Composition containing a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, and a method for evaluating the quality and a method for determining the enzyme reactivity thereof

Abstract

A compound comprising β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, wherein the purity measured by HPLC is 95% or higher, and the reactivity to lactate dehydrogenase is 30 U or higher.

Description

[Technical field]

[0001] The present invention relates to a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, as well as a method for evaluating the quality and a method for determining the enzymatic reactivity thereof. [Background technology]

[0002] β-nicotinamide mononucleotide (hereinafter sometimes referred to as "β-NMN") is a coenzyme NAD + It is an intermediate metabolic product in the biosynthesis of β-NMN. In recent years, it has been reported that β-NMN has the effect of improving insulin secretion in aged mice, the effect of dramatically improving insulin sensitivity and secretion in mouse models of type 2 diabetes caused by a high-fat diet and aging (see, for example, Patent Document 1), the effect of being involved in the control of circadian rhythms (see, for example, Patent Document 2), and the effect of significantly enhancing mitochondrial function in aged muscles. Furthermore, it has been reported that administration of β-NMN is useful for improving and preventing the symptoms of various diseases associated with aging, such as obesity, elevated blood lipid levels, decreased insulin sensitivity, memory loss, and deterioration of eye function such as macular degeneration (see, for example, Patent Document 3). Furthermore, administration of β-NMN increases NAD in the body. + By increasing the amount of sirtuin and activating the sirtuin gene, it is expected that the decline in physical functions associated with aging in the living body can be inhibited and delayed, thereby providing an anti-aging effect (see, for example, Patent Document 4).

[0003] On the other hand, when applying β-NMN to medicines, supplements, cosmetics, etc., it has been proposed to crystallize β-NMN in order to increase its purity and improve its storage stability (see, for example, Patent Documents 5 and 6). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 7,737,158 [Patent Document 2] US Patent Application Publication No. 2011 / 123510 [Patent Document 3] International Publication No. 2014 / 146044 [Patent Document 4] International Publication No. 2017 / 200050 [Patent Document 5] Special Publication No. 2018-534265 [Patent Document 6] International Publication No. 2018 / 047715 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have found that, although the β-nicotinamide mononucleotide they produced and commercially available β-NMN products have high purity as measured by high performance liquid chromatography (hereinafter sometimes referred to as "HPLC"), and even if the purity is increased by crystallization, the same effect cannot be obtained when administered to a living body at the same dosage. In other words, since there are β-NMNs with different physiological activities even if they have the same purity as measured by HPLC, it is necessary to provide β-NMN with higher physiological activity.

[0006] The present invention aims to meet such demands, break through the current situation, solve the above-mentioned conventional problems, and achieve the following objectives: That is, the present invention aims to provide a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, which has a high purity as measured by HPLC and exhibits high enzymatic reactivity and thus has high physiological activity, as well as a method for evaluating the quality of a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, and a method for determining the enzymatic reactivity thereof. [Means for solving the problem]

[0007] As a result of intensive research conducted by the inventors in order to achieve the above-mentioned object, they discovered that the difference in physiological activity between these products, even when the purity measured by HPLC is the same, is due to the difference in the enzymatic reactivity of β-NMN in these products, and thus completed the present invention.

[0008] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> A compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, characterized in that the compound has a purity of 95% or more as measured by HPLC and a reactivity to lactate dehydrogenase of 30 units or more. <2> The purity as measured by HPLC is 98% or more, and the reactivity to lactate dehydrogenase is 33 units or more. <1> It is a compound described in <3> The above in crystalline form <1> or <2> It is a compound described in <4> The lactate dehydrogenase is lactate dehydrogenase derived from mammalian skeletal muscle. <1> ~ <3> The compound according to any one of the above items. <5> The lactate dehydrogenase has the amino acid sequence of SEQ ID NO: 1. <4> It is a compound described in <6> The method according to claim 1, which is substantially free of nicotinamide dinucleotide. <1> ~ <5> The compound according to any one of the above items. <7> A method for evaluating the quality of a compound comprising β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, comprising the steps of: The method is characterized by comprising evaluating the quality of the compound consisting of the β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof using as indicators a purity of 95% or more when measured by HPLC and a reactivity to lactate dehydrogenase of 30 units or more. <8> A method for determining the enzymatic reactivity of a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, comprising: The method is characterized by comprising determining the reactivity of the β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof with lactate dehydrogenase using as indicators a purity of 95% or more when measured by HPLC and a reactivity with lactate dehydrogenase of 30 units or more. Effect of the Invention

[0009] According to the present invention, it is possible to solve the above-mentioned problems in the conventional art and achieve the above-mentioned object, and it is possible to provide a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, which has a high purity when measured by HPLC and exhibits high enzymatic reactivity and thus has high physiological activity, as well as a method for evaluating the quality of a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof and a method for determining the enzymatic reactivity. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram showing the reaction of β-NMN with lactate dehydrogenase. [Diagram 2] FIG. 2 is a graph showing the enzymatic reactivity of an example of a β-NMN compound of the present invention and a commercially available β-NMN product to lactate dehydrogenase (SEQ ID NO: 1), as measured in Test Example 1. [Figure 3A] FIG. 3A is a graph showing the enzymatic reactivity of an example of a β-NMN compound of the present invention and a commercially available β-NMN product to porcine lactate dehydrogenase (LDH) 1 (SEQ ID NO: 4), as measured in Test Example 3. [Figure 3B] FIG. 3B is a graph showing the enzymatic reactivity of an example of a β-NMN compound of the present invention and a commercially available β-NMN product to human lactate dehydrogenase (LDH) 1 (SEQ ID NO: 2), as measured in Test Example 3. [Figure 3C] FIG. 3C is a graph showing the enzymatic reactivity of an example of a β-NMN compound of the present invention and a commercially available β-NMN product to human lactate dehydrogenase (LDH) 5 (SEQ ID NO: 3), as measured in Test Example 3. [Figure 4A]FIG. 4A is a graph showing the relative activity of lactate dehydrogenase (SEQ ID NO: 1) for various concentrations of an example of a β-NMN compound of the present invention and commercially available β-NMN product 1, as measured in Test Example 4. [Figure 4B] FIG. 4B is a graph showing the relative activity of lactate dehydrogenase (SEQ ID NO: 1) for various concentrations of an example of a β-NMN compound of the present invention and commercially available β-NMN product 2, as measured in Test Example 4. [Figure 4C] FIG. 4C is a graph showing the relative activity of lactate dehydrogenase (SEQ ID NO: 1) for various concentrations of an example of a β-NMN compound of the present invention and commercially available β-NMN product 3, as measured in Test Example 4. [Figure 4D] FIG. 4D is a graph showing the relative activity of human lactate dehydrogenase (LDH) 1 (SEQ ID NO: 2) for various concentrations of an example of a β-NMN compound of the present invention and a commercially available β-NMN product 1, as measured in Test Example 4. [Figure 4E] FIG. 4E is a graph showing the relative activity of human lactate dehydrogenase (LDH) 1 (SEQ ID NO: 2) for various concentrations of an example of a β-NMN compound of the present invention and a commercially available β-NMN product 2, as measured in Test Example 4. [Figure 4F] FIG. 4F is a graph showing the relative activity of human lactate dehydrogenase (LDH) 1 (SEQ ID NO: 2) for various concentrations of an example of a β-NMN compound of the present invention and a commercially available β-NMN product 3, as measured in Test Example 4. [Figure 5A] FIG. 5A is a graph showing the cell damage rate calculated from the intracellular and extracellular AST activity when a β-NMN compound as an example of the present invention was used, as measured in Test Example 5. [Figure 5B] FIG. 5B is a graph showing the cell damage rate calculated from the intracellular and extracellular AST activity when β-NMN product 3 was used, as measured in Test Example 5. [Figure 6A] FIG. 6A is a graph showing the intracellular NAD content measured in Test Example 5 when a β-NMN compound as an example of the present invention was used. [Figure 6B]FIG. 6B is a graph showing the intracellular NAD content measured in Test Example 5 when β-NMN product 3 was used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (compound) The compound of the present invention is a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, and has a purity of 95% or more as measured by HPLC and a reactivity to lactate dehydrogenase of 30 units or more.

[0012] β-nicotinamide mononucleotide exists as two optical isomers, α and β, and the structure of β-nicotinamide mononucleotide (CAS number: 1094-61-7) according to the present invention is as follows. [ka]

[0013] The compound consisting of β-NMN or a pharmacologically acceptable salt thereof according to the present invention (hereinafter, sometimes referred to as "β-NMN compound") may be prepared by any method. For example, β-NMN artificially synthesized by chemical synthesis, enzymatic method, fermentation method, etc., and purified can be used as the active ingredient. In addition, since β-NMN is a component widely present in living organisms, β-NMN obtained by extraction and purification from natural raw materials such as animals, plants, and microorganisms can also be used as the active ingredient. Commercially available purified β-NMN may also be used.

[0014] As a chemical synthesis method for synthesizing β-NMN, for example, nicotinamide and L-ribose tetraacetate are reacted, and the resulting nicotinamide mononucleoside is phosphorylated to produce β-NMN. As an enzymatic method, for example, β-NMN can be produced from nicotinamide and 5'-phosphoribosyl-1'-pyrophosphate (hereinafter sometimes referred to as "PRPP") using nicotinamide phosphoribosyltransferase (hereinafter sometimes referred to as "NAMPT"), and β-NMN can also be produced from nicotinamide riboside using nicotinamide riboside kinase. As a fermentation method, for example, β-NMN can be produced from nicotinamide using the metabolic system of a microorganism expressing NAMPT.

[0015] The β-NMN may be a pharmacologically acceptable salt. The pharmacologically acceptable salt of β-NMN may be an inorganic acid salt or an organic acid salt having a basic site such as an amine. Examples of acids constituting such acid salts include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. The pharmacologically acceptable salt of β-NMN may be an alkali salt or an organic salt having an acidic site such as a carboxylic acid. Examples of bases that constitute such acid salts include alkali metal salts or alkaline earth metal salts, such as those derived from bases such as sodium hydride, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, trimethylammonia, triethylammonia, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, and tetramethylammonium hydroxide.

[0016] As the β-NMN compound according to the present invention, it may be in a crystalline form or amorphous (non-crystalline). However, in order to further suppress the mixing of impurities and enhance the stability of the compound, it is preferably a crystallized product subjected to a crystallization process using a solution containing alcohols such as a methanol solution or ethanol. There is no particular limitation on the crystallization method of the β-NMN compound, and known methods can be appropriately selected. For example, it can be carried out according to the methods described in JP-T-2018-534265 or WO 2018 / 047715.

[0017] <HPLC purity> Regarding the purity of the β-NMN compound according to the present invention, there is no particular limitation as long as the purity measured by HPLC (hereinafter sometimes referred to as "HPLC purity") is 95% or more, and it can be appropriately selected. However, 98% or more is preferable.

[0018] The HPLC purity in the present invention means the ratio of the peak area derived from NMN to the total amount of the peak areas of various peaks detected by measuring a sample containing β-NMN by HPLC. Specifically, it can be obtained from the following formula. - Formula - HPLC purity (%) = (peak area derived from β-NMN) / (total amount of measured peak areas) × 100

[0019] In the present invention, there is no particular limitation on the HPLC analysis method for measuring the HPLC purity as long as it is a method and conditions capable of efficiently separating and measuring β-NMN, and it can be appropriately selected. For example, Hypercarb can be used as the column. TM(length: 15 cm, inner diameter: 4.6 mm, particle size: 3 μm, manufactured by Thermo Fisher Scientific) as described in "Yoshino, et al., Cell Metabolism, 2011, vol. 14, p. 528-536.", or using a TSK-GEL ODS column (length: 15 cm, inner diameter: 4.6 mm, particle size: 5 μm, manufactured by Tosoh Corporation) as a column, the measurement can be performed according to the method described in "Vitamin, Vol. 64, No. 1, pp. 19-25, 1990." In the present invention, the measurement was performed according to the following method. The HPLC system used is Prominence (Shimadzu Corporation). Dissolve the β-NMN sample in distilled water to a concentration of 2 mM and use this as the sample solution. Apply 10 μL of the sample solution to a TSK-GEL ODS-80TS column (length: 15 cm, inner diameter: 4.6 mm, particle size: 5 μm, manufactured by Tosoh Corporation). The column-adsorbed β-NMN fraction is separated as follows: As the eluent, 50 mM Tris-acetate (pH 7.5) / methanol is used, and elution and separation are performed with a concentration gradient of 0-15% methanol adjusted to a flow rate of 0.7 mL / min, and the absorbance is measured at 260 nm.

[0020] <Enzyme reactivity> Furthermore, the reactivity of the β-NMN compound of the present invention to lactate dehydrogenase (hereinafter sometimes referred to as "enzymatic reactivity") is not particularly limited as long as it is 30 units or more and can be appropriately selected, but 33 units or more is preferable.

[0021] Lactate dehydrogenase (hereinafter sometimes referred to as "LDH") in the present invention is a dehydrogenase also known as L-lactate dehydrogenase (EC 1.1.1.27). As the lactic acid dehydrogenase, lactic acid dehydrogenase derived from mammalian skeletal muscle is preferred, and lactic acid dehydrogenase 5 (LDH5) (hereinafter sometimes referred to as "R-LDH5") consisting of a tetramer of subunits having the amino acid sequence of SEQ ID NO: 1 is more preferred. However, other lactic acid dehydrogenases of mammals, such as porcine-derived lactic acid dehydrogenase (LDH) 1, human-derived lactic acid dehydrogenase (LDH) 1, and human-derived lactic acid dehydrogenase (LDH) 5, can also be used. LDH catalyzes a dehydrogenation reaction using lactic acid as a substrate and converts it to pyruvic acid. At this time, β-NMN is converted to reduced β-NMN (see Figure 1).

[0022] In the present invention, the enzyme reactivity refers to a value obtained by measuring according to the following reagents and methods. <R1 reagent> · 80 mM Tris-HCl (pH 8.5) · 19 mM β-NMN (Assume that the purity of β-NMN in the measurement sample is 100%. Also, β-NMN is dissolved in an 80 mM sodium carbonate solution.) <R2 reagent> · 100 mM Tris-HCl (pH 8.5) · 100 mM L-lactic acid <Enzyme solution> · Adjust the enzyme solution so that the final concentration of LDH in the reaction solution is 245 U / mL. In the present invention, the activity unit (U) of LDH is the activity unit when NAD is used as a coenzyme, and the activity unit measured by the human LDH measurement method based on IFCC is used.

[0023] <Activity measurement> For the measurement of enzyme activity, a Hitachi 7180 automatic analyzer (manufactured by Hitachi High-Tech Corporation) is used. The measurement parameters are shown below. -Measurement parameters- · Analysis method ··· Rate A · Measurement wavelength (sub / main) ··· 405 nm / 340 nm · Reaction time ··· 10 minutes Metering points 20-24 Sample solution (enzyme solution) 18μL R1 Reagent 120μL R2 Reagent 87μL -Measurement procedure- 18μL of enzyme solution and 120μL of R1 reagent are mixed and incubated at 37℃ for 4.5 minutes (photometric points 1-16), after which 87μL of R2 reagent is added to start the reaction (photometric point 17). The absorbance of the sample measured 1-2 minutes after the start of the reaction (photometric points 20-24) is subtracted from the absorbance of the blank water at the same photometric points to calculate the change in absorbance per minute (ΔmAbs / min). The activity unit (unit of reactivity to lactate dehydrogenase R-LDH5) is defined as 0.1mAbs of absorbance change per minute.

[0024] It is preferable that the β-NMN compound of the present invention is substantially free of nicotinamide dinucleotide (hereinafter, sometimes referred to as "NAD"), because if NAD is contained, the reactivity to lactate dehydrogenase may not be accurately measured. In the present invention, "substantially free of nicotinamide dinucleotide" means that NAD in the β-NMN compound is not detected by the above-mentioned HPLC analysis. Note that this does not exclude an embodiment in which NAD is contained in the preparation or food or drink containing the β-NMN compound according to the present invention.

[0025] The β-NMN compound of the present invention not only has a high purity as measured by HPLC, but also has a high enzymatic reactivity to lactate dehydrogenase. Therefore, it has a high bioavailability compared to conventional β-NMN products (drug substances), and is of high quality as β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof and a product containing the same, and at the same time has a high physiological effect. Therefore, it can be used not only as a medicine containing β-NMN as an active ingredient, but also as a raw material for food and drink (including but not limited to supplements) and feed, or by administering or ingesting the same, and can provide a high effect.

[0026] (Method of evaluating the quality of compounds) The compound quality evaluation method of the present invention is a quality evaluation method for a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, and includes at least an evaluation step and, if necessary, further includes other steps such as a measurement step.

[0027] <Evaluation process> The evaluation step in the quality evaluation method of the present invention is a step of evaluating the quality of a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, using as indicators a purity of 95% or more as measured by HPLC and a reactivity to lactate dehydrogenase of 30 units or more. The purity index as measured by HPLC is not particularly limited as long as it is 95% or more and can be appropriately selected, but is preferably 98% or more. The index of reactivity to lactate dehydrogenase is not particularly limited and can be appropriately selected as long as it is 30 units or more, but is preferably 33 units or more.

[0028] -evaluation- In the evaluation step of the quality evaluation method of the present invention, when the compound to be evaluated, which is composed of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, has a purity of 95% or more as measured by HPLC and a reactivity to lactate dehydrogenase of 30 units or more, the quality of the evaluation object is evaluated to be good. More specifically, when the purity is 95% or more as measured by HPLC and the reactivity to lactate dehydrogenase of 30 units or more, the evaluation object is evaluated to be a compound composed of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof with high bioavailability. The purity and reactivity to lactate dehydrogenase measured by HPLC may be measured when carrying out the method for evaluating the quality of the compound of the present invention, or may be measured separately.

[0029] <Other processes> As other steps in the method for evaluating the quality of the compound of the present invention, there are no particular limitations as long as the effects of the present invention are not impaired, and they can be appropriately selected. For example, a measurement step and the like can be mentioned.

[0030] - Measurement step - The measurement step in the method for evaluating the quality of the compound of the present invention is a step of measuring the purity and the reactivity with respect to lactate dehydrogenase when measured by HPLC of a compound composed of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof to be evaluated. The purity and the reactivity with respect to lactate dehydrogenase when measured by the above HPLC can be measured in the same manner as described in the items of <HPLC purity> and <enzyme reactivity> of the above (compound).

[0031] (Method for determining the enzyme reactivity of a compound) The method for determining the enzyme reactivity of the compound of the present invention is a method for determining the enzyme reactivity of a compound composed of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, including at least a determination step, and further including other steps as necessary.

[0032] <Determination step> The determination step in the method for determining the enzyme reactivity of the compound of the present invention is a step of determining the enzyme reactivity of a compound composed of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, using as an index that the purity when measured by HPLC is 95% or more and the reactivity with respect to lactate dehydrogenase is 30 units or more. As an index for the purity when measured by the above HPLC, there are no particular limitations as long as it is 95% or more, and it can be appropriately selected, but 98% or more is preferable. As an index for the reactivity with respect to the above lactate dehydrogenase, there are no particular limitations as long as it is 30 units or more, and it can be appropriately selected, but 33 units or more is preferable.

[0033] - Determination - In the determination step of the method for determining the enzymatic reactivity of the compound of the present invention, when the compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof to be determined has a purity of 95% or more as measured by HPLC and a reactivity against lactate dehydrogenase of 30 units or more, it is determined that the enzymatic reactivity of the determination target is high. More specifically, when the purity as measured by HPLC is 95% or more and the reactivity against lactate dehydrogenase is 30 units or more, it is determined that the evaluation target is a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof having a high reactivity against lactate dehydrogenase. The purity measured by the HPLC and the reactivity against lactate dehydrogenase may be those measured when performing the method for determining the enzymatic reactivity of the compound of the present invention, or may be those measured separately.

[0034] <Other steps> As other steps in the method for determining the enzymatic reactivity of the compound of the present invention, there are no particular limitations as long as the effects of the present invention are not impaired, and they can be appropriately selected. For example, a measurement step and the like can be mentioned.

[0035] -Measurement step- The measurement step in the method for determining the enzymatic reactivity of the compound of the present invention is a step of measuring the purity of the compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof to be evaluated as measured by HPLC and the reactivity against lactate dehydrogenase. The purity measured by the HPLC and the reactivity against lactate dehydrogenase can be measured in the same manner as described in the items of <HPLC purity> and <enzymatic reactivity> of the above-mentioned (compound).

[0036] According to the method for evaluating the compound of the present invention and the method for determining the enzymatic reactivity of the compound, it is possible to evaluate and determine the bioavailability that cannot be evaluated only by the HPLC purity of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, and thereby provide a compound consisting of high-quality β-NMN or a pharmacologically acceptable salt thereof.

[0037] The present invention also includes a method for producing a compound consisting of high-quality β-NMN or a pharmacologically acceptable salt thereof, which includes the method for evaluating the compound or the method for determining the enzymatic reactivity of the compound. EXAMPLES

[0038] The present invention will be described below with reference to Production Examples and Test Examples, but the present invention is not limited to these Production Examples and Test Examples.

[0039] (Production Example 1) <Enzyme treatment of β-NMN> Approximately 100 mg of β-NMN reagent (Amorphous, manufactured by Oriental Yeast Co., Ltd.) was dissolved in 100 mM phosphate buffer (pH 6.5) and the pH was adjusted to 6.0 with 4N KOH, after which 10,000 U of skeletal muscle-derived R-LDH5 was added and reacted at 10°C for 10 minutes. The reaction solution was ultrafiltered, the filtrate was collected, and the β-NMN from which LDH had been removed was recovered. The recovered β-NMN-containing solution was freeze-dried to obtain a powdered β-NMN sample.

[0040] <Crystallization treatment> 5mL of an aqueous solution of water and ethanol mixed at a volume ratio of 1:2 was measured out into a test tube, and the β-NMN sample was dissolved therein to prepare a saturated aqueous solution of NMN. The aqueous solution was then left to stand at 25°C for 3 days, causing crystals to precipitate. The solution containing the crystals was then centrifuged, and the supernatant was removed. The obtained crystals were suspended in an excess amount of ethanol, then centrifuged, and the supernatant was removed. The solution was then heated and dried at 60°C for 1 hour to obtain the crystals.

[0041] (Production Examples 2 to 4) The β-NMN compound of the present invention was produced by carrying out enzyme treatment and crystallization treatment in the same manner as in Production Example 1, using a β-NMN reagent from a different production lot.

[0042] (Test Example 1) In addition to the β-NMN compound produced in Production Example 1, the following β-NMN products, which are commercially available products and all subjected to crystallization treatment, were used in this test example. · β-NMN Product 1 (fermentation production method product, manufactured by Company A) · β-NMN Product 2 (chemically synthesized product, manufactured by Company A) · β-NMN Product 3 (production method unknown, manufactured by Company B)

[0043] <Measurement of HPLC Purity> The HPLC purity of the β-NMN compound (Production Example 1) and β-NMN Products 1 to 3 of the present invention was measured by the following method. The obtained results are shown in Table 1 below. The measurement results are the average values of three measurements.

[0044] 〔Measurement of HPLC Purity〕 · As the HPLC instrument, an HPLC system Prominence (manufactured by Shimadzu Corporation) was used. · The β-NMN sample was dissolved in distilled water to a concentration of 2 mM, and this was used as the sample solution. · 10 μL of the sample solution was applied to a TSK-GEL ODS-80TS column (length: 15 cm, inner diameter: 4.6 mm, particle size: 5 μm, manufactured by Tosoh Corporation). · Separation of the β-NMN fraction adsorbed on the column was carried out by the following method. Using 50 mM Tris-acetic acid (pH 7.5) / methanol as the eluent, elution and separation were carried out with a concentration gradient of 0 - 15% methanol adjusted to a flow rate of 0.7 mL / min, and measurement was performed at an absorbance of 260 nm. · The HPLC purity was calculated from the total peak area of the obtained HPLC chart and the peak area of β-NMN by the following formula. -Formula- HPLC purity (%) = (peak area derived from β-NMN) / (total amount of measured peak areas) × 100

[0045] <Evaluation of Enzymatic Reactivity> The enzymatic reactivity of the β-NMN compound of the present invention (Production Example 1) and β-NMN products 1 to 3 with R-LDH5 (SEQ ID NO: 1) was evaluated by the following method. The results are shown in Table 1 and Figure 2. The measurement results are the average of measurements performed three times.

[0046] [Enzyme reactivity evaluation] -R1 Reagent- 80mM Tris-HCl (pH 8.5) 19mM β-NMN (The purity of β-NMN in the measurement sample was assumed to be 100%. β-NMN was also dissolved in 80 mM sodium carbonate solution.) -R2 Reagent- 100mM Tris-HCl (pH 8.5) · 100mM L-lactic acid -Enzyme solution- Adjust the enzyme solution so that the final concentration of R-LDH5 in the reaction mixture is 245U / mL.

[0047] -Activity measurement- The enzyme activity was measured using a Hitachi 7180 automatic analyzer (Hitachi High-Tech Corporation). The measurement parameters are as follows: --Measurement parameters-- · Analysis Method · Rate A · Measurement wavelength (secondary / main) ··· 405nm / 340nm Reaction time: 10 minutes Metering points 20-24 Sample solution (enzyme solution) 18μL R1 Reagent 120μL R2 Reagent 87μL

[0048] --Measurement procedure-- 18 μL of enzyme solution and 120 μL of R1 reagent were mixed and incubated at 37°C for 4.5 minutes (photometric points 1-16), after which 87 μL of R2 reagent was added to start the reaction (photometric point 17). Measurements were performed for 5 minutes after the start of the reaction, and the absorbance of the measurement sample at 1-2 minutes from the start (photometric points 20-24) was subtracted from the absorbance of water (blank) at the same photometric points to calculate the absorbance change per minute (ΔmAbs / min). One activity unit was defined as 0.1 mAbs, which is the absorbance change per minute.

[0049] [Table 1]

[0050] As can be seen from the results in Table 1, although the HPLC purity of the β-NMN compound in Production Example 1 and β-NMN Products 1 to 3 was 99% or higher, the enzymatic reactivity was low, at 39 units in Production Example 1 and 23 to 26 units in Products 1 to 3. In addition, no NAD peak was observed in HPLC for the β-NMN compound of Production Example 1 and β-NMN Products 1 to 3, and they contained substantially no NAD.

[0051] (Test Example 2) The β-NMN compounds produced in Production Examples 2 to 4 were measured for HPLC purity and enzyme reactivity using the same method as in Test Example 1. As a result, it was confirmed that the β-NMN compounds of the present invention have high HPLC purity and enzyme reactivity even when raw materials from different production lots are used. These results are shown in Table 2 below, along with the test results of Production Example 1. The measurement results are the average values ​​of three measurements. In addition, no NAD peak was observed in HPLC for any of the β-NMN compounds of Production Examples 2 to 4, and they contained substantially no NAD.

[0052] [Table 2]

[0053] (Test Example 3) <Evaluation of Enzymatic Reactivity with LDH from Other Animal Species> To confirm whether the same tendency is observed with LDH from other animal species and humans, for the β-NMN compound produced in Production Example 1 and β-NMN Products 1 to 3, which are the same as those used in Test Example 1, the enzymatic reactivity with LDH ((A) Porcine-derived LDH1, (B) Human-derived LDH1, (C) Human-derived LDH5) was evaluated. The evaluation of enzymatic reactivity was performed in the same manner as in Test Example 1, except that the following enzyme solutions were used. The obtained results are shown in FIGS. 3A to C (FIG. 3A: Porcine-derived LDH1, FIG. 3B: Human-derived LDH1, FIG. 3C: Human-derived LDH5). - Enzyme solution - (A) Porcine-derived LDH1 The enzyme solution was adjusted so that LDH had a final concentration of 45 U / mL in the reaction solution. (B) Human-derived LDH1 The enzyme solution was adjusted so that LDH had a final concentration of 45 U / mL in the reaction solution. (C) Human-derived LDH5 The enzyme solution was adjusted so that LDH had a final concentration of 245 U / mL in the reaction solution.

[0054] From the results of Test Example 3, as in Test Example 1, when porcine-derived LDH1, human-derived LDH1, and human-derived LDH5 were used, Products 1 to 3 had low enzymatic reactivity with respect to the β-NMN compound of Production Example 1.

[0055] (Test Example 4) <β-NMN Compound Concentration Dependence of LDH Activity Value> To confirm the β-NMN compound concentration dependence of the LDH activity value, using the β-NMN compound produced in Production Example 1 and β-NMN Products 1 to 3, which are the same as those used in Test Example 1, the enzymatic activity of LDH was measured by changing the β-NMN concentration in the R1 reagent. The enzyme activity of LDH was measured in the same manner as in Test Example 1 (Enzyme Reactivity Evaluation), except that the following R1 reagent and enzyme solution were used, and the value of the change in absorbance per minute (ΔmAbs / min) was calculated. The relative activity value (%) of each NMN at each concentration is shown in Figures 4A-F (Figures 4A-C: R-LDH5, Figures 4D-F: human-derived LDH1), assuming the LDH activity value when 200 mM of the β-NMN compound of Production Example 1 was used as 100%. The measurement results are the average value of three measurements. -R1 Reagent- 80mM Tris-HCl (pH 8.5) 0, 10, 20, 50, 100, or 200mM β-NMN (The purity of β-NMN in the measurement sample was assumed to be 100%. β-NMN was also dissolved in 100 mM sodium carbonate solution.) -Enzyme solution- (I)R-LDH5 The enzyme solution was adjusted so that the final concentration of LDH in the reaction mixture was 245 U / mL. (II) Human LDH1 The enzyme solution was adjusted so that the final concentration of LDH in the reaction mixture was 45 U / mL.

[0056] As a result of the measurements, when R-LDH5 was used, Product 1 was unable to measure enzyme activity when the β-NMN concentration was 200 mM, and the concentration dependency could not be tracked (Figure 4A). This is thought to be because an inhibitory reaction of unknown origin became stronger as the β-NMN concentration increased. In Product 2 (Figure 4B) and Product 3 (Figure 4C), the difference in activity value with respect to the β-NMN compound of Production Example 1 became larger as the β-NMN concentration increased. When human-derived LDH1 was used, the difference in activity value against the β-NMN compound of Production Example 1 increased as the β-NMN concentration increased for all of Product 1 (Figure 4D), Product 2 (Figure 4E) and Product 3 (Figure 4F).

[0057] (Test Example 5) <Effects of NMN on cultured cells> In order to evaluate the biological activity of the β-NMN compounds of Production Example 1 and β-NMN Product 3, cultured cells were cultured in a medium containing the β-NMN compound, and the changes in intracellular NAD were measured. The cultured cells were HEK293 cells that had been suspended. TM 293 Expression Medium (Thermo Fisher Scientific) was used. First, HEK293 cells were suspended in medium at 1 × 10 6 The cells / mL concentration was adjusted and 30 mL was dispensed into six 125 mL flasks. The β-NMN compounds of Production Example 1 and β-NMN Product 3 were added to each flask to a final concentration of 0 and 0.1 mM, respectively, and the culture was performed with shaking in a CO2 incubator at 37°C for 7 days without changing the medium. After 7 days of shaking culture, the cells and culture supernatant were collected for various analyses. First, to calculate the cell damage rate, intracellular and extracellular aspartate aminotransferase (AST) activity was measured using L-type Wako AST·J2 (Fujifilm Wako Pure Chemical Industries, Ltd.) in accordance with the method specified by the reagent, using a Hitachi 7180-type automatic analyzer (Hitachi High-Tech Corporation). In addition, to measure the intracellular NAD content, NAD was detected using an NAD / NADH measurement kit (Dojindo Laboratories, Ltd.). Specifically, the weight of the collected cells was measured, and NAD was measured for those extracted with the Extraction buffer included in the kit. The intracellular NAD content was calculated from the calibration curve obtained by measuring NAD solutions of various concentrations.

[0058] Figure 5A shows the cell damage rate calculated from the intracellular and extracellular AST activity when the β-NMN compound of Production Example 1 was used, and Figure 5B shows the cell damage rate when the β-NMN compound of β-NMN product 3 was used. Figure 6A shows the intracellular NAD content when the β-NMN compound of Production Example 1 was used, and Figure 6B shows the intracellular NAD content when the β-NMN compound of β-NMN product 3 was used. The measurement results showed that the cell damage rate due to nutrient depletion caused by culturing for 7 days without changing the medium was lower when the β-NMN compound of Production Example 1 was added than when the β-NMN compound of β-NMN product 3 was added, and the intracellular NAD content was higher when the β-NMN compound of Production Example 1 was added than when the β-NMN compound of β-NMN product 3 was added.

Claims

1. A composition comprising a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, characterized in that the purity of the compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof is 95% or more but less than 100% when the composition is measured by HPLC, and the reactivity of the composition to lactate dehydrogenase is 30 units or more.

2. The composition described in claim 1, wherein the purity of the compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof is 98% or more but less than 100% when the composition is measured by HPLC, and the reactivity of the composition to lactate dehydrogenase is 33 units or more.

3. 3. The composition of claim 1 or 2 in a crystalline form.

4. A composition described in any one of claims 1 to 3, wherein the lactate dehydrogenase is a lactate dehydrogenase derived from mammalian skeletal muscle.

5. The composition described in claim 4, wherein the lactate dehydrogenase has the amino acid sequence of SEQ ID NO:

1.

6. The composition according to any one of claims 1 to 5, which is substantially free of nicotinamide dinucleotide.

7. A method for evaluating the quality of a composition containing a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, comprising: measuring the composition by HPLC and calculating the purity of the compound consisting of the β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof; measuring the reactivity of the composition with lactate dehydrogenase and calculating the reactivity of the composition with lactate dehydrogenase; and an evaluation step of evaluating the quality of the composition using as indicators that the purity of the compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof is 95% or more and that the reactivity of the composition to lactate dehydrogenase is 30 units or more.

8. A method for determining the enzyme reactivity of a composition containing a compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, comprising: measuring the composition by HPLC and calculating the purity of the compound consisting of the β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof; measuring the reactivity of the composition with lactate dehydrogenase and calculating the reactivity of the composition with lactate dehydrogenase; and a determination step of determining the reactivity of the composition with lactate dehydrogenase using as indicators that the purity of the compound consisting of β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof is 95% or more and that the reactivity of the composition with lactate dehydrogenase is 30 units or more.

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