S(+) form of 4-(2,4-difluorophenyl)-2-(1h-indol-3-yl)-4-oxobutanoic acid

Abstract

The present invention relates to the use of the S(+) form of 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid or a physiologically-acceptable salt thereof as an active ingredient in a pharmaceutical composition or a food / beverage composition.

Description

S(+) form of 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid

[0001] The present invention relates to the use of the S(+) enantiomer of 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid (hereinafter sometimes referred to as "MA5") or a physiologically acceptable salt thereof as an active ingredient in a pharmaceutical composition or a food or beverage composition.

[0002] The present inventors have reported that the racemic form of 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid (i.e., MA5) has the effect of enhancing erythropoietin expression and treating mitochondrial diseases (Patent Document 1), the effect of inhibiting organ fibrosis (Patent Document 2), and the effect of preventing or improving hearing loss (Patent Document 3). Furthermore, the present inventors have reported that the R(-) form, which is the enantiomer of MA5, inhibits the production of oxidized nicotinamide adenine dinucleotide (NAD) (NAD ＋ It has been reported that the compound has the effect of increasing the amount of hydroxybenzoates (Patent Document 4).

[0003] International Publication No. 2014 / 080640 Pamphlet Japanese Patent Application Laid-Open No. 2015-189670 Japanese Patent Application Laid-Open No. 2019-116453 International Publication No. 2023 / 013756 Pamphlet

[0004] The inventors have found that the absolute configuration described in previous reports is incorrect by converting each enantiomer of MA5 into a benzylamine salt, which is easy to crystallize, and performing X-ray crystallography. ＋ It was confirmed that it is the S(+) form of MA5, not the R(-) form, that has the effect of increasing the amount of mitochondrial ATP and has mitochondrial function activation and anti-aging effects.

[0005] The present invention relates to a pharmaceutical composition, food or drink composition, etc. containing the S(+) isomer of MA5 as an active ingredient, and provides the following [1] to

[15] : [1] A pharmaceutical composition containing, as an active ingredient, a compound represented by the following formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer: [2] NAD in the subject ＋ [1] The pharmaceutical composition according to [1], which is used to increase the level of mitochondrial disease. [3] The pharmaceutical composition according to [1] or [2], which is for the treatment or prevention of mitochondrial disease. [4] The pharmaceutical composition according to [1] or [2], which is for the treatment or prevention of symptoms or diseases associated with aging. [5] The pharmaceutical composition according to [4], wherein the symptoms or diseases associated with aging are hearing loss. [6] The pharmaceutical composition according to any of [1] to [5], wherein the enantiomeric excess of the S(+) isomer is at least 97% or more. [7] A food or drink composition comprising, as an active ingredient, a compound represented by the following formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer. [8] NAD in the subject ＋ The food and drink composition according to [7], which is used to increase the level of mitochondrial disease. [9] The food and drink composition according to [7] or [8], which is intended for a subject in need of treatment or prevention of a mitochondrial disease.

[10] The food and drink composition according to [7] or [8], which is intended for a subject in need of treatment or prevention of an age-related symptom or disease.

[11] The food and drink composition according to

[10] , wherein the age-related symptom or disease is hearing loss.

[12] The food and drink composition according to any of [7] to

[11] , wherein the enantiomeric excess of the S(+) isomer is at least 97% or more.

[13] The pharmaceutical composition according to any of [1] to [6], or the food and drink composition according to any of [7] to

[12] , which is stored protected from light.

[14] The pharmaceutical composition according to any of [1] to [6], or the food and drink composition according to any of [7] to

[12] , which is stored at approximately neutral pH, preferably at pH 6.0 to 8.0.

[15] The pharmaceutical composition according to any one of [1] to [6], or the food and beverage composition according to any one of [7] to

[12] , wherein, when chloroform is contained, the content of the short-chain alcohol relative to the chloroform is less than 1%, and preferably, the short-chain alcohol and chloroform are not contained simultaneously.

[0006] In another embodiment, the present invention provides the following [1] to

[15] (the compound represented by formula (A-1) is as described above): [1] NAD＋ for use in the treatment or prevention of a subject in need of increasing NAD levels or in a subject ＋ [2] A compound represented by formula (A-1) or a physiologically acceptable salt thereof, substantially free of the R(-) isomer, for use in a method for increasing the level of NAD in a subject. [2] A compound represented by formula (A-1) or a physiologically acceptable salt thereof, substantially free of the R(-) isomer, for use in the treatment or prevention of mitochondrial disease. [3] A compound represented by formula (A-1) or a physiologically acceptable salt thereof, for use in the treatment or prevention of age-related symptoms or diseases. [4] The compound or a physiologically acceptable salt thereof for use according to [3], wherein the age-related symptom or disease is any one selected from the group consisting of hearing loss (more specifically, age-related hearing loss), visual impairment (more specifically, age-related visual impairment), anemia (more specifically, age-related anemia), muscle weakness (more specifically, age-related muscle weakness), hair thinning or hair loss (more specifically, age-related hair thinning or hair loss), and shortened lifespan (more specifically, age-related lifespan shortening), preferably hearing loss. [5] A compound or a physiologically acceptable salt thereof for use according to [3], wherein the age-related symptom or disease is any one selected from the group consisting of hearing loss (more specifically, age-related hearing loss), visual impairment (more specifically, age-related visual impairment), anemia (more specifically, age-related anemia), muscle weakness (more specifically, age-related muscle weakness), hair thinning or hair loss (more specifically, age-related hair thinning or hair loss), and shortened lifespan (more specifically, age-related lifespan shortening). ＋ [6] The compound or physiologically acceptable salt thereof for use according to any of [2] to [4], wherein the treatment or prevention is achieved by increasing the level of NAD. [6] The compound or physiologically acceptable salt thereof for use according to any of [1] to [5], wherein the enantiomeric excess of the S(+) isomer is at least 97% or more, preferably 97.5% or more, more preferably 98% or more, particularly preferably 98.5% or more, and even more preferably 99% or more, for example, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more. [7] Non-therapeutic use of a compound represented by formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer, wherein the compound represented by formula (A-1) or a physiologically acceptable salt thereof is NAD. ＋[8] Non-therapeutic use of a compound represented by formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer, wherein the compound represented by formula (A-1) or a physiologically acceptable salt thereof is used to improve or prevent mitochondrial disease. [9] Non-therapeutic use of a compound represented by formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer, wherein the compound represented by formula (A-1) or a physiologically acceptable salt thereof is used to improve or prevent symptoms or diseases associated with aging.

[10] The non-therapeutic use according to [9], wherein the age-related symptom or disease is any one selected from the group consisting of hearing loss (more specifically, age-related hearing loss), visual impairment (more specifically, age-related visual impairment), anemia (more specifically, age-related anemia), muscle weakness (more specifically, age-related muscle weakness), thinning hair or hair loss (more specifically, age-related thinning hair or hair loss), and shortened lifespan (more specifically, age-related shortened lifespan), preferably hearing loss.

[11] The non-therapeutic use according to [9], wherein the age-related symptom or disease is any one selected from the group consisting of hearing loss (more specifically, age-related hearing loss), visual impairment (more specifically, age-related visual impairment), anemia (more specifically, age-related anemia), muscle weakness (more specifically, age-related muscle weakness), thinning hair or hair loss (more specifically, age-related thinning hair or hair loss), and shortened lifespan (more specifically, age-related shortened lifespan), preferably hearing loss. ＋

[12] The non-therapeutic use according to any of [7] to

[11] , wherein the compound represented by formula (A-1) or a physiologically acceptable salt thereof has an enantiomeric excess of the S(+) form of at least 97.5% or more, more preferably 98% or more, particularly preferably 98.5% or more, even more preferably 99% or more, for example, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more.

[13] The compound for use according to any of [1] to [6] or a physiologically acceptable salt thereof, or the non-therapeutic use according to any of [7] to

[12] , wherein the compound represented by formula (A-1) or a physiologically acceptable salt thereof is present in a formulation stored in a light-shielded condition.

[14] The compound or physiologically acceptable salt thereof for use according to any one of [1] to [6], wherein the compound represented by formula (A-1) or a physiologically acceptable salt thereof is present in a formulation having a pH of approximately neutral, preferably 6.0 to 8.0, or the non-therapeutic use according to any one of [7] to

[12] .

[15] The compound or physiologically acceptable salt thereof for use according to any one of [1] to [6], wherein, when the compound represented by formula (A-1) or a physiologically acceptable salt thereof is present in a formulation containing chloroform, the content of the short-chain alcohol relative to the chloroform is less than 1%, and preferably, the compound represented by formula (A-1) or a physiologically acceptable salt thereof is present in a formulation that does not simultaneously contain a short-chain alcohol and chloroform.

[0007] In another embodiment, the present invention provides the following [1] to [5] (the compound represented by formula (A-1) is as described above): [1] A method for preventing NADPH in a subject in need thereof, comprising administering to the subject a compound represented by formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of an R(-) isomer. ＋[2] A method for treating, ameliorating, or preventing a mitochondrial disease, comprising administering to a subject in need thereof a compound represented by formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer. [3] A method for treating, ameliorating, or preventing an age-related symptom or disease, comprising administering to a subject in need thereof a compound represented by formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) isomer. [4] The method according to [3], wherein the age-related symptom or disease is any one selected from the group consisting of visual impairment, anemia, muscle weakness, thinning hair or hair loss, and shortened lifespan. [5] The method according to [3], wherein the age-related symptom or disease is hearing loss.

[0008] Other embodiments of the present invention include the use of the S(+) form of MA5 or a physiologically acceptable salt thereof in the manufacture of a therapeutic or preventive agent for mitochondrial disease; the use of the S(+) form of MA5 or a physiologically acceptable salt thereof in the manufacture of a therapeutic or preventive agent for age-related symptoms or diseases; and the use of the S(+) form of MA5 or a physiologically acceptable salt thereof in the manufacture of a therapeutic or preventive agent for NADPH in animal cells. ＋ to a subject in need of promoting the production of NAD in animal (subject) cells. ＋ a method for promoting the production of NAD in animal cells, the method comprising the step of culturing the animal cells in the presence of the S(+) form of MA5 or a physiologically acceptable salt thereof; ＋ Examples of the mitochondrial function activators include: a method for promoting the production of MA5 in vitro (hereinafter sometimes referred to as "the present promotion method"); an agent for activating mitochondrial function in animals, comprising the S(+) form of MA5 or a physiologically acceptable salt thereof; a method for activating mitochondrial function in animals, comprising the step of administering the S(+) form of MA5 or a physiologically acceptable salt thereof to a subject in need of mitochondrial function activation; an agent for improving motor dysfunction in animals, comprising the S(+) form of MA5 or a physiologically acceptable salt thereof; and a method for improving motor dysfunction in animals, comprising the step of administering the S(+) form of MA5 or a physiologically acceptable salt thereof to a subject in need of improvement of motor dysfunction.

[0009] The S(+) form of MA5, which is the compound of the present invention, differs from the racemic form of MA5 and the R(-) form of MA5 in that it inhibits NAD in animal cells. ＋ The S(+) form of MA5, unlike the R(-) form of MA5, has the effect of increasing the expression level of SIRT in animal cells. Furthermore, compared to the R(-) form of MA5, the S(+) form of MA5 is less likely to be metabolized into a toxic substance (glucuronide conjugate) in the body, is more likely to be retained in the blood, and has low cytotoxicity to animal cells.

[0010] On the other hand, NAD in animal cells ＋ It is known that NAD levels decrease with age and that it plays an important role in the development of age-related diseases. ＋ Increasing the amount of ATP has been reported to extend the lifespan of nematodes (C. elegans) and to protect against metabolic diseases in mice (see Nature. 2018 563(7731) 354-359). C. elegans is also used as a model organism for human aging (see Bulletin of the Institute for Advanced Life Sciences, Tokai University, Vol. 1, March 2017).

[0011] Therefore, when the S(+) form of MA5 is administered to animals, it inhibits NAD in animal cells without adverse effects such as side effects. ＋ Increasing the amount of ATP can be expected to result in increased lifespan and treatment or prevention of symptoms or diseases such as age-related symptoms or diseases and metabolic disorders.

[0012] Furthermore, the S(+) form of MA5 has the effect of improving mitochondrial function in cells, and is therefore useful as a mitochondrial function activator or a therapeutic or preventive agent for mitochondrial diseases.

[0013] Rat pancreatic β-cell line (INS-1E cell line) was cultured for 6 hours in the presence of 0.1% DMSO ("0" in the figure) or various concentrations (3 pM, 10 pM, 30 pM, 100 pM, 300 pM, 1 nM, or 3 nM) of three compounds (racemic MA5 [Fig. 1A], S(+) MA5 [Fig. 1B], or R(-) MA5 [Fig. 1C]). ＋Figure 1 shows the results of measuring the amount of DMSO (mean ± standard deviation [SE]) in the figure. In the figure, "*" and "**" indicate a statistically significant increase (p<0.05 and p<0.01, respectively) compared to the results in the absence of compound, and "#" and "##" indicate a statistically significant decrease (p<0.05 and p<0.01, respectively) compared to the results in the presence of 0.1% DMSO. Figure 1 shows the results of mass spectrometry analysis of the metabolites produced after incubation of human liver microsomes in the presence of three compounds (racemic MA5 ["racemi" in the figure], S(+) MA5 ["S" in the figure], or R(-) MA5 ["R" in the figure]) or in the absence of these compounds ("Control" in the figure). The human bladder epithelial cell line (HB1EpC cell line) was cultured in the presence of 0.1% DMSO ("0" in the figure) or various concentrations (30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 300 μM, or 1 mM) of three compounds (racemic MA5 [FIG. 3A], S(+) MA5 [FIG. 3B], or R(-) MA5 [FIG. 3C]), and the viable cell levels (mean ± SE) were analyzed. The "viable cell levels" on the vertical axis are the optical density (OD) at 450 nm measured by WST-8 assay. ４５０The figures show the measured values ​​of the viability of MA5 (mean ± SE). The "*" and "**" in the figure indicate statistically significant differences (p<0.05 and p<0.01, respectively) compared to the results for 0.1% DMSO. The figures show the results of measuring the viability of MA5 (mean ± SE) in a fibroblast cell line derived from a patient with Leigh encephalopathy (MT-ND3 mutant cell line) cultured in the presence of 0.1% DMSO ("0" in the figure) or various concentrations (0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, or 100 μM) of each enantiomer of MA5 (S(+) form ["S" in the figure] or R(-) form ["R" in the figure]). The "*" in the figure indicates statistically significant differences (p<0.05) compared to the results for 0.1% DMSO. The MT-ND3 mutant cell line was cultured in the presence of 0.1% DMSO ("0" in the figure) or various concentrations (0.1 μM or 1 μM) of three compounds (racemic MA5 ["racemi" in the figure], S(+) MA5 ["S" in the figure], or R(-) MA5 ["R" in the figure]), and the amount of ATP produced in the culture medium was measured. The "*" in the figure indicates a statistically significant difference (p<0.05) compared to the results for 0.1% DMSO. Fluorescence images of mitochondria in body-wall muscle cells of wild-type nematodes of ccIs4251 ("WT" in the figure; n = 18) or immt-1 / mitofilin mutants ("immt-1 / mitofilin mutant mock"; n = 41, "immt-1 / mitofilin mutant MA-5(racemi)"; n = 37, "immt-1 / mitofilin mutant MA-5S"; n = 61, and "immt-1 / mitofilin mutant MA-5R"; n = 34) reared in the presence of four types of substances (DMSO or 10 μM racemic MA5, S(+) MA5, or R(-) MA5). Figure 6 shows the results of an analysis of the level of mitochondrial swelling in body-wall muscle cells based on the fluorescence images of Figure 6. Figure 7 shows the results of an analysis of the level of mitochondrial swelling in body-wall muscle cells based on the fluorescence images of Figure 6. The difference between "a" and "b" in the figure indicates a statistically significant difference (p<0.05). "X" in the figure indicates the average value, and "◯" in the figure indicates an outlier.1 shows the results of measuring the concentrations of the S(+) form of MA5 and the R(-) form of MA5 in plasma after oral administration of the S(+) form of MA5 and the R(-) form of MA5 to cynomolgus monkeys. 2 shows the results of measuring the concentrations of the reduced metabolite of MA5 (compound represented by formula (B) [hereinafter also referred to as "Red-MA5"]) in plasma after oral administration of the S(+) form of MA5 and the R(-) form of MA5 to cynomolgus monkeys. Fibroblast cell lines derived from patients with Leigh syndrome (KCMC10 cell line) were cultured in the presence of 0.1% DMSO ("0" in the figure) or various concentrations (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, or 30 μM) of two compounds (racemic MA5 [Figure 11A] or Red-MA5 [Figure 11B]), and the viability levels (mean ± SE) were measured. An "*" in the figure indicates a statistically significant difference (p<0.05) compared to the results for 0.1% DMSO.

[0033] Figure 1 shows the results of Western blotting analysis of the expression levels of various SIRTs (SIRT1, SIRT2 isoform 1, SIRT2 isoform 2, and SIRT3) in a mouse inner ear cell line (HEI-OC1 cell line) cultured in the presence of 0.1% DMSO or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of the racemic MA5. The lower graph shows the results of expressing each band intensity in the above results as a relative value, with the band intensity in the 0.1% DMSO control set at 1. The HEI-OC1 cell line was cultured in the presence of 0.1% DMSO or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of the racemic MA5, and the expression levels of various SIRTs (SIRT5, SIRT6, and SIRT7) and β-actin ( FIG. 13D ) in the cells were analyzed by Western blotting. The lower graph shows the band intensity in the upper results, expressed relative to the band intensity in the control 0.1% DMSO, which was set at 1.

[0033] Figure 1 shows the results of Western blotting analysis of the expression levels of various SIRTs (SIRT1, SIRT2 isoform 1, SIRT2 isoform 2, and SIRT3) in the cells of the HEI-OC1 cell line, which was cultured in the presence of 0.1% DMSO or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of the S(+) form of MA5. The lower graph shows the band intensity in the above results, expressed as a relative value, with the band intensity in the 0.1% DMSO control set at 1.

[0033] Figure 1 shows the results of Western blotting analysis of the expression levels of various SIRTs (SIRT5, SIRT6, and SIRT7) and β-actin in the HEI-OC1 cell line, which was cultured in the presence of 0.1% DMSO or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of the S(+) form of MA5. The lower graph shows the band intensity in the upper results, expressed as a relative value, with the band intensity in the 0.1% DMSO control set at 1.

[0033] Figure 1 shows the results of Western blotting analysis of the expression levels of various SIRTs (SIRT1, SIRT2 isoform 1, SIRT2 isoform 2, and SIRT3) in the cells of the HEI-OC1 cell line, which was cultured in the presence of 0.1% DMSO or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of the R(-) form of MA5. The lower graph shows the results of expressing each band intensity in the above results as a relative value, with the band intensity in the 0.1% DMSO control set at 1. This figure shows the results of Western blotting analysis of the expression levels of various SIRTs (SIRT5, SIRT6, and SIRT7) and β-actin in the HEI-OC1 cell line, which was cultured in the presence of 0.1% DMSO or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of the R(-) form of MA5. The graph below shows the band intensities in the results above, expressed relative to the band intensity in the 0.1% DMSO control, which was set at 1. This figure shows the results of a binding test of the S(+) and R(-) forms of MA5 to NAMPT. The Kd value of the S(+) form of MA5 was 58.94 μM, but the R(-) form of MA5 did not bind to NAMPT, and the Kd value could not be measured.The fluorescence intensity of γH2AX (phosphorylated H2AX) is shown by immunostaining. The level of γH2AX was low only when the S(+) form of MA5 was added, demonstrating an inhibitory effect on DNA damage. The chromatogram of MA5 obtained using a fractionation device (LC-Forte) is shown. X-ray crystallography of the corresponding benzylamine salt determined that the absolute configuration of MA5 in the early elution fraction was S, and that of MA5 in the late elution fraction was R. In addition, measurement of the optical rotation of MA5 determined that the MA5 in the early elution fraction was dextrorotatory (+) and the MA5 in the late elution fraction was levorotatory (-).

[0014] The compound of the present invention is the S(+) enantiomer of a compound represented by the following formula (A-1) (i.e., a compound represented by the following formula (A) (4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid (MA5)) or a physiologically acceptable salt thereof.

[0015]

[0016]

[0017] The S(+) isomer of MA5 used in the present invention is substantially free of the R(-) isomer of MA5. "Substantially free of the R(-) isomer of MA5" means that the enantiomeric excess of the S(+) isomer is about 97% or more, preferably about 97.5% or more, more preferably about 98% or more, particularly preferably about 98.5% or more, and even more preferably about 99% or more, for example, about 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more.

[0018] "Enantiomeric excess" (also written as "ee") refers to the excess of one enantiomer over the other, and when the S-isomer is in excess, it is expressed by the following formula: ee = (A Ｓ -A Ｒ ) / (A Ｓ +A Ｒ ) A Ｒ and A Ｓ represent the mole fractions of the R- and S-configurations, respectively.

[0019] As used herein, "about" refers to a value that varies by plus or minus 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%, respectively, from the reference value. Preferably, the term "about" refers to a range of plus or minus 10%, 5%, or 1%, respectively, from the reference value.

[0020] As used herein, the term "physiologically acceptable salt" refers to a salt that is, within the scope of sound medical, pharmaceutical, or biological judgment, suitable for use in contact with animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Examples of physiologically acceptable salts include ammonium salts; alkali metal salts such as sodium salts, lithium salts, and potassium salts; alkaline earth metal salts such as aluminum salts, calcium salts, and magnesium salts; salts with organic bases such as dicyclohexylamine salts and N-methyl-D-glucamine; salts with amino acids such as arginine, lysine, and ornithine; salts formed with basic nitrogen-containing groups; and the like.

[0021] The compound of the present invention represented by formula (A-1) can be obtained according to an organic synthesis method using a known organic chemical reaction, for example, the method described in the section "1. Production of S(+) form of MA5" in the Examples to be described later.

[0022] The present invention provides a pharmaceutical composition or a food or beverage composition containing the S(+) isomer of MA5 or a physiologically acceptable salt thereof as an active ingredient. The pharmaceutical composition or food or beverage composition is substantially free of the R(-) isomer of MA5 or a physiologically acceptable salt thereof. "Substantially free of the R(-) isomer of MA5 or a physiologically acceptable salt thereof" means that the enantiomeric excess of the S(+) isomer is about 97% or more, preferably about 97.5% or more, more preferably about 98% or more, particularly preferably about 98.5% or more, and even more preferably about 99% or more, for example, about 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more.

[0023] In one embodiment, the pharmaceutical composition or food or drink composition is a compound that inhibits NAD in animal cells. ＋in a subject in need thereof, e.g., to promote the production of NAD in the subject ＋ Used to increase the level.

[0024] In another embodiment, the pharmaceutical composition or food or beverage composition is used to activate mitochondrial function in a subject, for example, to treat or prevent a mitochondrial disease in a subject in need thereof.

[0025] In another embodiment, the pharmaceutical composition or food or beverage composition is used to treat or prevent age-related symptoms or diseases in a subject in need thereof.

[0026] Examples of food and beverage compositions include health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally balanced foods, supplements, etc.), and health functional foods (specified health foods, nutritionally functional foods, functional food products, etc.).

[0027] As used herein, animals include mammals (human or non-human mammals), birds, reptiles, amphibians, fish, invertebrates, and the like. Other examples of animals include humans and livestock. Here, "livestock" refers to animals kept and bred by humans. Examples of such livestock include non-human mammals (e.g., rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; and carnivores such as dogs and cats), birds (e.g., chickens, quails, turkeys, pigeons, ducks, and geese), fish (e.g., carp and goldfish), and invertebrates (e.g., silkworms and honeybees).

[0028] In this specification, the pharmaceutical composition or food or beverage composition may contain additives. Examples of the additives include physiologically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonicity agents, additives, coating agents, solubilizers, lubricants, glidants, solubilizers, flavorings, sweeteners, solvents, gelling agents, and nutrients. Specific examples of such additives include water, saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0029] The S(+) form of MA5 exhibits physical properties different from those of the racemic form, and is particularly susceptible to discoloration and decomposition under light, acid, and alkaline conditions. Therefore, the pharmaceutical composition or food and beverage composition of the present invention is preferably stored protected from light. Furthermore, the pharmaceutical composition or food and beverage composition of the present invention is preferably stored near neutral, preferably at a pH of 6.0 to 8.0. Furthermore, since the S(+) form of MA5 is susceptible to decomposition in the presence of a short-chain alcohol and chloroform, the pharmaceutical composition or food and beverage composition of the present invention does not contain both a short-chain alcohol and chloroform; particularly, if chloroform is contained, the content of the short-chain alcohol relative to the chloroform is preferably less than 1%.

[0030] In this specification, the term "mitochondrial disease" refers to any symptom caused by a decrease in mitochondrial function, such as ATP production, regulation of apoptosis, and regulation of intracellular concentrations of calcium ions and iron, due to genetic mutations in cell nuclear DNA or mitochondrial DNA, and specific examples include CPEO (chronic progressive external ophthalmoplegia syndrome), MELAS (encephalomyopathy, lactic acidosis, and stroke-like syndrome), MERRF (MERF (ragged-red fiber, myoclonic epilepsy syndrome)), Leigh encephalopathy (subacute necrotizing encephalomyelopathy), Leber disease (Leber hereditary optic neuropathy [LHON]), Kearns-Sayre syndrome (KSS), Barth syndrome, Pearson disease, and Friedreich's ataxia (FRDA).

[0031] In this specification, examples of "symptoms or diseases associated with aging" include thinning hair or hair loss (more specifically, age-related thinning hair or hair loss), hearing loss (more specifically, age-related hearing loss), visual impairment (more specifically, age-related visual impairment), anemia (more specifically, age-related anemia), muscle weakness (more specifically, age-related muscle weakness), shortened lifespan (more specifically, age-related shortened lifespan), and the like.

[0032] As used herein, "NAD ＋ "Those who need to increase their levels," or "NAD ＋ Examples of "subjects in need of promoting the production of" include subjects in need of extending their lifespan (subjects requiring longevity); patients with neurological diseases (Parkinson's disease, depression, Alzheimer's disease, amyotrophic lateral sclerosis [ALS], etc.); patients with cardiovascular diseases (heart failure, arrhythmia, etc.); patients with muscle diseases (sarcopenia, inclusion body myositis, muscular dystrophy, etc.); patients with kidney diseases (chronic kidney disease, renal failure, diabetic nephropathy, nephritis, etc.); patients with metabolic diseases (diabetes, liver dysfunction, alcoholic liver disease, non-alcoholic fatty liver disease [NAFLD; non-alcoholic fatty liver disease]), non-alcoholic steatohepatitis [NASH; non-alcoholic steatohepatitis], thyroid / adrenal gland diseases, etc.); patients with digestive diseases (inflammatory bowel disease, etc.); cancer patients; patients with age-related symptoms or diseases (thinning hair or hair loss, hearing loss, vision impairment, anemia, muscle weakness, shortened lifespan, etc.); The pharmaceutical composition or food and drink composition of the present invention induces NAD in the cells of these subjects. ＋ levels increased (NAD ＋ As a result of this, lifespan can be extended and the above diseases and disorders can be prevented or treated.

[0033] As used herein, "subjects in need of mitochondrial function activation" may include, for example, patients suffering from diseases related to or caused by the weakening or failure of mitochondrial function, and specifically, patients suffering from diabetes, mitochondrial disease, brain disease, etc.

[0034] In the present specification, the administration form of the pharmaceutical composition or food and drink composition is not particularly limited, and examples thereof include oral administration in the form of powder, granules, tablets, capsules, syrup, suspension, etc., and parenteral administration in the form of injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection) of a dosage form such as a solution, emulsion, suspension, etc., or intranasal administration in the form of a spray.

[0035] In this specification, the administration (ingestion) amount of the S(+) form of MA5 or a physiologically acceptable salt thereof in a pharmaceutical composition or a food or beverage composition is determined appropriately depending on age, body weight, sex, symptoms, drug sensitivity, etc., and is, for example, in the range of 1 μg to 200 mg / kg (body weight) / day. The pharmaceutical composition or food or beverage composition can be administered once or multiple times (for example, 2 to 4 times) per day.

[0036] The present specification describes a method for producing NADPH in animal cells, which comprises culturing animal cells in the presence of the S(+) form of MA5 or a physiologically acceptable salt thereof. ＋ The culture temperature of animal cells in the method is usually within the range of 30 to 40°C, preferably about 37°C (36 to 38°C). ２ The concentration is usually in the range of about 1 to 10%, preferably about 5% (4 to 6%). ２ The concentration is usually within the range of about 10 to 40%, preferably about 21% (20 to 22%).

[0037] In the above-described method, examples of the culture medium used for culturing animal cells include serum-containing or serum-free culture medium, physiological aqueous solutions such as saline, phosphate-buffered saline, Tris-buffered saline, HEPES-buffered saline, Ringer's solution (lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc.), and 5% glucose aqueous solution. Here, serum-containing culture mediums include culture mediums for mammalian cell culture (DMEM, EMEM, IMDM, RPMI-1640, αMEM, F-12, F-10, M-199, AIM-V, etc.) containing 0.1 to 30 (v / v)% serum (fetal bovine serum; FBS, calf bovine serum; CS, etc.). Furthermore, examples of the serum-free culture medium include commercially available B27 supplement (minus insulin) (manufactured by Life Technologies), N2 supplement (manufactured by Life Technologies), B27 supplement (manufactured by Life Technologies), Knockout Serum, etc. Examples of such a medium include the above-mentioned culture medium for mammalian cell culture to which an appropriate amount (for example, 1 to 30%) of a serum substitute such as Replacement (manufactured by Invitrogen) has been added.

[0038] The present invention relates to a method for improving NADPH in a subject, comprising administering to the subject an S(+) form of MA5 or a physiologically acceptable salt thereof. ＋ The present invention provides a method for increasing the level of MA5, wherein the S(+) form of MA5 or a physiologically acceptable salt thereof is substantially free of the R(-) form of MA5 or a physiologically acceptable salt thereof.

[0039] In another embodiment, the present invention provides a method for treating or preventing a mitochondrial disease in a subject, comprising administering to the subject an S(+) form of MA5 or a physiologically acceptable salt thereof, wherein the S(+) form of MA5 or a physiologically acceptable salt thereof is substantially free of the R(-) form of MA5 or a physiologically acceptable salt thereof.

[0040] In another embodiment, the present invention provides a method for treating or preventing an age-related symptom or disease, comprising administering to a subject the S(+) form of MA5 or a physiologically acceptable salt thereof, wherein the S(+) form of MA5 or a physiologically acceptable salt thereof is substantially free of the R(-) form of MA5 or a physiologically acceptable salt thereof.

[0041] The phrase "substantially free of the R(-) form of MA5 or a physiologically acceptable salt thereof" means that the enantiomeric excess of the S(+) form is about 97% or more, preferably about 97.5% or more, more preferably about 98% or more, particularly preferably about 98.5% or more, and even more preferably about 99% or more, for example, about 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more.

[0042] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. The INS-1E cell line was cultured in an incubator (37°C, 21% O) in the presence of RPMI-1640 culture medium (GIBCO) containing 11.1 mM glucose, 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM glutamine, 50 μM β-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin (hereinafter simply referred to as "RPMI-1640 culture medium"). ２ , 5% CO ２) in the incubator. A fibroblast cell line derived from a patient with Leigh syndrome (MT-ND3 mutant cell line [Leigh syndrome, m.10191 T>C [p.Ser45Pro]]) was cultured in the incubator in DMEM low glucose (GIBCO) medium containing 1% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin (hereinafter referred to as "DMEM low glucose medium"). HBlEpC cell lines were cultured in the incubator in Human Bladder Epithelial Cell Basal medium (Cell Application) medium containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin (hereinafter referred to as "HBlEpC medium").

[0043] 1. Formation of the S(+) form of MA5 1-1 Formation of trans-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid

[0044]

[0045] In a 50 mL round-bottom flask under nitrogen, 1,3-difluorobenzene (0.51 g, 4.47 mmol) was dissolved in dichloromethane (20 mL), and maleic anhydride (0.43 g, 4.46 mmol) and aluminum chloride (1.20 g, 9.01 mmol) were added, followed by stirring at room temperature for 4 hours. The reaction mixture was adjusted to pH 1 with 1N hydrochloric acid (10 mL), and extracted three times with ethyl acetate (40 mL). The organic layer was washed with saturated brine and dehydrated over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the mixture was purified by recrystallization from benzene to give trans-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (0.57 g, yield 56%): melting point 114.8-119.6°C; １ H NMR (acetone-d ６ ): δ 7.98 (m, 1H), 7.71 (dd, J Ｈ－Ｆ =15.6, 3.4Hz, 1H), 7.23 (m, 2H), 6.75 (dd, J Ｈ－Ｆ =15.6, 1.2Hz, 1H); １３C NMR (acetone-d ６ ): δ 187.2 (d, J Ｃ－Ｆ =2.6Hz), 166.9(dd, J Ｃ－Ｆ =254.5, 12.3Hz), 166.4, 163.4(dd, J Ｃ－Ｆ =254.5, 12.9Hz), 140.0(d, J Ｃ－Ｆ =6.1Hz), 134.0(dd, J Ｃ－Ｆ = 10.9, 3.6 Hz), 133.0 (d, J Ｃ－Ｆ =1.6Hz), 123.3(dd, J Ｃ－Ｆ =12.4, 3.6Hz), 113.4(dd, J Ｃ－Ｆ =21.5, 3.6Hz), 105.8(dd, J Ｃ－Ｆ =27.3, 26.3Hz); IR (neat): 2917, 1697, 1661cm －１ ;FAB-MS m / z[M+H ＋ ] calcd for 213 (C １１ H １０ O ３ ), found 213.

[0046] 1-2 Production of racemic MA5

[0047]

[0048] In a 30 mL round-bottom flask, trans-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (0.39 g, 1.84 mmol) was dissolved in benzene (10 mL), and indole (0.26 g, 2.19 mmol) was added. The mixture was stirred at 80°C for 8 hours and then allowed to cool to room temperature. The reaction solution was evaporated under reduced pressure, and the residue was purified using silica gel column chromatography (chloroform:methanol=20:1) to obtain 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic acid (racemic form of MA5) (see Table 3). (0.30 g, yield 51%): melting point 180.2-184.6°C; １ H NMR (DMSO-d ６):δ 7.98(m,1H),7.65(d,J=7.9Hz,1H),7.37(d,J=8.1Hz,1H),7.42(m,1H),7.28(d,J=2.3Hz,1H),7.24(m,1H),7.09(t,J=7.1Hz,1H),7.01(t,J=7.5Hz,1H),4.34(dd,J=10.5,3.5Hz,1H),3.90(ddd,J Ｈ－Ｆ =18.5,10.6,2.4Hz,1H),3.30(ddd,J Ｈ－Ｆ =18.5,6.1,3.5Hz,1H); １３ C NMR(DMSO-d ６ ):δ 195.2(d,J Ｃ－Ｆ =4.1Hz),174.8,165.2(d,J Ｃ－Ｆ =253.0,13.4Hz),162.2(d,J Ｃ－Ｆ =255.5,13.4Hz),136.4,132.7(dd,J Ｃ－Ｆ =10.8,4.1Hz),126.3,123.3,122.2(dd,J Ｃ－Ｆ =12.3,3.6Hz),121.4,119.1,118.8,112.6(dd,J Ｃ－Ｆ =21.1,3.6Hz),111.9,111.8,105.4(dd,J Ｃ－Ｆ =26.1Hz),45.6(d,J Ｃ－Ｆ =6.3Hz),37.9;IR(neat):3382,2919,1678cm －１ ;HRFAB m / z[M+H] ＋ calcd for 332.1036(C １９ H １７ NO ３ ),found 312.1028.

[0049] 1-3 Separation of the S(+) isomer of MA5 The S(+) isomer of MA5 was separated from the synthesized racemic MA5 using a LC-Forte fractionator (YMC). The CHIRALPAK AD-H (column inner diameter: 2 cm, column length: 25 cm, Daicel Corporation) was used as the preparative column. Elution was performed at a flow rate of 10 mL / min over 40 minutes using a single mobile phase consisting of solution A (0.05% TFA / hexane), solution B (0.05% TFA / 2-propanol), and a 60 / 40 ratio of solution A / solution B. The mobile phase flow rate can also be increased to 20 mL / min, and the elution time can be halved to 20 minutes. The detection wavelength for MA5 was set at 220 nm. The retention times of the S(+) and R(-) isomers of MA5 (see Table 3) were 17.3 minutes and 29.8 minutes (flow rate 10 mL / min), respectively. Column separation yielded the S(+) isomer of MA5 with 100% optical purity. The chromatogram is shown in Figure 17.

[0050] 1-4 Determination of the absolute configuration of the S(+) isomer of MA5 The optical rotation of the collected MA5 from the early elution fraction and the MA5 from the late elution fraction was measured using a polarimeter P-2200 (manufactured by JASCO Corporation). The specific optical rotation of MA5 from the early elution fraction was [α] D +130.3 (c = 1.0, chloroform solution, 25 °C), the specific rotation of MA5 in the later eluted fraction is [α] D The pH was −128.6 (c=1.0, chloroform solution, 25° C.).

[0051] X-ray crystal structure analysis was performed using an AXS "SMART APEX II" (Bruker). Crystals of the benzylamine salt of each elution fraction MA5 were prepared for the purpose of analysis because they are suitable for single crystal preparation (see Tables 4 and 5). Crude crystals were obtained from an ethyl acetate solution of the early-elution fraction MA5 or the late-elution fraction MA5 and benzylamine in a 1:1 molar ratio, and then recrystallized from a water-methanol solution to obtain the benzylamine salt of each elution fraction MA5. The crystal structures were determined by anomalous scattering, and the absolute configuration of the benzylamine salt of the early-elution fraction MA5 was S, while the absolute configuration of the benzylamine salt of the late-elution fraction MA5 was R.

[0052]

[0053]

[0054] From the above results, it was determined that MA5 in the early elution fraction was the S(+) form, and MA5 in the late elution fraction was the R(-) form.

[0055]

[0056]

[0057]

[0058] 2. NAD in animal cells ＋ Evaluation of the amount of NAD in animal cells ＋ To examine the effect of the S(+) form of MA5 on the amount of erythrocytes, analysis was carried out according to the method described in the following section "2-1."

[0059] 2-1 Method: 2 x 10 cells were placed in a lysine-coated 96-well plate. ４ INS-1E cell lines (see Drug Metab. Pharmacokinet. 25 (3): 274-282 (2010)) were seeded and cultured in the presence of RPMI-1640 medium for 48 hours. After the medium was removed, 100 μL of RPMI-1640 medium containing 0.1% DMSO, a solvent for the compounds, or RPMI-1640 medium containing various concentrations (3 pM, 10 pM, 30 pM, 100 pM, 300 pM, 1 nM, or 3 nM) of three compounds (racemic MA5, S(+) MA5, or R(-) MA5) was added and cultured for 6 hours. Subsequently, intracellular NAD was measured using the NAD / NADH-Glo Assay (G9071, Promega). ＋ The amount was measured.

[0060] 2-2 Results When the INS-1E cell line was cultured in the presence of racemic MA5, the intracellular NAD levels were significantly lower than when cultured in the absence of racemic MA5. ＋ On the other hand, when the INS-1E cell line was cultured in the presence of the R(-) form of MA5, the amount of intracellular NAD ＋The amount of NAD decreased in a culture time-dependent manner (see Figure 1C). When INS-1E cells were cultured in the presence of the S(+) form of MA5, the amount of NAD in the cells decreased compared to when they were cultured in the absence of the S(+) form of MA5. ＋ The amount increased in a culture time-dependent manner (see FIG. 1B).

[0061] These results show that the R(-) form of MA5 inhibits NAD in animal cells. ＋ On the other hand, the S(+) form of MA5 acts to reduce the amount of NAD in animal cells. ＋ The effect of increasing the amount of NAD in animal cells ＋ This indicates that it has the effect of promoting the production of

[0062] 3. In vivo administration test 1 When the S(+) form of MA5 was administered in vivo, the occurrence of chiral inversion (conversion) and the change in plasma concentration of the S(+) form of MA5 were analyzed according to the method described in "3-1" below.

[0063] 3-1 Method Each enantiomer of MA5 (S(+) or R(-) isomer) was dissolved in a mixture of physiological saline and 0.1 N sodium hydroxide (9:1), and the solution was sterilized by filtration using a 0.22 μm filter to prepare a solution containing each enantiomer of MA5 at 2 mL / kg. Next, the solution containing each enantiomer of MA5 was administered intravenously in a single dose of 1 mg / kg to three male cynomolgus monkeys aged 4 to 5 years. As a control, a solution not containing each enantiomer of MA5 was also administered in the same manner. Blood samples were collected at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours after administration, and plasma samples were collected. The concentrations of each MA5 enantiomer in the plasma were measured by LC-MS / MS (liquid chromatography / tandem mass spectrometry), and the area under the blood concentration-time curve (AUC) (μg hr / mL) was calculated. The LC-MS / MS analysis conditions were as follows:

[0064] The ionization unit used was a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Fisher Scientific) equipped with an ESI probe, connected to a high-performance liquid chromatograph, NANOSPACESI-2 (Osaka Soda). The target substances were the S(+) form of MA5 (S-(+)-MA5) and the R(-) form of MA5 (R-(-)-MA5), and the stable isotopes, S-(+)-MA5-d ６ and R-(-)-MA5-d ６ Quantitative analysis was performed using an internal standard method. The analytical column was a CHIRALPAK AD-3R (150 x 2.1 mm id, 3.0 μm particle size, Daicel Corporation), and the guard column was a CAPCELL C8UG120 (10 x 2.0 mm id, 5.0 μm particle size, Osaka Soda Corporation). The column oven temperature was 35°C. The mobile phase was formic acid / water (0.1 / 100 [v / v]) (Solution A) / acetonitrile (Solution B), with a flow rate of 200 μL / min. Measurements were performed under isocratic conditions, with elution using a single composition of Solution A / Solution B (57 / 43 [v / v]). The Solution A / Solution B (57 / 43 [v / v]) was used until the analysis time was 9.5 minutes, and then the Solution A / Solution B (10 / 90 [v / v]) was used at 9.7 minutes. In addition, a system was installed in which the column was washed with solution A / solution B (10 / 90 [v / v]) from 9.7 to 11.7 minutes of analysis time, and the column was equilibrated with solution A / solution B (57 / 43 [v / v]) from 11.8 to 15 minutes. The retention times were 6.95 minutes for S-(+)-MA5 and 6.95 minutes for S-(+)-MA5-d. ６ is 6.80 minutes, R-(-)-MA5 is 8.69 minutes, R-(-)-MA5-d ６ was 8.56 minutes.

[0065] MS conditions were negative ion mode detection, and the spray voltage, vaporizer temperature, and ion transfer tube temperature were set to 2.5 kV, 250°C, and 250°C, respectively. The SRM channel for precursor ion > product ion was set to "m / z 328.0 > 116.2" for MA5, and "m / z 328.0 > 116.2" for MA5-d. ６ The collision energy for generating product ions was set to 12 eV for MA5, and 12 eV for MA5-d. ６ The voltage was set to 17 eV. Waveform analysis and quantitative calculation by the internal standard method were performed using Xcalibur (manufactured by Thermo Fisher Scientific).

[0066] 3-2 Results From immediately after administration of the S(+) form of MA5 (5 minutes after administration) until 24 hours after administration, when the S(+) form of MA5 had disappeared from the blood, the R(-) form of MA5 was not detected, and only the S(+) form of MA5 was detected. This result indicates that the S(+) form of MA5 does not undergo chiral conversion in vivo.

[0067] Furthermore, the AUC value when the R(-) isomer of MA5 was administered was 12 (μg hr / mL), whereas the AUC value when the S(+) isomer of MA5 was administered was 30 (μg hr / mL) (see Table 6). This result indicates that the S(+) isomer of MA5 is more than 2.5 times more likely to be retained in the blood than the R(-) isomer of MA5, suggesting that the dose of the S(+) isomer of MA5 can be lower than that of the R(-) isomer of MA5.

[0068]

[0069] 4. Metabolic Study Using Human Liver Microsomes Since the S(+) form of MA5 was more likely to be retained in the blood than the R(-) form of MA5 in vivo, the metabolic rates of the individual enantiomers of MA5 were analyzed according to the method described in "4-1" below.

[0070] The S(+) or R(-) isomer of MA5 was dissolved in acetonitrile (LC-MS grade, manufactured by Kanto Chemical Co., Ltd.) to prepare a 10 mM solution. 855 mg of sucrose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 mL of water to prepare a 250 mM sucrose solution. A 20 mg / mL solution of human liver microsomes (8-donor pool, manufactured by Sekisui XenoTech) was then diluted 4-fold with this solution to prepare a 5 mg / mL suspension of human liver microsomes. In a 1 mL tube, 50 μL of human liver microsome suspension was mixed with 5 μL of one of three test solutions (a sucrose solution containing 10 mM S(+) MA5, a sucrose solution containing 10 mM R(-) MA5, or a control vehicle [sucrose solution]), 5 μL of NADPH Regenerating System Solution B (Corning), 100 μL of UGT Reaction Mix Solution B (Corning), and 275 μL of water. The mixture was shaken for 30 seconds and preincubated at 37°C for 5 minutes. Subsequently, 25 μL of NADPH Regenerating System Solution A (Corning) and 40 μL of UGT Reaction Mix Solution A (Corning) were added, shaken for 30 seconds, and incubated at 37°C for 1 hour. The reaction was terminated by adding 500 μL of ice-cold acetonitrile solution containing 0.1% formic acid. The mixture was shaken for 30 seconds, sonicated for 10 minutes, and then centrifuged at 20,000 × g and 4°C for 10 minutes. 50 μL of the supernatant was collected in a new 1 mL tube and centrifuged at 2,000 rpm at room temperature (25-35°C) for 30 minutes, then frozen at -80°C. This was dried in a freeze dryer for 30 minutes and redissolved in 25 μL of 20% methanol (LC-MS grade, Kanto Chemical Co., Inc.) containing 0.1% formic acid. 2 μL of this solution was then subjected to LC / HRMS. The number of samples tested was n=3, and manipulations were carried out on ice whenever possible.

[0071] LC / HRMS Measurement: Nanospace si-2 (Osaka Soda) was used as the LC device. Develosil C30-ug-3 (2.1 x 150 mm, 3.0 μm particle size, Nomura Chemical) was used as the column. 10 mM NH ５ CO ３ / H ２ Methanol was used as the mobile phase B solution, and linear gradient elution was performed at a flow rate of 200 μL / min (15-65% B solution (0.0-22.0 min), 65-90% B solution (22.0-24.4 min), 100% B solution (24.5-29.0 min), 15% B solution (30.0-40.0 min)). The column oven was set to 40°C, the sample injection volume was 2 μL, and 10% methanol was used as the wash solution and 50% acetonitrile as the wash port solution.

[0072] The HRMS instrument used was a Q-Exactive (manufactured by Thermo Fisher Scientific). The ESI conditions were: spray voltage: 3000 V, sheath gas pressure: 40 psi, auxiliary gas pressure: 10 psi, capillary temperature / vaporizer temperature: 300°C. For detection, full scan-ddMS of both positive and negative ions was used. 2 The topN (topN) mode was selected. The parameters were as follows: Full scan; Resolution 70,000, AGC target 3e6, Maximum IT 100 ms, Scan range 80-1,200, ddMS 2 Resolution 17,500, AGC target 5e4, Maximum IT 50ms, topN N=20, Isolation window 4.0 m / z, NCE 20 (50% stepped NCE). Additional MS ２ Data are target MS for both positive and negative ions 2Measurement was performed in mode. The various parameters were as follows: Resolution 35,000, AGC target 6e5, Maximum IT 200 ms, Isolation window 4.0 m / z, NCE 20 (50% stepped NCE).

[0073] Data analysis Data analysis was performed using Compound Discoverer (Thermo Fisher Scientific) and Xcalibur (Thermo Fisher Scientific). For peak extraction, the Metabolism w / FlSh workflow was selected, and the structural information of MA5 was registered in "Compound." The lower peak intensity was set to 1 / 10,000 of the average peak intensity of MA5 in each sample. [M + Na] was added to the ion adduct of the metabolite prediction node. ＋ , [M + NH ４ ] ＋ , [M-H ２ O + H] ＋ [M + Cl] － In addition, the metabolic pathways include the tryptophan > kynurenine pathway [-C + O], AMP conjugation [+C １０ H １２ N ５ O ６ P], CoA conjugation [+C ２１ H ３４ N ７ O １５ P ３ S] has been added.

[0074] 4-2 Results When human liver microsomes were incubated in the presence of racemic MA5 or the R(-) form of MA5, glucuronide conjugates (+C6H8O6 [+1.7 min, +2.6 min]) were detected, whereas when human liver microsomes were incubated in the presence of the S(+) form of MA5, the glucuronide conjugates were barely detected or were detected at levels comparable to those of the control (see Figure 2). This result indicates that the R(-) form of MA5 is more than 10 times more likely to be glucuronidated than the S(+) form of MA5, supporting the results of "4. Metabolism study using human liver microsomes" above, i.e., that the S(+) form of MA5 is more likely to be retained in the blood in vivo than the R(-) form of MA5.

[0075] Furthermore, a 4-week repeated-dose toxicity study in rats and a 4-week repeated-dose toxicity study in monkeys were conducted as GLP-compliant non-clinical studies, and results showed that toxic effects on the urothelium were observed in the high-dose group. To clarify the cause, urinary metabolites were evaluated, and it was found that the urinary concentrations of the glucuronide conjugate of racemic MA5 in monkeys and the sulfate conjugate of racemic MA5 in rats correlated with the onset of toxicity, and were presumed to be the cause of the toxicity.

[0076] Since there are species differences in hepatic metabolic enzymes and renal transporters between monkeys and rats, and it has been revealed that human hepatic metabolism is similar to that of monkeys, it is presumed that the glucuronide conjugate is also the cause of side effects in humans. The above-mentioned metabolism test using human liver microsomes showed that the S(+) form of MA5 produced almost no glucuronide conjugates (less than one-tenth) compared to the R(-) form, so the S(+) form of MA5 can be said to be a safer compound with fewer side effects than the R(-) form of MA5.

[0077] 5. Cytotoxicity Evaluation 1 To evaluate the toxicity of the S(+) form of MA5 to animal cells, analysis was carried out according to the method described in the following section "5-1."

[0078] 5-1 Method 2 x 10 per well in a 96-well plate ３HBlEpC cell lines (Cell Application) were seeded and cultured in 0.1% DMSO (compound solvent) or various concentrations (30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 300 μM, or 1 mM) of three compounds (racemic MA5, S(+) MA5, or R(-) MA5) were added to the HBlEpC culture medium and cultured for 48 hours. A control experiment was also performed without any compound ("-" in Figure 3). The viability (number of viable cells) after culture was measured by WST-8 assay using Cell Count Reagent SF (Nacalai Tesque).

[0079] 5-2 Results When the HBlEpC cell line was cultured in the presence of MA5 racemate or enantiomers (S(+) or R(-)), the cell viability was higher than when cultured in the presence of MA5 racemate. In particular, the cell viability was higher with the S(+) form of MA5 than with the R(-) form of MA5 (see Figure 3). This result indicates that the S(+) form of MA5 has lower cytotoxicity to animal cells than the racemate or the R(-) form of MA5.

[0080] 6. Cytotoxicity Evaluation 2 To evaluate the toxicity of the S(+) form of MA5 to animal cells, analysis was carried out according to the method described in the following section "6-1."

[0081] 6-1 Method: 2 x 10 cells per well in a 96-well plate ３MT-ND3 mutant cell lines were seeded and cultured in DMEM low-glucose medium for 2 days. Then, 0.1% DMSO (the compound solvent) or various concentrations (0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, or 100 μM) of each enantiomer of MA5 (S(+) or R(-) isomer) were added. After further culture for 5 days, cell viability was measured. Specifically, cell viability was measured by the MTT assay using Cell Counting Kit-8 (Dojindo Laboratories). Specifically, 100 μL of Cell Count Reagent SF was added to each well, incubated for 2 hours, and after 3 seconds of agitation using a microplate reader, the absorbance at 450 nm (reference 750 nm) was measured (see Figure 4). As a control, an experiment was also carried out in which nothing was added ("-" in FIG. 4).

[0082] 6-2 Results When the MT-ND3 mutant cell line was cultured in the presence of high concentrations (30 μM or 100 μM) of the S(+) isomer of MA5, no decrease in the viable cell level was observed (see Figure 4A). On the other hand, when the MT-ND3 mutant cell line was cultured in the presence of high concentrations (30 μM or 100 μM) of the R(-) isomer of MA5, a significant decrease in the viable cell level was observed (see Figure 4B). This result indicates that the S(+) isomer of MA5 is less cytotoxic to animal cells than the R(-) isomer of MA5, supporting the results of "5. Cytotoxicity Evaluation 1" above.

[0083] 7. Evaluation of ATP production ability in animal cells It has been reported that the racemic form of MA5 has the effect of enhancing ATP production ability in animal cells (see, for example, the pamphlet of International Publication No. 2014 / 080640). Therefore, to confirm whether there is a difference in the enhancing effect of ATP production ability between the S(+) form of MA5 and the R(-) form of MA5, analysis was performed according to the method described in the following section "7-1."

[0084] 7-1 Method: 2 x 10 cells per well in a 96-well plate ３MT-ND3 mutant cell lines were seeded and cultured in the presence of DMEM low-glucose culture medium for 24 hours, after which 0.1% DMSO, the solvent for the compounds, or various concentrations (0.1 μM or 1 μM) of three compounds (racemic MA5, S(+) MA5, or R(-) MA5) were added. After culturing for 6 hours, the concentration of ATP produced in the culture medium was measured using a GloMa 96 Microplate Luminometer (Promega) with a "cellular" ATP measurement reagent (Toyo B-Net).

[0085] 7-2 Results No difference was observed in the level of ATP production in animal cells between the S(+) form of MA5 and the R(-) form of MA5 (see FIG. 5).

[0086] 8. Evaluation of Lifespan To confirm that the S(+) form of MA5 has the effect of extending the lifespan of an organism, analysis is performed, for example, according to the methods described in the following sections [Method 1] and [Method 2].

[0087] [Method 1] [1] Neurodegeneration model flies (obtained from the Bloomington Drosophila Stock Center) were mated, and the resulting eggs were raised at 18°C. [2] One to three days after they emerged as adults, they were placed in an incubator at 29°C. [3] Vials containing 5% sucrose solution or 5% sucrose solution containing three compounds (racemic MA5, S(+) MA5, or R(-) MA5) at various concentrations (100 nM or 1000 nM) were prepared, and 20 male and female flies were transferred into each vial and allowed to consume the respective solutions (protected from light, at 29°C for 20 hours). [4] The flies were transferred to another vial containing regular food and allowed to consume the regular food (for one day). [5] The following day, the above procedures [3] and [4] were repeated. [6] During steps [3] and [4] above, remove dead flies and record their number and lifespan. When the vial is empty (i.e., when there are no live flies in the vial), the total number of dead flies recorded is used as the original count to calculate the number of surviving flies. The average lifespan of flies is calculated based on the lifespan of each dead fly and the total number of surviving flies. The reason for shifting the temperature from 18°C ​​to 29°C is because a temperature-sensitive gene expression system is used, which does not express neurodegenerative proteins during development but only after emergence into adults (expressing neurodegenerative proteins in all neurons during development is often lethal). Flies are used as model organisms for human disease (see "Hum Mol Genet. 2019. PMID: 31227826") and human aging (see "FEBS Open Bio. 2022. PMID: 34854258"). Therefore, if the lifespan extension effect of the S(+) form of MA5 is confirmed in flies, it can be said that this effect can also be applied to humans.

[0088] [Method 2] [1] 28-day-old α-klotho homo KO mice (male [♂], 4 weeks old, n=3, 2 replicates) were obtained from CLEA Japan and allowed to acclimate for 2 days. [2] On the 30th day after birth, three compounds (racemic MA5, S(+) MA5, or R(-) MA5) suspended in water were orally administered to the KO mice at a dose of 10 mg / kg body weight / 100 μL / day. [3] The weights of the KO mice were measured once a week and recorded until death was confirmed. Additionally, as a control, weights of KO mice that were not orally administered the three compounds were similarly measured until death was confirmed.

[0089] 9. Evaluation of Mitochondrial Function Mitochondrial swelling in muscle cells has been reported in immt-1-deficient mutant nematodes (hereafter referred to as "immt-1 / mitofilin mutants"), a nematode orthologue of the mitochondrial inner membrane protein mitofilin (see "Journal of Cellular Physiology. 224 (2010) 748-756" and "Molecular Biology of the Cell. 22 (2011) 831-841.") Therefore, we analyzed whether mitochondrial swelling was alleviated when three compounds (racemic MA5, S(+) MA5, or R(-) MA5) were administered to the immt-1 / mitofilin mutant.

[0090] Specifically, the immt-1 / mitofilin mutant of ccIs4251 (Pmyo-3::GFP::LacZ::NLS, Pmyo-3::mitochondrial-GFP+dpy-20(+), Cell 139 (2009) 623-633), which was genetically engineered to visualize mitochondrial DNA and genomic DNA with GFP, was cultured on a control culture plate (E. coli OP-50) sprayed with DMSO (solvent). Larvae were reared on the culture plates containing either NGM agar or the three compounds (racemic MA5, S(+) MA5, or R(-) MA5) at a final concentration of 10 μM for 5 days (i.e., until 4-day-old adults) from the L4 stage. Body wall muscle cells were observed in 4-day-old adults (see Figure 6), and the number of abnormal mitochondria (swelling mitochondria) and the percentage of body wall muscle cells containing abnormal mitochondria were measured (see Figures 7 and 8). As a control, wild-type worms (ccIs4251, obtained from Caenorhabditis Genetics Center, CGC) were reared on the culture plates and analyzed in the same manner.

[0091] When immt-1 / mitofilin mutants were reared in the presence of the S(+) form of MA5, the percentage of body-wall muscle cells with five or more swelling mitochondria per cell was significantly reduced compared to when they were reared in the presence of the control DMSO or the R(-) form of MA5 (Fig. 7), and the number of swelling mitochondria per cell was also significantly reduced (Fig. 8).Furthermore, when immt-1 / mitofilin mutants were reared in the presence of the R(-) form of MA5, the percentage of body-wall muscle cells with five or more swelling mitochondria per cell and the number of swelling mitochondria per cell remained almost unchanged or were only slightly reduced compared to when they were reared in the presence of the control DMSO (Figs. 7 and 8).

[0092] These results indicate that the R(-) form of MA5 has almost no effect on alleviating mitochondrial swelling, while the S(+) form of MA5 has the effect of alleviating mitochondrial swelling. In other words, this indicates that the S(+) form of MA5 has the effect of improving mitochondrial function in nematodes. Furthermore, since nematodes are used as a model organism for analyzing human mitochondrial function (see the literature "Nature. 2018. PMID: 30356218"), it can be said that the S(+) form of MA5 has been shown to be useful as a mitochondrial function activator and a therapeutic or preventive agent for mitochondrial disease in humans.

[0093] 10. In Vivo Administration Test 2 In "3. In Vivo Administration Test 1" above, it was confirmed that the S(+) form of MA5 administered intravenously was more easily retained in the blood than the R(-) form of MA5 administered intravenously. This study confirmed whether similar results would be obtained when the compound was administered orally. Specifically, according to the method described in "3-1 Method" above, a solution containing each enantiomer of MA5 (S(+) form or R(-) form) was orally administered once at a dose of 100 mg / kg to male cynomolgus monkeys, and the concentrations of each enantiomer of MA5 and the reduced metabolite of each enantiomer of MA5 (compound represented by formula (B) [i.e., Red-MA5]) in plasma were measured.

[0094]

[0095] As a result, after oral administration, the concentration of the S(+) form of MA5 in plasma was higher than that of the R(-) form of MA5 (see Figure 9). Furthermore, the reduced metabolite (compound represented by formula (B), i.e., Red-MA5) in plasma was detected when the R(-) form of MA5 was orally administered, but was hardly detected when the S(+) form of MA5 was orally administered (see Figure 10).

[0096] These results indicate that when the S(+) form of MA5 is orally administered, it is more likely to be retained in the blood than when the R(-) form of MA5 is orally administered, and that the S(+) form of MA5 is less metabolized than the R(-) form of MA5, so the S(+) form of MA5 can exert its effects in the body more sustainably than the R(-) form of MA5.

[0097] 11. Cytotoxicity Evaluation 3 To evaluate the toxicity of Red-MA5 to animal cells, analysis was performed according to the method described in the following section "11-1."

[0098] 11-1 Method 3 x 10 per well in a 96-well plate ３ KCMC10 cells were seeded and cultured for 24 hours in the presence of 0.1% DMSO (the compound solvent) or various concentrations (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, or 30 μM) of two compounds (racemic MA5 or Red-MA5). Cell viability was measured by MTT assay using Cell Counting Kit-8 (Dojindo Laboratories). Cell Count Reagent SF (100 μL) was added to each well, incubated for 2 hours, and then agitated for 3 seconds using a microplate reader. Absorbance at 450 nm (reference 750 nm) was measured (see Figure 11). A control experiment was also performed ("-" in Figure 11).

[0099] 11-2 Results When the KCMC10 cell line was cultured in the presence of 30 μM racemic MA5, no decrease in viable cell levels was observed (see FIG. 11A), whereas when cultured in the presence of 30 μM Red-MA5, a significant decrease in viable cell levels was observed (see FIG. 11B). This result indicates that MA5 is less cytotoxic than Red-MA5. In other words, taken together with the results of "10. In vivo administration test 2" above, this indicates that the S(+) form of MA5 administered in vivo is less cytotoxic than the R(-) form of MA5 administered in vivo.

[0100] 12. Evaluation of SIRT expression level in animal cells ＋ It is known that there is a positive correlation between the amount of MA5 and the expression level of SIRT (see "Nat Rev Nephrol. 2017. PMID: 28163307"). Furthermore, a correlation between increased SIRT expression in inner ear cells and improvement of hearing loss has been reported (see "Front CellDev Biol. 2021. PMID: 34869361," "Exp Cell Res. 2022 Jul 11:113280," and "Hindawi Neural Plasticity Volume 2021, Article ID 5520794"). Therefore, it is thought that the S(+) form of MA5 acts as a NAD+ receptor in the cell. ＋ To investigate whether increasing the amount of SIRT increases the expression level in inner ear cells and is useful for improving hearing loss, analysis was performed according to the method described in section "12-1" below.

[0101] 12-1 Method 1 × 10 cells were placed in a dish containing 10 mL of culture medium (low-glucose DMEM medium [glucose concentration 1.0 g / dL] supplemented with 10% FBS by weight). ６ A mouse inner ear cell line (HEI-OC1 cell line) was seeded and cultured in a cell / dish. One day later, 0.1% DMSO (the compound solvent) or various concentrations (1 μM, 3 μM, 10 μM, or 30 μM) of three compounds (racemic MA5, S(+) MA5, or R(-) MA5) were added, followed by 24-hour culture. After culture, the cells were harvested and disrupted by standard methods. The expression levels of various SIRTs (SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7) in the cells were analyzed by Western blotting (see Figures 12-1 to 14-2).

[0102] 12-2 Results When the HEI-OC1 cell line was cultured in the presence of the R(-) isomer of MA5, the expression levels of various SIRTs (SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7) in the cells were almost unchanged compared to when the cell line was cultured in the absence of the R(-) isomer of MA5 (see Figure 14 [Figures 14-1 and 14-2]). However, when the HEI-OC1 cell line was cultured in the presence of the S(+) isomer of MA5, the expression levels of various SIRTs in the cells increased compared to when the cell line was cultured in the absence of the S(+) isomer of MA5 (see Figure 13 [Figures 13-1 and 13-2]). This result is consistent with the results of the above-mentioned "2. NAD in animal cells" ＋ Taking into account the results of "Evaluation of the amount of NAD in cells," the S(+) form of MA5 ＋ As a result of increasing the amount, the expression level of SIRT in the cells increased, indicating that it is useful for improving hearing loss.

[0103] 13. Evaluation of Animal Motor Function To confirm that the S(+) form of MA5 has the effect of improving motor dysfunction in animals, mice described in the following sections "Aged Mice" and "Young Mice" are used and analyzed according to the methods described in the following sections "Method 1" to "Method 3."

[0104] [Aged mice] Water containing one of three compounds (racemic MA5, S(+) MA5, or R(-) MA5) was orally administered at a dose of 50 mg / kg body weight / day to five aged mice (92 weeks old) for nine weeks. As a control, water was orally administered to six aged mice for nine weeks.

[0105] [Young mice] Water containing three compounds (racemic MA5, S(+) MA5, or R(-) MA5) was orally administered to six young mice (4 months old) at a dose of 10 mg / kg body weight / day (low dose) or 50 mg / kg body weight (high dose) for nine weeks. As a control, water was orally administered to six young mice for nine weeks.

[0106] [Method 1: Treadmill Test] [1] Practice treadmill testing using a mouse treadmill device (MK-690, manufactured by Muromachi Kikai Co., Ltd.; the same applies below). For aged mice, the test was performed over a total of three days: Day 1: 10 minutes x 2 at 8 m / min, Day 2: 10 minutes x 2 at 10 m / min, and Day 3: 10 minutes x 2 at 12 m / min. On the other hand, for juvenile mice, the test was performed over a total of five days: Day 1: 10 minutes x 2 at 8 m / min, Day 2: 10 minutes x 2 at 9 m / min, Day 3: 10 minutes x 2 at 10 m / min, Day 4: 10 minutes x 2 at 11 m / min, and Day 5: 10 minutes x 2 at 12 m / min. [2] The actual treadmill test was performed for up to two hours using the mouse treadmill device under the following conditions. The speed starts at 5 m / min, and is increased by 5 m / min every 3 minutes, eventually reaching 28 m / min (i.e., 5 m / min → 10 m / min → 15 m / min → 20 m / min → 25 m / min → 28 m / min). [3] If the animal does not move forward even when its tail is stimulated with a brush, and does not move on the electrified rod for more than 10 seconds, it is judged to have run out of energy. If the animal has been running for more than 2 hours, it is stopped and recorded as 2 hours.

[0107] [Method 2: Reverse lattice hanging test] The reversible lattice hanging test for mice is performed using a wire mesh with a diameter of 37 cm and a square of 8 mm. A 12 cm plastic wall is constructed around the wire mesh to prevent the mouse from escaping. The mouse is placed on the wire mesh for 1 minute to allow it to acclimate. With the mouse in the center of the wire mesh, the mesh is turned over and the time (in minutes) until the mouse falls is measured. The wire mesh is held at a height of 60 cm from the ground. To prevent injury to the mouse if it falls, bedding used for raising the mouse is placed underneath. If the mouse falls in less than 30 seconds, it is considered to be an accidental fall and additional measurements are taken until the time reaches 30 seconds or more. Two measurements are taken on separate days and the average value is used. The mouse's weight is also measured and the "Hanging Impulse Score" (time spent trapped [min] x weight [min]) is calculated.

[0108] [Method 3: Grip Strength] Mouse grip strength measurements are performed using a mouse grip strength measuring device (GPM-101, Melquest). For measuring the grip strength of the forelimbs, a forelimb grip is used, and for measuring the grip strength of the limbs, a wire mesh for the limbs is set up and used. Measurement of the forelimb grip strength is started first, and measurements are made in the horizontal and vertical directions. For the horizontal measurement, the mouse's forelimbs are held on the grips and pulled horizontally until the hands are released. For the vertical measurement, the mouse's forelimbs are held on the grips and pulled vertically until the hands are released. The maximum value is measured after five consecutive attempts. For the limb measurement, with the mouse's limbs placed on the wire mesh, the tail is pulled horizontally until the hands are released, and the maximum value is measured after five consecutive attempts.

[0109] 14. Nampt binding test Mammalian NAD ＋ The binding of the S(+) form of MA5 and the R(-) form of MA5 to nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in the synthesis system, was measured using Octet K2.

[0110] A 0.01% DDM PBS solution was prepared as the assay buffer. 1 μL of biotin Nampt was added to 1000 μL of assay buffer. This was diluted 2-fold to prepare a 0.4 ng / μL ligand solution. A 100 mM MA5 S(+) DMSO solution was diluted from 300 μM to 1000 μM, and a 100 mM MA5 R(-) DMSO solution was diluted from 500 μM to 1200 μM to prepare the respective analyte solutions. 200 μL each of assay buffer, 1 μM biotin solution, ligand solution, 100 μM biocytin solution, and analyte solution was added to a 96-well plate, and the plate was set in an Octet K2 for measurement. Data were analyzed using the accompanying software. Kd values ​​were plotted using Prism 9, using the concentration of the analyte solution and the Req (nm) value calculated by analysis.

[0111] It was confirmed that the S(+) form of MA5 binds to Nampt, but the R(-) form does not bind to Nampt (Figure 15). ＋ Since MA5 is known to be an enzyme that controls the synthesis of NAD, the above results suggest that the S(+) form of MA5＋ This is consistent with the results showing increased levels.

[0112] 15. Inhibitory Effect of Camptothecin on DNA Damage. Rat fetal cardiac myoblast H9c2 cells were maintained and cultured in DMEM medium (10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin). Camptothecin (CPT) at a final concentration of 10 μM, CPT at a final concentration of 10 μM and MA5 (racemic, S(+)-, or R(-)-isomer) at a final concentration of 30 μM, or DMSO (control) was added. After 24 hours of culture, the medium was removed, and γH2AX (phosphorylated H2AX), an indicator of DNA damage, was detected by immunostaining. Immunostaining was performed using mouse anti-γH2AX antibody (Thermo Fisher) as the primary antibody and CF568-conjugated goat anti-mouse IgG antibody (Biotium Inc.) as the secondary antibody.

[0113] As a result of immunostaining, a decrease in the fluorescence intensity of phosphorylated H2AX was observed only in the sample to which the S(+) form of MA5 was added (Figure 16), confirming that MA5 has the effect of suppressing DNA damage caused by CPT. The R(-) form did not show this effect. ＋ It is known that an increase in NAD has a DNA repair effect. Therefore, the above results suggest that the S(+) form of MA5 acts as a NAD ＋ This suggests that DNA damage caused by CPT can be suppressed by increasing its level.

[0114] 16. Physical Properties of the S(+) Form of MA5 Comparing the physical properties of the S(+) form of MA5 with those of the racemic form, the S(+) form of MA5 is readily soluble in acetone, acetonitrile, ethyl acetate, chloroform, dichloromethane, methanol, ethanol, and isopropanol. The S(+) form of MA5 is also somewhat soluble in a 50% aqueous solution of acetonitrile, but is slightly insoluble in a 50% aqueous solution of methanol. Meanwhile, the racemic form of MA5 is somewhat soluble in a chloroform-methanol mixed solvent, a dichloromethane-methanol mixed solvent, and acetone, but is slightly insoluble in ethyl acetate, chloroform, dichloromethane, and methanol.

[0115] Furthermore, the S(+) form of MA5 discolors and decomposes under light, acid, and alkaline conditions. Specifically, solutions of the S(+) form of MA5 in ethanol, isopropanol, acetone, acetonitrile, ethyl acetate, 50% aqueous acetonitrile, and 0.1% formic acid-containing acetonitrile are colorless and transparent and do not discolor even after refrigerated storage for approximately one week. However, solutions of the S(+) form of MA5 dissolved in methanol, dichloromethane, chloroform, 50% aqueous methanol, and 0.1% formic acid-containing methanol are colorless and transparent on the day of preparation, but develop a light yellow color after refrigerated storage for approximately one week. Furthermore, solutions of the S(+) form of MA5 dissolved in a mixed solvent of alcohol (methanol, ethanol, isopropanol) and chloroform developed a yellow color the day after preparation.

[0116] Halogenated solvents produce hydrogen chloride as a small amount of decomposition product, so for example, chloroform contains 0.4-0.8% ethanol as a stabilizer. It has been confirmed that discoloration decomposition is promoted in mixed solvents of alcohol and chloroform, and it is thought that the presence of small amounts of acid and alcohol may contribute to the decomposition.

[0117] When a solution of the S(+) form of MA5 was spotted on silica gel TLC and allowed to stand for a while before development, decomposition products appeared, but when developed immediately, no decomposition products were produced. Decomposition products were hardly produced in an ethyl acetate solution, but many decomposition products were produced in an alcohol-chloroform solution.

[0118] The TLC showed that the condition was slightly acidic, which may have promoted decomposition. Furthermore, the combination of alcohol (short-chain alcohol) and chloroform is more likely to promote decomposition.

[0119] The present invention contributes to the prevention or treatment of symptoms or diseases associated with aging, metabolic diseases, mitochondrial diseases, and the like, and to the extension of lifespan.

Claims

1. A pharmaceutical composition comprising as an active ingredient a compound represented by the following formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) form:

2. NAD in subjects ＋ The pharmaceutical composition of claim 1, which is used to increase levels.

3. The pharmaceutical composition according to claim 1 or 2, which is for the treatment or prevention of a mitochondrial disease.

4. The pharmaceutical composition according to claim 1 or 2, which is for the treatment or prevention of symptoms or diseases associated with aging.

5. The pharmaceutical composition according to claim 4, wherein the age-related symptom or disease is hearing loss.

6. The pharmaceutical composition according to claim 1, wherein the enantiomeric excess of the S(+) form is at least 97% or more.

7. A food or beverage composition comprising as an active ingredient a compound represented by the following formula (A-1) or a physiologically acceptable salt thereof, which is substantially free of the R(-) form:

8. NAD in subjects ＋ The food and beverage composition according to claim 7, which is used to increase the level of 9. A food or beverage composition according to claim 7 or 8, intended for a subject in need of treatment or prevention of a mitochondrial disease.

10. A food or beverage composition according to claim 7 or 8, intended for a subject in need of treatment or prevention of symptoms or diseases associated with aging.

11. The food or beverage composition according to claim 10, wherein the symptom or disease associated with aging is hearing loss.

12. The food and beverage composition according to claim 7, in which the enantiomeric excess of the S(+) form is at least 97% or more.

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