing, modulating or strengthening the immune response to antigen in human or animal subjects, wherein the subject is elderly or immunocompromised subjects.

[0076] According to another aspect of the invention there is provided a compound of formula (I) for use in (i) enhancing an immune response to an antigen, and / or (ii) enhancing, modulating or potentiating an immune response to an antigen in a human or animal subject, wherein the immune response is to a vaccine.

[0077] The immune response involves several components and compounds of formula (I) are expected to show benefits in one or more of these components. These include: (i) Improved antibody titers; (ii) improving immune cell responses, e.g., in CD4 T cells, CD8 T cells, dendritic cells, macrophages, natural killer (NK) cells and / or B cells; and / or (iii) Improved cytokine response.

[0078] In one embodiment the improvement in antibody titers will be at least 5%, such as at least 10%, such as at least 15% or 20%, such as at least 25% or 30%.

[0079] In a further embodiment the improvement in seroconversion rate will be 5% or more, such as 10% or more, such as 15% or more or 20% or more, such as 25% or more or 30% or more.

[0080] In another embodiment the improvement in seroprotection will be 5% or more, such as 10% or more, such as 15% or more or 20% or more, such as 25% or more or 30% or more.

[0081] In another embodiment, the peak antibody titers will be at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher.

[0082] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing antibody titers in response to vaccination in a subject animal, the method comprising treating the animal with an effective amount of a compound of formula (I).

[0083] Vaccination may be against a pathogenic bacterium or virus that causes disease in a human or animal subject. Thus, according to a further aspect of the present invention, there is provided a compound of formula (I) for use in (i) raising an immune response to an antigen and / or (ii) enhancing, modulating or strengthening an immune response to an antigen in a human or animal subject, the immune response being to a vaccine against influenza, chickenpox, diphtheria, meningococcus, measles, mumps, whooping cough, pneumococcus, polio, rubella, tetanus, tuberculosis, Zika, Ebola, typhoid and / or yellow fever.

[0084] According to a further aspect of the present invention, a) administering a compound of formula (I) as an immune enhancer; b) administering the vaccine.

[0085] According to a further aspect of the present invention, a) administering a urolithin as an immune enhancer; b) administering the vaccine.

[0086] For the avoidance of doubt, the compound of formula (I) and the urolithin may be administered in either order together with the vaccine or they may be administered simultaneously.

[0087] Particularly preferred vaccines are against influenza viruses. Influenza viruses occur in several types: influenza A, influenza B, influenza C, and influenza D. Thus, according to a further aspect of the invention, there is provided a method or use according to the invention, wherein the vaccination is against influenza A, influenza B, influenza C or influenza D. In one embodiment of the invention, the vaccination is against influenza A. In a further embodiment of the invention, the vaccination is against influenza B.

[0088] Influenza A occurs as several subspecies (serotypes). In a further embodiment of the invention there is provided a method or use according to the invention, wherein the vaccination is against influenza A, the serotypes being H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7 and / or H7N9, in particular H1N1 and / or H3N2.

[0089] In one embodiment of the invention, the compound of formula (I) is administered simultaneously with the vaccine in the method or use of the invention. In another embodiment, the compound of formula (I) is administered before the vaccine in the use or method of the invention, and optionally continues to be administered after vaccination. The compound of formula (I) may be administered several weeks or days before the vaccine, such as 1 to 12 weeks, such as 1 to 8 weeks or 1 to 4 weeks. Alternatively, the compound of formula (1) may be administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks before the vaccine.

[0090] Vaccine can be delivered to subject in any form that vaccine can induce immune response.Examples of administration route include parenteral, subcutaneous, intradermal, oral, intranasal, sublingual, by inhalation, topical, rectal, oral and vaginal.Therefore, according to a further aspect of the present invention, the method or use of the present invention is provided, wherein vaccination is administered via one or more of parenteral, subcutaneous, intradermal, oral, intranasal, sublingual, by inhalation, topical, rectal, oral and vaginal routes.

[0091] When used in the methods and uses of the present invention, the compound of formula (I) is believed to be particularly effective in elderly subjects and children.Accordingly, according to a further aspect of the present invention, there is provided a method or use of the present invention, wherein the subject is a child, an elderly person, or an elderly person.

[0092] An elderly person may be over 50 years old, such as over 55 years old, such as over 60 years old, such as between 50 and 65 years old.

[0093] An elderly patient may be aged over 65 years, such as over 70 years, such as over 75 years, such as over 80 years, such as between 65 and 80 years, such as between 65 and 90 years, such as between 90 and 100 years, such as over 100 years.

[0094] A child may be aged from birth to about 18 years, such as from birth to about 1 year, such as from birth to about 10 years, such as from birth to about 4-5 years.

[0095] The compound of formula (I) is believed to be particularly effective in immunocompromised subjects, such as those infected with or at risk of infection with HIV, hepatitis and tuberculosis, when used in the methods and uses of the present invention.According to a further aspect of the present invention, there is thus provided a method or use of the present invention, wherein the subject is an immunocompromised subject, such as those infected with HIV, hepatitis and / or tuberculosis.

[0096] The compound of formula (I) enhances the immune response to antigens, particularly in response to vaccination, and thus acts as an immune enhancer. The compound of formula (I) can be used with one or more additional immune enhancer components to further enhance the immune response. Thus, according to a further aspect of the present invention, there is provided an immune enhancer composition comprising a compound of formula (I) and one or more additional immune enhancer components. Such compositions include compositions intended to be administered before vaccination, and compositions in which the compound of formula (I) is administered first and one or more additional immune enhancer components are administered after a period of time. The administration of the immune enhancer can be stopped at the time of vaccination or delivered simultaneously with and after vaccination. The period of time can vary from a few hours to several weeks.

[0097] The compounds of formula (I) can be administered in a vaccine-based treatment protocol, with or without one or more additional immune enhancing components. Thus, according to a further aspect of the present invention, there is provided a treatment protocol comprising: (i) a compound of formula (I); (ii) a vaccine; and (iii) optionally one or more immune enhancing components.

[0098] According to a further aspect of the present invention, there is provided a treatment protocol in which the vaccine comprises an inactivated or attenuated microorganism or a protein or peptide antigen derived therefrom. The inactivated or attenuated microorganism is selected from an inactivated or attenuated virus or an inactivated or attenuated bacterium. For the avoidance of doubt, the protein or peptide antigen may be derived from an active or non-attenuated virus or bacterium.

[0099] In a treatment protocol, the compounds of formula (I) can be administered in several ways, such as: - primary prevention: giving a compound of formula (I) prior to vaccination to stimulate / sensitize the immune system and prepare it for vaccination; and / or - secondary prevention: giving a compound of formula (I) in conjunction with vaccination / immunization (as an immune booster - e.g. oral administration as a tablet in parallel with subcutaneous vaccination) to boost the immune response; and / or - Tertiary prevention: a compound of formula (I) given after the vaccination / influenza response has been initiated to alleviate or manage the symptoms of an influenza episode.

[0100] According to a further aspect of the invention, the vaccine is an inactivated or attenuated microorganism or antigen selected from one or more of influenza, chickenpox, diphtheria, meningococcus, measles, mumps, whooping cough, pneumococcus, polio, rubella, tetanus, tuberculosis, Ebola, typhoid and / or yellow fever, or a protein, subunit or peptide derived therefrom, preferably influenza or a protein, subunit or peptide derived therefrom.

[0101] Increasing the number of functional hematopoietic stem cells is beneficial for the overall renewal of the hematopoietic system, resulting in improved immune function; reduced incidence of bone marrow failure, such as after irradiation or bone marrow transplantation; and treatment of anemia. This can be used to improve recovery from the cytotoxic effects of chemotherapy treatments used to treat cancer (such as leukemia and lymphoma), to treat and / or prevent bone marrow suppression as a result of chemotherapy treatments, and to improve bone marrow transplantation and reduce transplant-related mortality. Thus, according to a further aspect of the present invention, a method for increasing the number of functional hematopoietic stem cells is provided, for example: (i) Prior to patient treatment and / or donor bone marrow harvest; (ii) in post-irradiation and / or post-bone marrow transplant treatment; (iii) in the treatment of the cytotoxic effects of chemotherapeutic treatment; and / or (iv) There is provided a compound of formula (I) for use in the treatment and / or prevention of myelosuppression as a result of chemotherapy treatment.

[0102] According to a further aspect of the present invention, (i) Prior to patient treatment and / or donor bone marrow harvest; (ii) in post-irradiation and / or post-bone marrow transplant treatment; (iii) in the treatment of the cytotoxic effects of chemotherapeutic treatment; and / or (iv) There is provided a compound of formula (I) for use in the treatment and / or prevention of myelosuppression as a result of chemotherapy treatment.

[0103] Hematopoietic stem cells are also a source of new red blood cells.Thus, according to a further aspect of the present invention there is provided a compound of formula (I) for use in the prevention and / or treatment of anemia.

[0104] The compound of formula (I) can also be used in patients being treated with irradiation, for example in the treatment of cancer.Accordingly, according to a further aspect of the present invention, there is provided the use of the compound of formula (I) as an immune enhancing agent during irradiation treatment in patients.The compound of formula (I) can be used to enhance the immune system before or during irradiation, so as to limit the damage to the immune system during treatment.Alternatively, or in addition, the compound of formula (I) can be used after irradiation to enhance the recovery of the immune system.

[0105] The compounds of formula (I) can be used as immune enhancers during bone marrow cell transplantation, before harvesting bone marrow cells for subsequent transplantation, and / or after transplanting the bone marrow cells back into the patient to enhance bone marrow repopulation. The compounds of formula (I) can be used ex vivo in expanding bone marrow cells before returning them to the patient after immunosuppressive therapy, such as cytotoxic chemotherapy for cancer treatment.

[0106] The compounds of formula (I) may be used ex vivo or in vivo to increase the numbers and function of immune cells used in cancer immunotherapy, including natural killer cells, macrophages, and activated T cells.

[0107] Compounds of formula (I) may be used ex vivo in the expansion of CAR-T, TCR-T or other engineered T cells before returning them to the patient for subsequent cancer immunotherapy (Kershaw, MH, Westwood, JA, & Darcy, PK Gene-engineered T cells for cancer therapy. Nature Reviews Cancer, 2013).

[0108] Compounds of formula (I) may be used ex vivo to increase the number and function of mesenchymal stem cells prior to return to the patient for cartilage regeneration.

[0109] The compounds of formula (I) may be used to stimulate the differentiation of pluripotent stem cells, induced pluripotent stem cells (iPS), embryonic stem cells, and adult stem cells into keratinocytes before being returned to the patient for skin grafting.

[0110] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for preventing and / or treating the cytotoxic effects of chemotherapy. According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for preventing and / or treating myelosuppression, for example myelosuppression following cytotoxic chemotherapy.

[0111] Depletion of neural stem cells (NSCs) has been implicated in the loss of smell during aging.Accordingly, according to a further aspect, there is provided a compound of formula (I) for use in a method for treating loss of smell, for example loss of smell during aging.

[0112] According to a further aspect of the invention there is provided a compound of formula (I) in a method of promoting the proliferation of inner ear hair progenitor cells.

[0113] Furthermore, NSCs are stimulated to proliferate in response to seizures, suggesting that the proliferation of NSCs leads to increased neurogenesis to repair brain damage or neuronal loss.According to a further aspect of the present invention, therefore, a compound of formula (I) is provided for use in a method for promoting brain repair.A further aspect of the present invention provides a compound of formula (I) or its use in a method for preventing, inhibiting or delaying neuronal loss.A further aspect of the present invention provides a compound of formula (I) for use in a method for preventing and / or treating seizures.

[0114] Increasing the number of functional neural stem cells can promote neurogenesis and support learning, such as adaptive learning.According to a further aspect of the present invention, therefore, there is provided a compound of formula (I) for use in a method for increasing neurogenesis.According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for supporting learning, such as adaptive learning.

[0115] A decrease in melanocyte stem cell function and / or a decrease in the number of melanocyte stem cells is associated with hair greying during aging. Thus, according to a further aspect of the present invention, there is provided a compound of formula (I) in a method for preventing, inhibiting or delaying hair colour loss or hair greying, such as hair colour loss or hair greying during aging.

[0116] The decline in hair follicle stem cell function and / or the decrease in the number of hair follicle stem cells may contribute to hair loss and baldness.According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for (i) preventing, inhibiting or delaying hair loss and / or baldness, (ii) promoting and / or improving hair growth; and / or (iii) promoting and / or improving hair growth after transplantation.

[0117] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for regenerating and / or increasing the rate of renewal of hair follicle stem cells.

[0118] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of differentiation of hair follicle stem cells.

[0119] According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for increasing hair growth.The rate of hair growth can be measured by the rate of hair shaft formation or the rate of hair follicle formation that has the activity of forming hair shaft.In some embodiments, the effect can include the change in thickness of hair shaft.

[0120] According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for increasing hair follicle number.In one embodiment, the compound of formula (I) promotes at least 5%, 10%, 15%, 20%, 25% increase in hair follicle number (or follicular unit) over a period of several weeks to several months.For example, at least 10% increase in hair follicle number (or follicular unit) over a period of several weeks to several months.For example, 50% (or more) increase in hair follicle number (or follicular unit) over a period of several weeks to several months.

[0121] According to another aspect of the present invention, the compound of formula (I) is provided for reducing hair loss.In one embodiment, hair loss is reduced by at least 5%, 10%, 15%, 20%, 25% over a period of several weeks to several months.For example, hair loss is reduced by at least 10% over a period of several weeks to several months.For example, hair loss is reduced by 50% (or more) over a period of several weeks to several months.

[0122] According to a further aspect of the invention there is provided a compound of formula (I) for use in conditions which would benefit from increased hair follicle stem cell growth and / or hair growth, such as alopecia areata, androgenetic alopecia, male pattern baldness and chemotherapy-induced alopecia.

[0123] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for the treatment of androgenetic alopecia.

[0124] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for the treatment of alopecia.

[0125] According to a further aspect of the present invention there is provided a compound of formula (I) for improving existing hair. Improving existing hair includes increasing hair thickness.

[0126] According to a further aspect of the present invention, there is provided a composition comprising a compound of formula (I) for (i) preventing, inhibiting or delaying hair loss and / or baldness; (ii) promoting and / or improving hair growth; and / or (iii) promoting and / or improving hair growth after transplantation, characterized in that the composition is a tonic, lotion, serum, shampoo, conditioner, spray, gel or cream. In a further embodiment, the compound of formula (I) or a salt thereof is present in the composition at a concentration in the range of 0.01 μM to 100 mM, 0.01 μM to 10 mM, 0.01 μM to 1 mM, 0.01 μM to 100 μM, 0.1 μM to 500 μM, 0.1 μM to 100 μM, or 1 μM to 50 μM.

[0127] The reduced renewal rate of beta cells in pancreatic islets increases the likelihood of type 2 diabetes during aging.According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for preventing, inhibiting, delaying, and / or reversing the reduced renewal rate of beta cells in pancreatic islets.According to a further aspect of the present invention, there is provided a compound of formula (I) for use in increasing the renewal rate of beta cells in pancreatic islets.

[0128] A reduction in muscle stem cell number and function reduces the ability of muscle to regenerate after injury. Thus, according to a further aspect of the present invention there is provided a compound of formula (I) for use in a method for increasing muscle stem cell number.

[0129] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of promoting the regenerative capacity of muscle.

[0130] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of promoting the regenerative capacity of muscle stem cells.

[0131] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for promoting and / or improving muscle regeneration, for example following injury or surgery.

[0132] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of promoting and / or improving muscle regeneration in an elderly or geriatric patient, for example following injury or surgery.

[0133] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method for promoting and / or improving muscle regeneration following exercise.

[0134] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing muscle stem cell numbers.

[0135] The effects on muscle stem cells can be identified by FACS (fluorescence activated cell sorting). Two examples of approaches to select muscle stem cell populations and observe the beneficial effects of the present invention on muscle stem cells are to employ the use of the following marker combinations: 1.CD31 negative, CD45 negative, SCA negative, VCAM positive muscle stem cells 2.CD31 negative, CD45 negative, CD11b negative, SCA negative, integrin α7 positive, CD34 positive muscle stem cells.

[0136] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD31 negative, CD45 negative, SCA negative, VCAM positive muscle stem cells.

[0137] According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of increasing the number of CD31 negative, CD45 negative, CD11b negative, SCA negative, integrin α7 positive, CD34 positive muscle stem cells.

[0138] According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for promoting muscle regeneration after injury. According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for increasing the regenerative capacity of muscle. According to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for muscle healing after injury. The compound of formula (I) can be used ex vivo in the proliferation of muscle stem cells before returning to the patient for subsequent muscle regeneration therapy. Thus, according to a further aspect of the present invention, there is provided a compound of formula (I) for use in a method for muscle stem cell proliferation, for example an ex vivo method for muscle stem cell proliferation.

[0139] The compounds of formula (I) are also believed to have a role in differentiating stem cells into more mature phenotypes, such as progenitor cells, and terminally differentiated cells, such as muscle cells, hair cells, hematopoietic cells and neuronal cells. Without being bound by theoretical theory, it is believed that this is by acting as a cofactor for other differentiation inducers.

[0140] Thus, according to a further aspect of the invention there is provided a compound of formula (I) for use in a method of inducing differentiation of a stem cell, such as a pluripotent stem cell. According to a further aspect of the invention there is provided a compound of formula (I) for use in a method of inducing differentiation of a progenitor cell.

[0141] Examples of cells whose differentiation is promoted by the compounds of formula (I) include pluripotent stem cells, induced pluripotent stem cells (iPS), embryonic stem cells, and adult stem cells. Further examples of cells include hematopoietic stem cells, muscle stem cells, skin stem cells, liver stem cells, neural stem cells, melanocyte stem cells, hair follicle stem cells, mesenchymal stem cells, adipose-derived stem cells, limbal stem cells (corneal stem cells), cardiac stem cells, intestinal stem cells, epithelial stem cells, autologous stem cells, and allogeneic stem cells.

[0142] According to a further aspect of the present invention, a) a compound of formula (I); and b) one or more differentiation-inducing factors.

[0143] Examples of differentiation-inducing factors include growth differentiation factors, retinoids such as retinoic acid, butyrates such as sodium butyrate, activin, fibroblast growth factors (such as basic FGF), transforming growth factors (such as TGF-β), myostatin, insulin growth factor 1, vascular endothelial growth factor A, bone morphogenetic proteins, interleukin 3, interleukin 6, stem cell factor, epidermal growth factor, colony simulating factors (such as GM-CSF and M-CSF), and glucagon-like peptide type 1.

[0144] Friedreich's ataxia is an autosomal recessive disease that affects a gene (FXN) on chromosome 9 that produces a key protein called frataxin. Frataxin plays a role in mitochondria, and it is believed that the compound of formula (I) plays a role in mitigating the effects of this biochemical pathology. According to a further aspect of the present invention, there is provided a compound of formula (I) for the treatment of Friedreich's ataxia.

[0145] According to a further aspect of the invention there is provided a compound of formula (I) for use in the method of the invention in a patient or subject in need thereof. [Brief description of the drawings]

[0146] [Figure 1]Diagram illustrating the hematopoietic system: A) shows all the different blood cell lineages derived from hematopoietic stem cells, and B) illustrates the cell surface markers that can be utilized to define the different subpopulations of progenitor cells and the relative contribution of each population to the total bone marrow (%BM). In humans, a similar set of human protein markers has been identified for both HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49+) and MPPs (Lin-CD34+CD38-CD45RA-CD90-CD49f-). Hematopoietic stem cells (HSCs) are the most quiescent cells and are the source of various multipotent progenitor cells (MPPS). [Diagram 2] FIG. 1 shows a set of plots of bone marrow cells isolated from aged C57BL / 6J mice analyzed by flow cytometry. The plots show an example of the gating strategy used to select a population containing progenitor LSK (c-Kit+, Lin-, Sca 1+) cells. In this particular example, urolithin A treatment significantly increases the population of progenitor cells, from 3.16% of lineage-negative cells in control mice to 5.27% in aged mice treated with urolithin A. [Diagram 3] Figure 1 shows the gating strategy used to select different progenitor populations, and the effect of Urolithin A treatment on these different hematopoietic progenitor populations. The various progenitor populations present in the LSK (c-Kit+, Lin-, Sca 1+) population of cells were separated based on expression of CD150, CD48, CD34, and CD135. Urolithin A treatment increases the % of LSK cells, the least committed progenitor cells (HSC+MPP1), from 53.1% to 61.7%. This increased population of HSC+MPP1 progenitors is further enriched for HSC cells from 73.8% (untreated) to 81.7% (Urolithin A). [Figure 4] FIG. 1 shows the effect of Urolithin A on the most primitive hematopoietic stem cell population. Urolithin A treatment in aged animals resulted in an increase in the most primitive progenitor hematopoietic stem cell (HSC) population, both as a % of total cells and as cell number. The increase in total stem cell number was statistically significant, p<0.05. [Diagram 5] FIG. 1 shows the design of a phase 2 clinical trial to evaluate the effect of Urolithin A on antibody titers in response to influenza vaccination. [Figure 6] Figure 1 shows the effect of Urolithin A on pro- and anti-inflammatory cytokines. Urolithin A treatment in aged animals led to a significant increase in the IL-10 over IL-12(p40) ratio (p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0147] The term "about" refers to a tolerance of ±20% of the relevant value, such as ±15% of the relevant value, such as ±10% of the relevant value or ±5% of the relevant value.

[0148] The term "immune enhancer" refers to any substance that acts to accelerate, prolong, or enhance an antigen-specific immune response when used in combination with a particular vaccine antigen, and / or any substance that improves immune health so that an individual can better mount an immune response against infection or cancer.

[0149] The term "antigen" refers to a protein, subunit, peptide or other compound that elicits an immune response when recognized as non-self by a subject. Such immune response may be via innate and / or adaptive mechanisms to mobilize a response against an invading pathogen, toxin, or allergen. Antigens may generally be derived from pathogenic organisms, such as pathogenic bacteria or viruses. Antigens may also be derived from malignant cells.

[0150] The term "immune cell" refers to any cell that is involved in the defense of a subject against infection by microorganisms such as bacteria and viruses. This term also relates to any cell that is involved in the defense of a subject against cancer cells. Immune cells include phagocytes such as neutrophils and macrophages, cytotoxic natural killer (NK) cells, granulocytes, macrophages, dendritic cells, memory cells, B cells and T cell subsets including macrophages.

[0151] The term "inflammation" refers to the chronic low-grade inflammation associated with the aging process.

[0152] The term "immunodeficiency" refers to a condition in which the immune system's ability to fight infectious disease and / or cancer is impaired or completely absent. A person suffering from any type of immune deficiency is said to be immunocompromised.

[0153] The term "immunosenescence" refers to the gradual deterioration of the immune system brought about by natural aging, both in the host's ability to respond to infection and in the development of long-term immune memory, particularly following vaccination.

[0154] The term "salt" refers to any pharma- ceutically acceptable or non-pharma-ceutically acceptable salt. Only pharma-ceutically acceptable salts are suitable for administration to a subject, although non-pharma-ceutically acceptable salts may be used as intermediates in the formation of pharma-ceutically acceptable salts.

[0155] The term "seroconversion", as used herein, refers to an increase in an individual in the amount of serum antibodies against the immunogen against which the individual was vaccinated, following vaccination, e.g., following vaccination with a common influenza strain (H1N1, H3N2, Type B), where the post-vaccination antibody level is equal, e.g., at least two-fold to at least four-fold the antibody level measured prior to vaccination.

[0156] The term "seroprotection," as used herein, refers to the presence in an individual of serum antibodies to an immunogen against which the individual has been vaccinated, in an amount equal to or greater than a protective threshold that confers protection against an infectious agent.

[0157] The term stem cell senescence refers to the steady and irreversible loss (without therapeutic intervention) of proliferative capacity despite continued viability and metabolic activity.

[0158] The term "subject" as used herein refers to an individual having or at risk of having a disease, such as humans, cows, cats, dogs, donkeys, pigs, horses, mice, rats, etc., particularly humans. A subject may be a patient in need of treatment according to the invention. The term "subject" may include non-mammalian subjects, such as reptiles.

[0159] The term "thymic involution" refers to the shrinkage of the thymus gland with age, resulting in changes in thymic structure and a loss of tissue mass.

[0160] The term "vaccine" as used herein refers to an immunogenic composition for in vivo administration to an animal or human subject to confer protection against disease, particularly viral or bacterial disease. The term "vaccine" also refers to anti-cancer vaccines.

[0161] The present disclosure provides compositions and methods involving the administration of a urolithin, such as a compound of formula (I), i.e., urolithin A, that provide beneficial health effects.

[0162] Compounds of formula (I) and salts thereof Urolithins are metabolic products produced by the action of the gut microbiota of mammals, including humans, on ellagitannins and ellagic acid. Ellagitannins and ellagic acid are compounds commonly found in foods such as pomegranates, nuts and berries. Ellagitannins are minimally absorbed into the gut itself. Urolithins are a class of compounds having the representative structure (I) shown above. The structures of some particularly common urolithins are set forth in Table 1 below with reference to structure (I). [Table 1] In practice, for commercial scale production, it is convenient to synthesize urolithins, synthetic routes being described, for example, in WO2014 / 004902 and WO2015 / 100213.

[0163] Urolithins of any structure according to structure (I) may be used in the methods of the disclosure.

[0164] In one embodiment of the uses and methods of the present disclosure, suitable compounds are compounds of formula (I) where A, C, D and Z are independently selected from H and OH, and B, W, X and Y are all H, preferably at least one of A, C, D and Z is OH.

[0165] Particularly suitable compounds are naturally occurring urolithins. Thus, Z is preferably OH, and W, X, and Y are preferably all H. When W, X, and Y are all H, A and B are both H, and C, D, and Z are all OH, the compound is urolithin C. When W, X, and Y are all H, A, B, and C are all H, and D and Z are both OH, the compound is urolithin A. Preferably, the urolithin used in the methods of the disclosure is urolithin A, urolithin B, urolithin C, or urolithin D. Most preferably, the urolithin used is urolithin A.

[0166] According to one embodiment, there is provided a method of the invention wherein the compound of formula (I) is urolithin A.

[0167] According to one embodiment, there is provided a method of the invention, wherein the compound of formula (I) is urolithin B.

[0168] According to one embodiment, there is provided a method of the invention, wherein the compound of formula (I) is urolithin C.

[0169] According to one embodiment, there is provided a method of the invention wherein the compound of formula (I) is urolithin D. [ka]

[0170] The term pharma- ceutically acceptable salts applied to compounds of formula (I) defines non-toxic organic or inorganic acid addition salts of the free base compounds that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, or allergic reaction, and that are commensurate with a reasonable benefit / risk ratio. Suitable salts according to the invention include those formed with organic or inorganic bases. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, such as potassium and sodium salts, alkaline earth metal salts, such as calcium and magnesium salts, and salts with organic bases, such as dicyclohexylamine, N-methyl-D-gluco- mine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di- or tri-lower alkylamines, such as ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine, or mono-, di- or trihydroxy lower alkylamines, such as mono-, di- or triethanolamine.

[0171] These salts may exist in either hydrated or substantially anhydrous form.Crystal forms of the compounds of formula (I) are also contemplated for use in the uses or methods of the present invention.

[0172] Administration / Dosage Regimen The disclosed method includes orally administering to a subject a compound of formula (I) or a salt thereof at a daily dose in the range of 1.7-6.0 mmol per day for a period of 2-16 weeks prior to vaccination, such as 1.7-2.7 mmol per day for a period of 2-16 weeks prior to vaccination, or 2.8-6.0 mmol per day for a period of 2-16 weeks prior to vaccination. As discussed below, administration of urolithin A in the range of 250 mg to 1000 mg (corresponding to about 1.1-4.4 mmol) is preferred, which results in a surprisingly good pharmacokinetic profile compared to the much higher dosage of 2000 mg. In one embodiment, the dose is 250 mg / day, in an alternative embodiment, the dose is 500 mg / day, and in another embodiment, the dose is 1000 mg / day.

[0173] In a further embodiment, the administered dose is: -250 mg once or twice daily; -500 mg once or twice daily; -750 mg once or twice daily; -1000mg once or twice daily; -1250 mg once or twice daily; or -1500 mg once or twice daily.

[0174] The disclosed method involves daily administration of a compound of formula (I) or a salt thereof, or a composition comprising the compound or salt. In some embodiments, the compound or composition is administered once per day, i.e., the compound or composition is administered at least once per 24-hour period. In other embodiments, the compound or composition comprising the compound is administered multiple times per day, for example, twice per day, or three or four times per day. In such cases, the daily dosage is divided into multiple doses thereof. In one embodiment, administration is once per day, in a second embodiment, administration is twice per day, and in a third embodiment, administration is three times per day.

[0175] The disclosed method entails daily administration of a compound of formula (I) or a salt thereof, or a composition comprising the compound or salt, for a period of 2 to 16 weeks. In some embodiments, the method may involve daily administration of a compound of formula (I) or a salt thereof for even longer periods, such as at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, 4 months, 6 months, or at least 1 year. In some embodiments, the method includes daily administration of a compound or a salt thereof for a period of up to 3 months, up to 6 months, up to 1 year, up to 2 years, or up to 5 years. In some embodiments, the methods include administering the compound or salt daily for a period ranging from 21 days to 5 years, 21 days to 2 years, 21 days to 1 year, 21 days to 6 months, 21 days to 12 weeks, 28 days to 5 years, 28 days to 2 years, 28 days to 1 year, 28 days to 6 months, 28 days to 4 months, 28 days to 12 weeks, 6 weeks to 2 years, 6 weeks to 1 year, 8 weeks to 1 year, or 8 weeks to 6 months.

[0176] The disclosed method entails daily administration of a compound of formula (I) or a salt thereof in an amount of from 0.7 mmol per day to 2.7 mmol per day, or from 0.7 mmol twice daily to 2.7 mmol twice daily. In some embodiments, the amount administered is in the range of 2.0 to 2.5 mmol. In some embodiments, the amount administered is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, or 2.7 mmol. In other embodiments, the amount administered is about 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 mmol. In some preferred embodiments, the methods involve administration of about 2.2 mmol of a compound of formula (I) or a salt thereof (e.g., of urolithin A) per day or 2.2 mmol twice daily. The exact weight of the compound administered will depend on the molecular weight of the compound used. For example, urolithin A has a molecular weight of 228 g / mol (so that 2.20 mmol is 501.6 mg) and urolithin B has a molecular weight of 212 g / mol (so that 2.20 mmol is 466.4 mg).

[0177] In further embodiments, the disclosed methods entail daily administration of a compound of formula (I) or a salt thereof in an amount of 2.8 mmol per day to 6.0 mmol per day or twice daily. In some embodiments, the amount administered is in the range of 4.0 to 4.8 mmol. In some embodiments, the amount administered is about 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 mmol. In some preferred embodiments, the methods involve administration of about 4.4 mmol of a compound of formula (I) or a salt thereof (e.g., Urolithin A) per day or twice daily. The exact weight of the compound administered will depend on the molecular weight of the compound used. For example, Urolithin A has a molecular weight of 228 g / mol (so that 4.40 mmol is 1003.2 mg) and Urolithin B has a molecular weight of 212 g / mol (so that 4.40 mmol is 932.8 mg).

[0178] In some embodiments, the methods involve administration of Urolithin A in an amount within the range of 400-600 mg per day, or 400-600 mg twice daily. In preferred embodiments, the methods involve administration of Urolithin A in the range of 450-550 mg per day or twice daily, more preferably about 500 mg.

[0179] In other embodiments, the methods involve administration of Urolithin A in an amount in the range of 700-1300 mg / day twice daily, or in the range of 750-1250 mg / day, or in the range of 800-1200 mg / day, or in the range of 850-1150 mg / day, or in the range of 900-1100 mg / day twice daily. In preferred embodiments, the methods involve administration of Urolithin A in an amount in the range of 950-1150 mg / day or twice daily, more preferably about 1000 mg / day or twice daily.

[0180] In some preferred embodiments, the methods involve administering Urolithin A to the subject in an amount ranging from 4.5 to 11 mg / kg / day, such as 4.5 to 8.5 mg / kg / day. In other embodiments, the methods involve administering Urolithin A to the subject in an amount ranging from 5 to 9 mg / kg / day. In other embodiments, the methods involve administering Urolithin A to the subject in an amount ranging from 6.0 to 8 mg / kg / day.

[0181] In other preferred embodiments, the methods involve administering Urolithin A to the subject in an amount ranging from 9 to 18 mg / kg / day, such as from 9 to 17 mg / kg / day. In another embodiment, the methods involve administering Urolithin A to the subject in an amount ranging from 10 to 17 mg / kg / day. In another embodiment, the methods involve administering Urolithin A to the subject in an amount ranging from 11 to 16 mg / kg / day.

[0182] A combined dosage regimen of 500 mg and 1000 mg doses may be advantageous. For example, a twice daily dosage regimen combining a first dose of 1000 mg and a second dose of 500 mg several hours later. The 500 mg dose may be 6-18 hours after the 1000 mg dose, for example 8-12 hours after the 1000 mg dose. For example, about 12 hours after the 1000 mg dose. Thus, according to a further aspect of the present invention, there is provided a treatment of diseases with a compound of formula (I) comprising a twice daily dosage regimen comprising a first dose of 1000 mg followed by a second dose of 500 mg, the two doses being spaced 6-18 hours apart.

[0183] The compound of formula (I) or its salt, or the composition containing the compound of the salt, may be administered at any suitable time, for example, in the morning or evening after sleep. In some embodiments, it may be preferable to carry out the method at about the same time(s) every day, for example, within 15, 30, 60, or 120 minutes from a given time point.

[0184] Immune boosters An immune enhancer is any compound or composition that increases the strength and / or duration of the immune response to a foreign antigen compared to that elicited by the antigen alone. Therefore, the important functional characteristics of an immune enhancer include the ability to enhance the appropriate immune response to the target antigen, long-term safety in a wide range of applications, and flexibility in use with different antigen / disease applications.

[0185] The compounds of formula (I) may be administered with one or more additional immune enhancing agents.

[0186] In a further alternative embodiment of the invention, the immune enhancing agent is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly 1:C and derivatives thereof.

[0187] In a further embodiment of the invention, the immune enhancing agent is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and / or trehalose dicorynemycolate (TDM).

[0188] In another embodiment of the invention, the immune enhancing agent is an aluminum-based immune enhancing agent suitable for use in human subjects.

[0189] Immune enhancers (immunostimulants or immunomodulators) have long been used to improve immune responses to vaccine antigens. The incorporation of immune enhancers into vaccine formulations aims to enhance, accelerate, and prolong specific immune responses to vaccine antigens. The benefits of immune enhancers include enhanced immunogenicity of weaker antigens, reduced antigen doses required for successful immunization, reduced frequency of booster immunizations required, and improved immune responses in vaccines in the elderly and immunocompromised. Optionally, immune enhancers can also be used to optimize the desired immune response, for example, in terms of immunoglobulin classes and induction of cytotoxic or helper T lymphocyte responses. In addition, certain immune enhancers can be used to promote antibody responses at mucosal surfaces. Aluminum hydroxide and aluminum or calcium phosphate are routinely used in human vaccines. More recently, vaccines containing antigens incorporated into IRIV (immunostimulatory reconstituted influenza virosomes), and the emulsion-based immune enhancer MF59, have been licensed in various countries. Immune enhancers can be classified according to their origin, mechanism of action, and physical or chemical properties. The most commonly described immune enhancer classes are gel-type, microbial, oil emulsion and emulsion-based, particulate, synthetic, and cytokine. The final vaccine product may contain more than one type of immune enhancer. They may be combined together with a single antigen or all antigens present in the vaccine, or each immune enhancer may be combined with one specific antigen. The origin and nature of immune enhancers currently used or developed are very diverse. For example, aluminum-based immune enhancers consist of simple inorganic compounds, PLGs are polymeric carbohydrates, virosomes can be derived from heterologous virus particles, MDPs are derived from bacterial cell walls, saponins are of plant origin, e.g. QS-21, squalene is derived from shark liver, and recombinant endogenous immunomodulators are derived from recombinant bacteria, yeast or mammalian cells. There are several immune enhancers approved for veterinary vaccines, such as mineral oil emulsions, which are too reactive for human use. Similarly, complete Freund's immune enhancer is one of the most potent immune enhancers known, but is not suitable for human use.

[0190] composition The disclosed method preferably involves oral administration of the compound of formula (I) or a salt thereof. Any suitable oral composition containing the compound of formula (I) or a salt thereof may be used. Thus, the use of various compositions containing the compound of formula (I) and suitable for oral administration is envisioned. Thus, in some embodiments, the compound of formula (I) or a salt thereof is administered in the form of an oral composition containing the compound of formula (I) or a salt thereof and one or more excipients suitable for oral administration. The oral composition may include compositions having the form of pills, tablets, capsules, caplets, drops, lozenges, granules, powders for suspension, oral solutions, oral suspensions, oral emulsions, syrups, etc.

[0191] The compositions containing the compounds of formula (I) may take any physical form suitable for the intended use, for example, they may be in the form of a solid (e.g., a bar), a semi-solid (e.g., a soft gel), or a liquid (including an emulsion). In some cases, the composition may be in the form of a viscous fluid or a paste. If the composition is, for example, a bar, it may be of any suitable type and it may contain ingredients conventionally used for the preparation of snack bars. The semi-solid form may likewise contain excipients conventional in the art. The excipients may, for example, provide the desired hardness, shelf life, and taste, so that the composition has an acceptable taste, an attractive appearance, and good storage stability. The semi-solid form may be in the form of a paste. If the composition is a soft gel, it may, for example, be provided in a capsule with a shell. The shell may be of a conventional type, for example, a soft gelatin-based shell. As an example, the composition may be provided inside a hard capsule-type shell. The liquid composition may be in the form of a medicament for oral intake, a dietary supplement, or a beverage, respectively. The liquid formulation may be a solution, an emulsion, a slurry, or other semi-liquid. Excipients in liquid compositions can provide, for example, shelf life, appearance, taste, and texture, so that the composition has acceptable taste, attractive appearance, and good storage stability. At certain dilution levels, it may be necessary for the subject to shake the drink before drinking it in order to maintain a uniform suspension of the active ingredient.

[0192] In some preferred embodiments, the method includes administering a compound of formula (I) or a salt thereof (e.g., urolithin A) in micronized form. Micronization allows the compound of formula (I) to disperse or dissolve more quickly. Micronization can be achieved by methods established in the art, such as compression force milling, hammer milling, universal or pin milling, or jet milling (e.g., spiral jet milling or fluidized bed jet milling). Jet milling is particularly suitable. When a micronized compound is used, the compound preferably has a D of less than 100 μm. 50More preferably, the compound has a particle size of less than 75 μm, such as less than 50 μm, such as less than 25 μm, such as less than 20 μm, such as less than 10 μm. 50 More preferably, the compound has a size in the range of D from 0.5 to 50 μm, such as from 0.5 to 20 μm, for example from 0.5 to 10 μm, for example from 1.0 to 10 μm, for example from 1.5 to 7.5 μm, for example from 2.8 to 5.5 μm. 50 Preferably, the compound has a D of less than 100 μm. 90 More preferably, the compound has a size of less than 75 μm, such as less than 50 μm, such as less than 25 μm, such as less than 20 μm, such as less than 15 μm. 90 The compound preferably has a size in the range of D 5 to 100 μm, such as 5 to 50 μm, for example 5 to 20 μm, for example 7.5 to 15 μm, for example 8.2 to 16.0 μm. 90 Preferably, the compound has a D in the range of 0.5 to 1.0 μm. 10 Preferably, the compound of formula (I) or a salt thereof (e.g., Urolithin A) has a D in the range of 8.2 to 16.0 μm. 90 , D in the range of 2.8 to 5.5 μm 50 , and D in the range of 0.5 to 1.0 μm 10 has.

[0193] In a further embodiment, the compound of formula (I) or salt thereof has a size distribution selected from one of the following: (i) D in the range of 0.5 to 50 μm 50 Size and D in the range of 5 to 100 μm 90 size, (ii) The compound has a D in the range of 8.2 to 16.0 μm. 90 Size, D in the range of 2.8 to 5.5 μm 50 Size and D in the range of 0.5 to 1.0 μm 10 Has size; (iii) The compound of formula (I) has a D in the range of 0.5 to 20 μm. 50 Size and D in the range of 5 to 50 μm 90 Has size; (iv) The compound of formula (I) has a D 50 Size and D less than 75 μm 90 Has size; (v) The compound of formula (I) has a D 50 Size and D less than 50 μm 90 Has size; (iv) The compound of formula (I) has a D of less than 10 μm 50 Size and D less than 20 μm 90 Has size; (v) The compound of formula (I) has a D of less than 10 μm. 50 Size and D less than 15 μm 90 having a size; or (vi) The compound of formula (I) has a D of 10 μm 50 Size and D of 20 μm 90 Has a size.

[0194] Compositions comprising a compound of formula (I) or a salt thereof and a medium chain triglyceride In some preferred embodiments, the compound of formula (I) or salt thereof (e.g., urolithin A) is administered in the form of a composition comprising a) a medium chain triglyceride, and b) a compound of formula (I) or salt thereof. In those embodiments, preferably the compound of formula (I) (e.g., urolithin A) is in micronized form.

[0195] By selecting suitable medium chain triglycerides and excipients, the physical form of the composition can be tailored to the requirements of the product in question. For example, in some embodiments, the composition can be a pharmaceutical composition. In some embodiments, the composition can be a nutritional composition.

[0196] In many cases, the composition comprising a compound of formula (I) and a medium chain triglyceride will have the consistency of a viscous liquid or paste and may be provided as a one-off supplement to a subject's general diet (e.g., in a bar, gel, or softgel capsule, hard capsule, or diluted in a drink) or it may be provided as part of or as the entirety of a meal.

[0197] Where the methods of the present disclosure involve the use of a composition comprising medium chain triglycerides, the medium chain triglycerides typically constitute at least 1% w / w of the composition, such as at least 5% w / w, such as at least 10% w / w, such as at least 15% w / w. The medium chain triglycerides preferably constitute 20% w / w or more of the composition, such as 25% w / w or more by weight of the composition, such as 30% w / w or more by weight. For example, the medium chain triglyceride may constitute 1 to 40% w / w of the composition, 2 to 40% w / w of the composition, 5 to 40% w / w of the composition, 10 to 40% w / w of the composition, 1 to 99% w / w of the composition, 5 to 99% w / w of the composition, 10 to 99% w / w of the composition, 20 to 99% w / w of the composition, 5 to 90% w / w of the composition, 10 to 90% w / w of the composition, for example, 20 to 90% w / w of the composition, 20 to 80% w / w of the composition, for example, 30 to 80% w / w of the composition, for example, 30 to 70% w / w of the composition, for example, 30 to 60% w / w of the composition, for example, 30 to 50% w / w of the composition, for example, 30 to 40% w / w of the composition, for example, 30 to 35% w / w of the composition. For example, the medium chain triglycerides may constitute 40-70% w / w of the composition, such as 50-70% w / w of the composition, for example 55-65% w / w of the composition.

[0198] In such compositions, the compound of formula (I) typically constitutes 0.1-80% w / w of the composition, such as 0.1-60% w / w, for example 0.25-50% w / w. For example, the compound of formula (I) may constitute 0.5-50% w / w of the composition. If the composition is provided as part or whole of a meal, the compound of formula (I) may, for example, constitute 0.25-5% w / w of the composition, for example 0.3-3% w / w of the composition. If the composition is provided as a one-time supplement to the subject's general diet, the urolithin typically constitutes 20-80% w / w of the composition, such as 20-40% w / w of the composition, for example 25-35%. For example, the urolithin may constitute 26-34% w / w of the composition, such as 28-33% w / w of the composition, for example 29-32% w / w of the composition, for example 29-31% w / w of the composition.

[0199] In such compositions, the weight ratio of the medium chain triglyceride component to the compound of formula (I) is generally in the range of 0.01:1 to 100:1, such as 0.5:1 to 100:1, for example 0.5:1 to 50:1, such as 0.5:1 to 5:1, or such as 1:1 to 75:1, for example 1:1 to 50:1, such as 1:1 to 20:1, for example 1:1 to 10:1, such as 1:1 to 2.5:1, for example 1:1 to 2:1, such as 1:1 to 1.5:1. The weight ratio may be in the range of 0.01:1 to 10:1, for example 0.1:1 to 10:1, or 0.01:1 to 5:1, for example 0.01:1 to 0.1:1.

[0200] In some preferred embodiments, the methods of the disclosure involve administration of a softgel capsule comprising a filling comprising a compound of formula (I) or a salt thereof (e.g., urolithin A) and one or more medium chain triglycerides. In those embodiments, preferably the compound of formula (I) or a salt thereof (e.g., urolithin A) is micronized. In embodiments in which a softgel capsule is used, the shell components may be manufactured using conventional ingredients.

[0201] Medium chain triglycerides have the formula CH2(OR 1 )-CH(OR2 )-CH2(OR 3 )(wherein, R 1 , R 2 , and R 3 is a medium chain fatty acid radical), generally of the formula -C(=O)(CH2) n CH3, where n ranges from 4 to 10, e.g., 6 to 8. Medium chain fatty acids are fatty acids with an aliphatic tail of 6 to 12 carbon atoms. The aliphatic end is primarily saturated. Specific medium chain fatty acids include caproic acid (hexanoic acid, C6:0), caprylic acid (octanoic acid, C8:0), capric acid (decanoic acid, C10:0) and lauric acid (dodecanoic acid, C12:0). Myristic acid (tetradecanoic acid, C14:0) may also be present in small amounts. The most commonly used medium chain triglycerides generally have a mixture of caprylic and capric triglycerides and contain 95% or more saturated fatty acids. The medium chain triglyceride component present in the preferred compositions used in the methods of the present disclosure may consist of a homogenous single medium chain triglyceride compound type, or, more commonly, the medium chain triglyceride component is a mixture of two or more different medium chain triglyceride compounds.

[0202] The European Pharmacopoeia describes medium chain triglycerides as fixed oils extracted from the hard, dried fraction of the endosperm of Cocos nucifera L. (coconut) or the dried endosperm of Elaeis guineenis Jacq. (African oil palm). Both the European Pharmacopoeia and the USPNF have specifications for medium chain triglycerides that require the presence of certain fatty acids to be as follows: caproic acid (C6) ≤ 2.0%, caprylic acid (C8) 50.0-80.0%, capric acid (C10) 20.0-50.0%, lauric acid (C12) ≤ 3.0%, and myristic acid (C14) ≤ 1%.

[0203] Medium chain triglycerides for use in preferred compositions include mixtures of fatty acid chains and triglycerides present in the following ratios: C6≦5% or less, C8 50-70%, C10 30-50%, and C12≦12% or less, for example C6≦0.5% or less, C8 55-65%, C10 35-45%, and C12≦1.5% or less.

[0204] The medium chain triglycerides used in the preferred compositions may be from any known or other suitable source.

[0205] The composition used in the method of the present disclosure can advantageously comprise one or more phospholipids.The particularly preferred phospholipid is phosphatidylcholine.The advantage brought about by phosphatidylcholine can be at least partially due to their amphiphilic properties, for example due to their properties as emulsifier.

[0206] A particularly useful source of phospholipids, especially phosphatidylcholine, is lecithin, and the compositions used in the methods of the present disclosure advantageously comprise lecithin. Lecithin, when present in the composition, typically constitutes at least 0.5% w / w of the composition, preferably at least 1% w / w of the composition. Lecithin preferably constitutes 10% w / w or more of the composition, such as 20% w / w or more by weight of the composition, such as 30% w / w or more by weight. For example, lecithin may constitute 0.5-80% w / w of the composition, such as 1-80% w / w of the composition, such as 20-80% w / w, such as 40-80% w / w, or alternatively, such as 0.5-75% w / w, such as 1-40% w / w of the composition, such as 30-40% w / w of the composition, such as 30-35% w / w of the composition, such as 30-75% w / w of the composition. Alternatively, the lecithin may constitute 0.5-5% w / w of the composition, such as 1-5% w / w of the composition, such as 1-3% w / w of the composition, such as 0.5-2% w / w, such as 1-2% w / w of the composition. The weight ratio between lecithin (if present) and urolithin is generally in the range of 0.02:1 to 3:1, such as 0.03:1 to 1.2:1, such as 1:1 to 1.2:1, such as 1.1:1 to 1.2:1.

[0207] "Lecithin" refers to any group of fatty substances occurring in animal and plant tissues, including phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). Commercially available lecithin from soybean and sunflower contains the phospholipids phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. Lecithin can be obtained by chemical extraction from its source in non-polar solvents such as hexane, ethanol, acetone, petroleum ether, or benzene, or by mechanical extraction. In particular, lecithin can be obtained by extraction from sources such as soybean, egg, milk, rapeseed, cottonseed, and sunflower. Commercially available lecithin for use in edible preparations can be easily purchased.

[0208] Commercially available lecithin that can be used in the compositions described herein typically contains the following major components: 33-35% soybean oil, 20-21% inositol phosphatides, 19-21% phosphatidylcholine, 8-20% phosphatidylethanolamine, 5-11% other phospholipids, 5% free carbohydrates, 2-5% sterols, and 1% moisture.

[0209] Commercially available lecithins that may be used in the compositions described herein may be enriched with phosphatidylcholine, e.g., having at least 5% w / w phosphatidylcholine in the lecithin, e.g., having at least 10% w / w phosphatidylcholine in the lecithin, e.g., having at least 15% w / w phosphatidylcholine in the lecithin, e.g., having at least 20% w / w phosphatidylcholine in the lecithin, e.g., having at least 25% w / w phosphatidylcholine in the lecithin, e.g., having at least 30% w / w phosphatidylcholine in the lecithin, e.g., having at least 32% w / w phosphatidylcholine in the lecithin, e.g., having at least 40% w / w phosphatidylcholine in the lecithin.

[0210] Lecithins may also be modified by one or more of the following processes to adjust their properties: alcohol extraction of specific phospholipids to produce lecithins with modified ratios of different phospholipids; acetone extraction to remove oil and obtain powdered or granular phospholipid blends; spray drying onto proteins as carriers; spray chilling with synthetic emulsifiers such as high melting mono- and di-glycerides to produce flaky or powdered products; enzymatic modification (phospholipases, generally and especially phospholipase A2), partial hydrolysis to produce lecithins with particularly pronounced emulsifying behavior; hydrolysis of fatty acid groups with acids and alkalis; acetylation; and hydroxylation of fatty acid chains and amino groups.

[0211] In some embodiments, the methods include administration of a composition comprising a compound of formula (I) or a salt thereof, a medium chain triglyceride, and an emulsifier (eg, lecithin).

[0212] A pharmaceutical composition containing a compound of formula (I) or a salt thereof may, for example, contain an additional pharma- ceutical active compound.

[0213] The additional components in the composition may be compounds that do not provide a health benefit to the subject, but instead improve the composition in some other way, for example, its taste, texture, or shelf life, as described above.Thus, the composition may further contain one or more compounds selected from emulsifiers, colorants, preservatives, gums, hardening agents, thickeners, sweeteners, and flavors.

[0214] Suitable emulsifiers, colorants, preservatives, gums, hardeners, and thickeners are well known in the art of emulsion and other semi-liquid production.The emulsifier may include one or more of phosphatidylcholine, lecithin, polysorbate such as polysorbate 60 or polysorbate 80 (Tween-60 and Tween-80), and glycerol monostearate (GMS).Glycerol monostearate is also known as glyceryl monostearate.

[0215] Stabilizers may be used in the compositions described herein. Many compositions are stable suspensions that do not require additional stabilizers. A stable suspension is one that does not undergo phase separation over time. For certain compositions, stability may be improved by including additional stabilizers. Stabilizers suitable for use in the compositions of the present invention include glycerol monostearate (GMS), silicon dioxide, and vegetable shortening. An exemplary stabilizer is GMS, and preferred compositions of the present invention contain GMS. Its properties also make GMS an excellent solvent for phospholipids, such as those found in lecithin. GMS exists in two polymorphic forms, the alpha form, which is dispersible and foamy and is useful as an emulsifier or preservative. The beta form is suitable for wax matrices. The alpha form is converted to the beta form when heated at 50°C.

[0216] GMS is classified into two separate grades, 40-55 percent monoglyceride and 90 percent monoglyceride. The 40-55 percent monoglyceride, as defined by the European Pharmacopoeia, describes GMS as a mixture of monoacylglycerols, primarily monostearoylglycerol, combined with some di- and tri-glycerols. In particular, the 40-55 grade contains 40-55% monoacylglycerol, 30-45% diacylglycerol, and 5-15% triacylglycerol. The 99 percent grade contains more than 90% monoglycerides. The monoglycerides in commercial GMS products are a mixture of variable ratios of glyceryl monostearate and glyceryl monopalmitate. The European Pharmacopoeia further divides Glyceryl Monostearate 40-55 into three types, depending on the ratio of stearic acid esters in the mixture. Type 1 contains 40.0-60.0% stearic acid, and the sum of palmitic acid and stearic acid is 90% or less. < Type 2 contains 60.0-80.0% stearic acid, and the sum of palmitic acid and stearic acid is 90% or less. < Type 3 contains 90.0-99.0% stearic acid, and the sum of palmitic acid and stearic acid is 96% or less. < Any form of GMS may be used in the composition.

[0217] In some embodiments, the methods involve administration of a composition comprising a medium chain triglyceride, a compound of formula (I) or a salt thereof (e.g., urolithin A), and a stabilizer, such as glycerol monostearate. In some embodiments, the methods involve administration of a composition comprising an emulsifier and a stabilizer.

[0218] Metal chelating or sequestrant agents such as the sodium calcium salt of ethylenediaminetetraacetic acid (EDTA) may also be used. Other components that may be included in the formulations of the present invention may include polyethylene glycol, silicon dioxide, vegetable shortening, and beeswax.

[0219] Flavorings can be beneficial in the compositions used in the methods described herein.In liquid or semi-liquid compositions, fruit flavors can be provided, for example, by including fruit sauce or puree.Typical flavors include strawberry, raspberry, blueberry, apricot, pomegranate, peach, pineapple, lemon, orange, and apple.Generally, fruit flavorings include fruit extracts, fruit jams or fruit purees, together with sweeteners, starches, stabilizers, natural and / or artificial flavors, colorants, preservatives, water, and any combination of citric acid or other acids suitable for controlling pH.

[0220] Unit dose compositions for use in the methods described herein preferably contain 250 mg or 500 mg of a compound of formula (I), e.g., 250 mg or 500 mg of Urolithin A. The unit dose may be, for example, in the form of a snack bar having a weight in the range of 25 g to 150 g, in the form of a beverage provided in a container such as a bottle or pouch sufficient to hold a single dose (e.g., 50 to 500 mL, 100 to 300 mL, e.g., 250 mL to 500 mL). In a further preferred alternative, the unit dose is in the form of a softgel capsule containing, for example, 250 mg of Urolithin A.

[0221] Representative compositions are shown in the table below. [Table 2] The present invention further provides a composition comprising a compound of formula (I) and a vaccine, and optionally one or more additional immune enhancing agents and / or one or more pharma- ceutically acceptable excipients. In one embodiment, the components of the composition are provided separately. In another embodiment, the components are provided in admixture. In another embodiment, the vaccine is provided separately from the compound of formula (I) in admixture with one or more additional immune enhancing agents. In another embodiment, the vaccine is provided in admixture with the compound of formula (I) and the one or more additional immune enhancing agents are provided separately. In another embodiment, the vaccine is provided in admixture with one or more additional immune enhancing agents and the compound of formula (I) is provided separately.

[0222] The pharmaceutical composition may take the form of a kit of parts, which may comprise a composition of the invention together with instructions for use and / or unit dosage forms of a number of different components. Thus, in one embodiment of the invention, there is provided a kit for inducing an immune response against an antigen, comprising: a first component comprising a compound of formula (I); - a second ingredient containing an additional immune booster; - optionally one or more further components including one or more further immune enhancing agents.

[0223] In a further embodiment of the invention there is provided a kit for inducing an immune response to an antigen comprising: a first component comprising a compound of formula (I); - with a second component containing a vaccine; - optionally one or more further components including one or more further immune enhancing agents.

[0224] In a further embodiment of the invention there is provided a kit for inducing an immune response to an antigen comprising: a first component comprising a compound of formula (I); - with a second component containing a vaccine; - with a third ingredient containing an additional immune booster; - optionally one or more further components including one or more further immune enhancing agents.

[0225] Such kits of the invention are optionally provided with instructions describing the order and interval of administration of the components. In one embodiment, the components are administered simultaneously. In another embodiment, the components are administered sequentially with one or more days or one or more weeks between each component.

[0226] In one embodiment, the kit of the invention comprises a vaccine and a compound of formula (I), and optionally one or more additional immune enhancing agents provided separately. In another embodiment, the kit of the invention comprises a mixture of a vaccine and a compound of formula (I), and optionally one or more additional immune enhancing agents. EXAMPLES

[0227] The invention will now be described with reference to the following non-limiting examples.

[0228] Example 1: Preparation of Urolithin A Urolithin A (4) was prepared in two steps starting from 2-bromo-5-methoxybenzoic acid 1 and resorcinol 2. The pure compound was obtained as a pale yellow powder. [ka]

[0229] Step 1: A mixture of 2-bromo-5-methoxybenzoic acid 1 (27.6 g; 119 mmol; 1.0 equiv.), resorcinol 2 (26.3 g; 239 mmol; 2.0 equiv.) and sodium hydroxide (10.5 g; 263 mmol; 2.2 equiv.) in water (120 mL) was heated at reflux for 1 h. A 5% aqueous solution of copper sulfate (3.88 g CuSO4·5H2O in 50 mL water; 15.5 mmol; 0.1 equiv.) was then added and the mixture was refluxed for an additional 30 min. The mixture was cooled to room temperature and the solid was filtered through a Büchner filter. The residue was washed with cold water to give a pale red solid, which was triturated with hot MeOH. The suspension was left at 4 °C overnight. The resulting precipitate was filtered and washed with cold MeOH to give the title compound 3 as a pale brown solid.

[0230] Step 2: To a suspension of 3 (10.0 g, 41 mmol; 1.0 equiv.) in anhydrous dichloromethane (100 mL) was added dropwise a 1 M solution of boron tribromide in anhydrous dichloromethane (11.93 mL of pure BBr3 in 110 mL of anhydrous dichloromethane; 124 mmol; 3.0 equiv.) at 0° C. The mixture was left at 0° C. for 1 h and then warmed to room temperature. The solution was stirred at that temperature for 17 h. Then, ice was added generously to the mixture. The yellow precipitate was filtered and washed with cold water to give a yellow solid, which was heated at reflux in acetic acid for 3 h. The hot solution was quickly filtered and the precipitate was washed with acetic acid and then with diethyl ether to give the title compound 4 as a yellow solid. 1 H and 13 C NMR was consistent with the structure of 4.

[0231] Example 2: Dosage Forms of Urolithin A Urolithin A was formulated into a softgel capsule containing the following components: [Table 3] [Table 4]

[0232] Example 3: Effect of Urolithin A on Stem Cell Populations To determine whether Urolithin A treatment had any effect on stem cell populations, hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) were isolated from aged C57BL / 6J mice fed a standard rodent diet or a mixed diet containing Urolithin A mixed with food to reach a dosage of 50 mg / kg / day delivered to the mice. Mice were treated with Urolithin A starting at 16 months of age and received 34 weeks of treatment. At the end of the treatment period, mice were sacrificed and bone marrow was harvested from femurs and tibias. Cells were extracted from bones after removing muscle and connective tissue, followed by the creation of single cell suspensions.

[0233] To determine the effect of Urolithin A treatment on the different progenitor cells of the hematopoietic system, flow cytometry was used to determine the number and percentage of each progenitor cell type. Flow cytometry allowed the selection and quantification of cells with different patterns of protein expression on the cell surface. Using labeled monoclonal antibodies, it was possible to identify various progenitor populations based on their expression of cell surface proteins. To isolate undifferentiated cells, including progenitor populations, bone marrow cells were stained with a cocktail of monoclonal antibodies against cell surface proteins specific for more differentiated cell types. Antibodies against these proteins, also known as lineage markers, were conjugated to Alexa647 [AlexaFluor 647 dye is a bright far-red fluorescent dye used as a label in fluorescence microscopy and other applications]. To distinguish the progenitor populations of various lineages, isolated bone marrow cells were stained with monoclonal antibodies against the following progenitor cell surface protein markers, including the following labels: CD34 (RAM34)-FITC; CD135 (Flt3 A2F10)-PE; CD48 (HM48.1)-Biotin+PE Texas Red Streptavidin; CD117 (ckit 2B8) PerCp-Cy5.5; Sca1 (D7)-PE-Cy7. Cell analysis of stained cells was performed on a Becton Dickson Flow cytometer (Franklin Lake, NJ, USA) and collected data was analyzed using Flowjo™ software, an analytical platform for single-cell flow cytometry analysis. Bone marrow was isolated from three mice that received Urolithin A treatment and six control untreated mice.

[0234] To identify the different populations of hematopoietic progenitor cells present in the isolated bone marrow cells, the isolated bone marrow cells were selected based on a combination of their cell surface markers. A so-called gating strategy was employed, where a specific cell population is selected (gated) based on the pattern of cell surface protein expression, and then this subselected population is further examined for its cell surface protein expression. Figures 2 and 3 show examples of the gating strategy employed in all samples examined. Uncommitted progenitor cells were investigated by selecting cells that were negative for the cell surface proteins found in the differentiated cells (lineage markers) mentioned above (Figure 2B). From this population of lineage-negative cells, cells that stained positive for the cell surface markers CD117 (c-kit) and Sca1 were selected. This subpopulation of lineage-negative, CD117 and Sca1 positive cells (LKS cells) comprises progenitor stem cells (Figure 2C). Figure 2C shows that in aged animals treated with Urolithin A, the population of LKS progenitors present in lineage-negative cells was substantially increased from 3.16% in control-treated aged mice to 5.27% in aged mice treated with Urolithin A.

[0235] LKS cells can be further classified into four distinct progenitor cell types: HSC (LT-HSC), MPP1 (ST-HSC), MPP2, and MPP3 or LMPP with decreasing levels of quiescence (Figure 1B). The most quiescent and uncommitted cells are HSC cells. To distinguish these cell populations within the LKS cells, cells were examined for expression of CD150 and CD48. The most primitive quiescent cell population, HSC+MPP1, is positive for the cell surface marker CD150 and negative for the cell surface marker CD48. As shown in Figure 3 (first set of graphs), in this example, Urolithin A treatment significantly increased this population, with 61.7% of the LKS cells being HSC+MPP1 cells in treated animals compared to 53.1% in untreated animals. This was found to be true for all animals treated with Urolithin A (n=3) when compared to untreated mice (n=5), and the increase in this progenitor population was statistically significant (Table 2). HSC+MPP1 progenitor cells are stem cells capable of reconstituting all the different blood cell lineage populations. HSC+MPP1 progenitor cells can be further subdivided into two different types of hematopoietic stem cells: a long-term renewal population (HSC or LT-HSC) that enters the cell cycle less frequently and helps maintain the stem cell population, and another population, MPP1 or ST-HSC, that cycles more frequently and is involved in maintaining the blood cell lineages. These two populations can be distinguished by selecting (gating) HSC+MPP1 cells and examining the expression of the cell surface marker CD34 on these cells. The most primitive stem cell populations, HSC or LT-HSC, do not express CD34 (Figure 1B). As shown in Figure 3 (second set of graphs), urolithin A treatment increases the % of HSC+MPP1 cells that are HSC in aged animals from 73.8% to 81.7%. Importantly, this increase in the most primitive stem cell population led to an even more significant increase in the final total number of HSCs present in the hindlimb, both as a % of total bone marrow cells and as cell numbers, as shown in Figure 4, with aged animals treated with Urolithin A having significantly increased HSC cell numbers versus untreated aged animals. [Table 5]

[0236] Example 4: Preclinical study to evaluate improvement in antibody titers in response to vaccination following Urolithin A administration Experimental Overview Adult female BALB / c are randomly assigned to experimental groups and allowed to acclimate for one week. Starting on day 0, animals are administered the food mixture according to the following schedule: On days 0, 14, and 28, intramuscular (im) challenges with PR8 antigen are performed. Starting on day 0, animals are monitored daily for non-specific clinical signs. Starting on day 0, animals are weighed three times a week. On day 28, blood samples are taken to perform hemagglutination inhibition assays (HAI) and processed to isolate serum for antibody analysis (total IgG targeting PR8).

[0237] On day 42, spleens are excised, processed into single cell suspensions, and seeded for T cell proliferation assays. Cells are incubated overnight in the presence or absence of PR8 (antigen) or in triplicate with a positive control. Ex vivo immune cells taken from spleens (systemic reservoir of immune cells in the body) of vehicle (group 1 in the table below) treated mice were stimulated with or without UA / vehicle for 24 hours and examined for cell proliferation. Cell proliferation is the primary measurement of this in vitro assay to assess the enhancement of immune responses and is quantified by the incorporation of tritiated thymidine. Cell culture supernatants are saved for multiplex analysis of cytokines. [Table 6]

[0238] Example 5: Phase 2 clinical trial to evaluate improvement in antibody titers in response to influenza vaccination following administration of Urolithin A A phase 2 trial will be conducted to evaluate the effect of Urolithin A on antibody titers in elderly people in response to influenza vaccination. The protocol is outlined in Figure 2.

[0239] Patients will be recruited into the study with the following key inclusion criteria: 1) 65-90 years old 2) Both genders (male and female) 3) 18.5~30kg / m 2 body mass index (BMI); and 4) Ability to give informed consent and complete the study and its visits.

[0240] Patients will be recruited with the following main exclusion criteria: 1) Participation in another clinical trial investigating a vaccine or drug is planned during the current study period. 2) Having received any vaccination within 4 weeks prior to the study period. 3) Received influenza vaccination within 6 months prior to the study vaccination period. 4) Known or suspected congenital or acquired immunodeficiency, or receiving immunosuppressive therapy such as anticancer chemotherapy or radiation therapy within the past year. 5) Systemic hypersensitivity to eggs, chicken proteins, or any of the vaccine components. 6) Current alcohol or drug addiction. 7) Chronic illness at any stage that, in the opinion of the investigator, may prevent the conduct or completion of the study. 8) Have a hearing or visual impairment. 9) Severe cognitive impairment (MMSE<18) or diagnosis of major depression. 10) Progressive neurodegenerative neurological diseases: e.g. Alzheimer's disease. 11) Hospitalization in the past 6 weeks. 12) self-reported thrombocytopenia, a contraindication to intramuscular vaccination; 13) Hemorrhagic disorder contraindicated to intramuscular vaccination in the investigator's judgment, or administration of anticoagulants within 3 weeks prior to vaccination.

[0241] Clinical Trial Protocol The role of UA in promoting immune health during aging to improve vaccination responses will be evaluated in a double-blind, randomized, and placebo-controlled clinical trial. The primary outcome of this study is to demonstrate a statistically higher fold change / seroconversion in geometric mean titers (GMTs) of antibodies following vaccination with major influenza strains (H1N1, H3N2, and B) in the UA-treated elderly group compared to those vaccinated with the placebo-treated group. Healthy elderly subjects will be screened based on the inclusion and exclusion criteria listed above and randomized into either a placebo group (n=45-75 subjects) or an active group receiving Urolithin A (n=45-75 subjects). The placebo and Urolithin A are, for example, in the form of soft gels. Subjects will receive the intervention for a total of 4 weeks after randomization. After 4 weeks of intervention with either placebo or active treatment, all subjects will be evaluated in serum for pre-vaccination antibody titers and immune health. Subjects are then challenged with influenza vaccine and antibody titers to influenza vaccination are assessed again at weeks 3 and 6 in serum collected at these time points. The clinical trial design is carried out as shown in FIG.

[0242] Clinical Trial Endpoints The primary endpoint of the clinical trial is the post-vaccination fold change in seroconversion (i.e., the geometric mean titer of antibody response assessed by hemagglutination inhibition (HAI) t against the three major virulent influenza strains H1N1, H3N2, and type B) comparing placebo to UA treatment. The expected seroconversion change of UA treatment compared to the placebo group can be in the range of 40-60% compared to the placebo-treated elderly group, which can be in the range of 15-30% seroconversion, for example. The geometric mean titers of the major influenza strains can be, for example, 1.5-2 times higher in UA-treated elderly subjects compared to the placebo intervention.

[0243] Key secondary endpoints include seroprotection (incidence of protection from influenza) and cellular T cell memory responses (cytokines, T cell memory).

[0244] Example 6: Preclinical studies to evaluate the improvement in anti-inflammatory profile following Urolithin A administration Urolithin A is mixed in the AIN-93G Growing Rodent Diet (Research Diet, New Brunswick, NJ, USA) at a concentration of 0.5 g / kg of food, corresponding to a dose of 50 mg / kg / day. Pellets with or without UA are fed to 22-month-old male C57BL / 6J mice (Janvier, Saint Berthevin, France) for 6 weeks. A total of 32 mice are housed individually and randomized to 16 animals per group according to their motor abilities. Animals that die naturally during the experiment are excluded from the calculations. These criteria were established before the start of the experiment. Animals are euthanized after 6 weeks of treatment after an overnight fast and then blood samples are taken by the vena cava. Blood samples are centrifuged at 1,500 x g for 15 min and the plasma supernatant is transferred to a clean tube. Plasma is analyzed for cytokines using the MILLIPLEX MAP Mouse Cytokine / Chemokine Magnetic Bead Panel (MCYTOMAG-70K).

[0245] Figure 6 shows the results of interleukin-10 (IL-10), interleukin-12 subunit beta, also known as IL-12(p40), and the ratio of IL-10 / IL-12(p40). IL-10 is an important anti-inflammatory cytokine (Fiorentino DF, et al., IL-10 inhibits cytokine production by activated macrophages. J Immunol. 1991 Dec 1; 147(11): 3815-22; Fiorentino DF et al., IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J Immunol. 1991 May 15; 146(10): 3444-51). Interleukin-12 (IL-12) is a potent pro-inflammatory cytokine that enhances the cytotoxic activity of T lymphocytes and resting natural killer cells. As shown in Figure 6, the ratio of IL-10 to IL-12(p40) is significantly increased. This result indicates that UA treatment in aged mice can reduce the so-called age-associated inflammation by shifting the cytokine profile to a more anti-inflammatory phenotype (Daniela Frasca and Bonnie B. Blomberg. Inflammageing decreases adaptive and innate immune responses in mice and humans. Biogerontology. 2016; Franceschi C et al., Inflammageing: a new immune-metabolic viewpoint for age-related diseases. Nature Reviews Endocrinology 2018).

[0246] Example 7: Preclinical study to evaluate the improvement of hair regrowth with Urolithin A Hair is formed in the hair follicle, a highly proliferative tissue due to the constant renewal of stem cells and their further differentiation. These dynamic mechanisms require very high bioenergetic capacity and therefore increasing evidence indicates the role of mitochondria during this process, especially during the differentiation stage (Kloepper, JE et al. J. Invest. Dermatol. 135, 679-689 (2015)). Human stem cells derived from human hair follicles rely mainly on aerobic glycolysis for energy generation (Kealey, Williams R, PM Ski. Pharmacol. 7, 41-46 (1994)), but upon differentiation they switch to oxidative phosphorylation (OXPHOS), which is entirely dependent on mitochondrial function (Tang, Y. et al. PeerJ 4, e1821 (2016); Hamanaka, RB et al. Sci. Signal. 6, (2014)).

[0247] In this study, C57BL6 / J male 3-month-old mice are used. The telogen dorsal skin hair is carefully depilated. For details on the identification of hair phase, see Muller-Rover, S. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J.Invest.Dermatol.117, 3-15 (2001). The animals are randomly assigned to three different experimental groups. One day after depilation, 100 μl of placebo control, UA concentration 1 and UA concentration 2 are applied daily to the target area (the appropriate UA formulation is determined in advance). To quantify the degree of hair growth, the back area of ​​the mice is imaged daily and further analyzed with NIH ImageJ software (https: / / imagej.nih.gov / ij / ). The level of pigmentation in this area is measured taking into account the background color of the mouse's skin. The mice are sacrificed 0, 7, 15 and 30 days after treatment. The skin is harvested, placed into a cryomold containing Optimal Cutting Temperature (OCT) compound, and frozen at -80 °C for further histological analysis.

[0248] To analyze the cycle of hair follicle regeneration, frozen skin sections are sectioned longitudinally using a cryostat and stained with hematoxylin and eosin. Images are acquired with an Olympus microscope. To quantify the hair cycle, individual hair follicles of each mouse are classified based on the guidelines (Muller-Rover, S. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Invest. Dermatol. 117, 3-15 (2001)). The percentage of hair follicles in a specific anagen phase is calculated for each group. Hair follicles in the same area are counted in the sections of each mouse. Furthermore, the total number of hair follicles in the same area is quantified.

[0249] Example 8a: In vivo testing of the ability of Urolithin A to increase muscle stem cell numbers. To test the ability of UA to increase muscle stem cell numbers, mouse models of muscle degeneration are used, including but not limited to models of muscle atrophy, muscular dystrophy and other neuromuscular degenerative disorders, such as those models described in more detail in the table below. [Table 7] Mice are fed a standard diet with or without UA at doses ranging from 10mpk / day to 250mpk / day. The number of animals per condition and the duration of treatment vary depending on the model used. At the end of the experiment, mice are euthanized and muscles are harvested and digested with collagenase type II (Sigma) and collagenase / dispase (Roche). The muscle slurry is then filtered through a cell strainer and the isolated cells are washed and stained with antibodies including MuSC (muscle stem cell) markers CD31 (1:800, eBioscience, eFluor450 conjugate); CD45 (1:200, eBioscience, eFluor450 conjugate); Sca-1 (1:1000, eBioscience, PE-Cy7 conjugate); and VCAM-1 (1:200, MBL) for 40 minutes at 4°C. Secondary staining is performed with propidium iodide (PI, Sigma). Stained cells are analyzed and sorted using a FACSAria II instrument (BD Biosciences). Muscle stem cells are defined as CD31-negative, CD45-negative, SCA-negative, VCAM-positive muscle stem cells. Debris and dead cells are excluded by forward scatter, side scatter and PI gating.

[0250] Example 8b: In vivo testing of Urolithin A to increase muscle stem cell numbers in aged mice Middle-aged and old male C57BL / 6 mice are fed standard chow pellets with or without UA and muscle stem cell numbers are assessed as described in Example 8a.

[0251] Example 8c: Ex vivo testing of Urolithin A to increase muscle stem cell numbers C57BL / 10ScSn-Dmd mdx Muscles from J mice and 20-month-old male C57BL / 6 mice are isolated, washed, and digested in collagenase solution. Single myofibers are liberated using a glass pipette, treated with DMSO or UA (final concentrations ranging from 5 µM to 100 µM) for various time frames, and stained with muscle stem cell markers. Microscopic images are acquired by confocal microscopy, and the number of muscle stem cells per myofiber is determined using ImageJ software.

[0252] Example 9: Ex vivo testing of Urolithin A to increase the number of endometrium hair progenitor cells Cochleae from neonatal mice (1-3 days old) are dissected in Hanks' Balanced Salt Solution (HBSS) and the organ of Corti (sensory epithelium) is separated from the stria vascularis (ion transporting epithelium) and modiolus (neural tissue). The organ of Corti is then treated with Cell Recovery Solution (Corning) for 1 h to separate the cochlear epithelium from the underlying mesenchyme. The epithelium is then harvested and treated with TrypLE (Life Technologies) for 15-20 min at 37 °C. Single cells obtained by mechanical trituration are filtered (40 mm) and suspended in Matrigel (Corning) domes for 3D culture. Cells were incubated in a serum-free 1:1 mixture of DMEM and F12 supplemented with Glutamax (GIBCO), N2, B27 (Invitrogen), EGF (50 ng / mL; Chemicon), bFGF (50 ng / mL; Chemicon), IGF-1 (50 ng / mL; Chemicon), and DMSO or UA. Medium was changed every other day.

[0253] To differentiate stem cell colonies, remove the growth medium and leave the colonies in 3D culture. Use a serum-free 1:1 mixture of DMEM and F12 supplemented with Glutamax (GIBCO), N2, and B27 (Invitrogen) and DMSO or UA to test the effect of compounds on differentiation.

[0254] To evaluate the effect of UA on the proliferation of inner hair progenitor cells, cells positive for Lgr5 are quantified after 10 days in the media of multiple conditions. Cell colonies are dissociated into single cells using TrypLE (GIBCO). Cell counts are performed with a hemocytometer. Cells are then stained with propidium iodide (PI) and analyzed for expression of Lgr5 using a flow cytometer.

[0255] To quantify hair cell production and evaluate the effect of UA on inner hair progenitor cell differentiation, Atoh1 positive cells are quantified on days 0 and 10 of the differentiation treatment. Cell colonies are incubated in CellRecovery Solution (Corning) to release the colonies from the Matrigel and dissociated into single cells using TrypLE. The total number and percentage of Atoh1+ cells are quantified by FACS.

[0256] Example 10. UA induces proliferation and differentiation of neural stem cells. Several central nervous system (CNS) diseases are characterized by the loss of neurons in specific regions of the brain, and therefore there is growing interest in identifying therapeutic strategies to repopulate damaged areas with functional neurons. One attractive approach is the use of neural stem cells (NSCs). NSCs are undifferentiated cells that have the capacity to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. These cells play a crucial role during CNS development and for the maintenance of the adult brain. NSCs can be isolated from mammalian embryonic brains, allowing the study of strategies that regulate their proliferation and differentiation into mature neurons (Marsh, SE & Blurton-Jones, M. Neurochem.Int. 106, 94-100 (2017)).

[0257] Proliferation assay: To evaluate the effect of UA on NSC proliferation and differentiation, embryonic rat NSCs are used. Rat NSCs are isolated according to standard procedures (Zhenxian Han, Qian Xu, Changfu Li, HZ Genesis 55, (2017)). Sprague-Dawley pregnant rats are euthanized after isoflurane overdose on day 16 of gestation, and rat embryonic brains are harvested. Cortices are removed and mechanically dissociated in Hank's medium (calcium and magnesium free), then centrifuged at 1000 rpm for 10 min. The pellet is then resuspended in Dubelco's modified Eagle's medium (DMEM). Cultures are maintained in DMEM / F12 supplemented with N2, EGF / BFGF (10 ng / mL), and PDGF / NT3 (5 ng / mL). NSC cells are seeded at a density of 30000 cells / well in a 96-well plate format.

[0258] Proliferation assay: To test the effect of UA on NSC proliferation, we use a neurosphere formation assay. Cells are seeded in culture medium and, after the determined treatment, neurospheres are stained with Ki-67 antibody, which specifically labels proliferating cells. Images are acquired using an Olympus microscope and the number of Ki-67 positive cells is quantified in a blinded manner.

[0259] Differentiation assay: To assay the effect of UA on NSC differentiation into neurons, cells are seeded in 6-well plates. After UA treatment, cells are stained with an antibody against the neuronal marker Tuj-1, followed by the addition of a secondary antibody. Images are acquired using an Olympus microscope, and the number of Tuj-1 positive cells is quantified in a blinded fashion.

Claims

1. A compound of formula (I) for use in a method for modulating stem cell function. 【Chemistry 1】 (In the formula, A, B, C, D, W, X, Y, and Z are each independently selected from H and OH. or a salt thereof.

2. 2. The compound of formula (I) according to claim 1, wherein the modulation of stem cell function is an increase in stem cell number and / or promotion of stem cell renewal.

3. 2. A compound of formula (I) according to claim 1, wherein the modulation of stem cell function is, for example, preventing, reducing or delaying senescence of stem cells during aging.

4. 2. The compound of formula (I) according to claim 1, wherein said modulation of stem cell function is the prevention, reduction or delay of inflammation associated with aging.

5. 2. The compound of formula (I) according to claim 1, wherein the modulation of stem cell function is the promotion of stem cell differentiation.

6. The compound of formula (I) according to any one of claims 1 to 5, wherein the stem cells are selected from one or more of the following cells: pluripotent stem cells, induced pluripotent stem cells (iPS), embryonic stem cells, and adult stem cells.

7. 6. The compound of formula (I) according to any one of claims 1 to 5, wherein the stem cells are selected from one or more of the following cells: hematopoietic stem cells, muscle stem cells, skin stem cells, liver stem cells, neural stem cells, melanocyte stem cells, hair follicle stem cells, mesenchymal stem cells, adipose-derived stem cells, limbal stem cells (corneal stem cells), cardiac stem cells, intestinal stem cells, epithelial stem cells, autologous stem cells and allogeneic stem cells.

8. The compound of formula (I) according to any one of claims 1 to 5, wherein said stem cells are selected from CD34 negative hematopoietic stem cells and CD34 positive hematopoietic stem cells.

9. The modulation of stem cell function, (i) promoting muscle stem cell proliferation, e.g., ex-vivo methods of muscle stem cell proliferation; (ii) promoting the regenerative capacity of muscle stem cells; (iii) promoting the regeneration of hair follicle stem cells; (iv) promoting differentiation of hair follicle stem cells; (v) preventing, inhibiting, and / or reversing the decline in the rate of beta cell renewal in pancreatic islets; (vi) promoting the proliferation of inner ear hair progenitor cells; and / or (vii) prevention, reversal or delay of olfactory decline during aging.

10. For use in methods of increasing the number of functional hematopoietic stem cells, e.g. (i) prior to treatment and / or bone marrow harvest in the donor; (ii) post-irradiation and / or post-bone marrow transplant treatment; (iii) treatment of the cytotoxic effects of chemotherapeutic treatment; (iv) Compounds of formula (I) as defined in claim 1 for use in the treatment and / or prevention of myelosuppression as a result of chemotherapy treatment.

11. 10. A compound of formula (I) as defined in claim 1 for use in a method for treating or preventing anemia.

12. 13. A compound of formula (I) as claimed in claim 1 for use in a method for (i) preventing, inhibiting or delaying hair loss and / or baldness, (ii) promoting and / or improving hair growth; and / or (iii) promoting and / or improving hair growth after transplantation.

13. A compound of formula (I) as claimed in claim 1 for use in the ex-vivo expansion of stem cells prior to return to the patient for subsequent regenerative therapy.

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