Composition containing a reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced 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

To provide β-NADH precursor compounds consisting of β-NMNH or a pharmacologically acceptable salt thereof, which have high purity as measured by HPLC, have a molar absorption coefficient at a wavelength 340 nm that is a predetermined value indicating β-NMNH, and exhibits high catalytic activity as a coenzyme when converted to β-NADH, thereby having high physiological activity; as well as to provide methods for evaluating the quality and methods for determining enzymatic reactivity of the β-NADH precursor compound comprising β-NMNH or a pharmacologically acceptable salt thereof.SOLUTION: Provided is a β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof, which has a purity of 95% or higher as measured by HPLC, and has a catalytic activity for lactate dehydrogenase that converts pyruvate to lactate of 85% or more when converted to β-NADH by nicotinamide mononucleotide adenylyltransferase.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced 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] Oxidized 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] Furthermore, 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).

[0004] On the other hand, it has been reported that reduced β-nicotinamide mononucleotide (hereinafter sometimes referred to as "β-NMNH") is converted by nicotinamide mononucleotide adenylyltransferase (hereinafter sometimes referred to as "Nmnat") into reduced β-nicotinamide adenine dinucleotide (hereinafter sometimes referred to as "β-NADH") (see, for example, Non-Patent Document 1) and that NMNH functions as an NADH precursor in cells (see, for example, Non-Patent Document 2), but the physiological action of β-NMNH has not been clarified. [Prior art documents] [Patent documents]

[0005] [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 [Non-patent literature]

[0006] [Non-Patent Document 1] Biochemistry, 2007 April, Vol.46 No.16, pp.4912-4922 [Non-Patent Document 2] Nature metabolism, 2020 April, Vol. 2, pp.364-379 Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have found that, even though chemically produced β-NMNH and enzymatically produced β-NMNH have high purity as measured by high performance liquid chromatography (hereinafter sometimes referred to as "HPLC") and have a molar absorption coefficient (also referred to as molecular absorption coefficient) at a wavelength of 340 nm that is a predetermined value indicating β-NMNH, there is a difference in the enzymatic activity as a coenzyme, i.e., the activity as a catalyst (catalytic activity), when converted to β-NADH, and further, when β-NMNH is administered to a living body, the same physiological action cannot be obtained even at the same dosage. In other words, there are β-NMNHs with different physiological activities even if they have the same purity as measured by HPLC and have a molar absorption coefficient at a wavelength of 340 nm that is a predetermined value indicating β-NMNH, and therefore it is necessary to provide β-NMNH with a higher physiological activity.

[0008] 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 β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof, which has a high purity when measured by HPLC, has a molar absorption coefficient at a wavelength of 340 nm that is a predetermined value indicating β-NMNH, and exhibits high catalytic activity as a coenzyme when converted to β-NADH, and thus has high physiological activity, as well as a method for evaluating the quality and a method for determining the enzymatic reactivity of a β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof. [Means for solving the problem]

[0009] As a result of intensive research to achieve the above-mentioned object, the present inventors have found that, even if the purity as measured by HPLC and the molar absorption coefficient at a wavelength of 340 nm are equivalent, the catalytic activity when converted to β-NADH and the physiological activity of β-NMNH itself differ due to the difference in catalytic activity when β-NMNH in these compounds is converted to β-NADH, and have completed the present invention.

[0010] 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 β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof, characterized in that the compound has a purity of 95% or more as measured by HPLC, and when converted to β-NADH by Nmnat, has a catalytic activity of 85% or more for lactate dehydrogenase, which converts pyruvate to lactate. <2> The purity as measured by HPLC is 98% or more, and the catalytic activity for lactate dehydrogenase is 90% or more. <1> It is a compound described in <3> The molar absorption coefficient at a wavelength of 340 nm is 5,500 [L / (mol cm)] or more. <1> or <2> It is a compound described in <4> The molar absorption coefficient at a wavelength of 340 nm is 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less. <3> It is a compound described in <5> The lactate dehydrogenase is a lactate dehydrogenase derived from mammalian heart. <1> ~ <4> The compound according to any one of the preceding claims. <6> The lactate dehydrogenase has the amino acid sequence of SEQ ID NO: 2. <5> It is a compound described in <7> The above, wherein Nmnat is a thermostable Nmnat (Thermococcus kodakaraensis TK0067 gene). <1> ~ <6> The compound according to any one of the preceding claims. <8> The method according to claim 1, which is substantially free of nicotinamide dinucleotide. <1> ~ <7> The compound according to any one of the preceding claims. <9> A method for evaluating the quality of a β-NADH precursor compound comprising β-NMNH or a pharmacologically acceptable salt thereof, comprising: The method comprises evaluating the quality of a β-NADH precursor compound comprising the β-NMNH or a pharmacologically acceptable salt thereof, using as indicators a purity of 95% or more as measured by HPLC and a catalytic activity of 85% or more for lactate dehydrogenase, which converts pyruvate to lactate, when converted to β-NADH by Nmnat. <10> A method for determining the enzymatic reactivity of a β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof, comprising: The method is characterized by comprising determining the reactivity of a reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced 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 catalytic activity of 85% or more with lactate dehydrogenase, which converts pyruvate to lactate, when converted to reduced β-nicotinamide adenine dinucleotide (β-NADH) by nicotinamide mononucleotide adenylyltransferase (Nmnat). Effect of the Invention

[0011] According to the present invention, the above-mentioned conventional problems can be solved and the above-mentioned object can be achieved, and it is possible to provide a β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof, which has a high purity when measured by HPLC, has a molar absorption coefficient at a wavelength of 340 nm that is a predetermined value indicating β-NMNH, and exhibits high catalytic activity as a coenzyme when converted to β-NADH, and thus has high physiological activity, as well as a method for evaluating the quality and a method for determining the enzymatic reactivity of a β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof. [Brief description of the drawings]

[0012] [Figure 1A] FIG. 1A shows an HPLC elution pattern of β-NMNH of Example 1 before the conversion reaction with Nmnat in Test Example 3-1. [Figure 1B]FIG. 1B is a diagram showing an HPLC elution pattern after the conversion reaction of β-NMNH of Example 1 with Nmnat in Test Example 3-1. [Figure 2A] FIG. 2A shows the HPLC elution pattern of 1,4-dihydroNMN of Comparative Example 1 in Test Example 3-1 before the conversion reaction by Nmnat. [Figure 2B] FIG. 2B shows the HPLC elution pattern of 1,4-dihydroNMN of Comparative Example 1 after the conversion reaction by Nmnat in Test Example 3-1. [Figure 3A] FIG. 3A is a graph showing the results when lactate dehydrogenase derived from pig heart was used in Test Example 3-2. [Figure 3B] FIG. 3B is a graph showing the results when rabbit skeletal muscle-derived lactate dehydrogenase was used in Test Example 3-2. [Figure 4] FIG. 4 is a graph showing the results of Test Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] (compound) The compound of the present invention is a β-NADH precursor compound (hereinafter, sometimes referred to as "β-NMNH compound") consisting of β-NMNH or a pharmacologically acceptable salt thereof, which has a purity of 95% or more as measured by HPLC, and when converted to β-NADH by Nmnat, has a catalytic activity of 85% or more for lactate dehydrogenase that converts pyruvate to lactate. The compound of the present invention preferably has a molar absorption coefficient at a wavelength of 340 nm that is a predetermined value indicating β-NMNH.

[0014] The β-NMNH compound may be prepared by any method. For example, β-NMNH artificially synthesized by chemical synthesis, enzymatic method, or the like and purified can be used as an active ingredient. In addition, β-NMNH obtained by extraction and purification from natural raw materials such as animals, plants, and microorganisms can also be used as an active ingredient. Commercially available purified β-NMNH may also be used.

[0015] As a chemical synthesis method for synthesizing β-NMNH, for example, β-NMNH can be produced by reacting β-NMN with a chemical agent that reduces β-NMN (e.g., sodium dithionite, hyposulfite, thiourea dioxide, etc.). As an enzymatic method, for example, β-NMNH can be produced from NADH using NADH pyrophosphatase (EC 3.6.1.22).

[0016] Details of NADH pyrophosphatase are described at https: / / www.brenda-enzymes.org / enzyme.php?ecno=3.6.1.22. NADH pyrophosphatase is known to utilize both the coenzyme NAD (oxidized β-nicotinamide adenine dinucleotide) and its reduced form NADH as substrates.

[0017] The amount of NADH pyrophosphatase used is not particularly limited and can be appropriately selected, but is usually 100 to 10,000 U / L, preferably 1,000 to 5,000 U / L. In the present invention, an activity unit (U) of NADH pyrophosphatase is defined as a catalytic amount that hydrolyzes 1 μmole of NAD per minute. The reaction conditions when using the NADH pyrophosphatase are not particularly limited and can be appropriately selected.

[0018] The foregoing β-NMNH may be a pharmacologically acceptable salt. The pharmacologically acceptable salt of β-NMNH may be an inorganic acid salt or an organic acid salt having a basic site such as an amine. Examples of the acid constituting such an acid salt 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, p-toluenesulfonic acid, and the like. Further, 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 the base constituting such an acid salt include alkali metal salts or alkaline earth metal salts, and 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.

[0019] <HPLC purity> Regarding the purity of the β-NMNH 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, but 98% or more is preferable.

[0020] The HPLC purity in the present invention means the ratio of the peak area derived from NMNH to the total amount of the peak areas of various peaks detected by measuring a sample containing β-NMNH by HPLC. Specifically, it can be obtained from the following (Formula 1). -(Formula 1)- HPLC purity (%) = {(β-NMNH peak area) / (total peak area measured)} x 100

[0021] In the present invention, the HPLC analysis method for measuring the HPLC purity is not particularly limited as long as the method and conditions are such that β-NMNH can be efficiently separated and measured, and can be appropriately selected. For example, a column using Nucleosil R 4000-7 PEI (length: 12.5 cm, inner diameter: 4.0 mm, particle size: 7 μm, manufactured by Machray-Nagel) was used, and the method described in "THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 43, pp. 36334-36341, October 28, 2005" was used, or an XBrigde Amide column (length: 15 cm, inner diameter: 10.0 mm, manufactured by Waters) was used as a column, and the method described in "bioRxiv, 2020, Reduced Nicotinamide Mononucleotide (NMNH) Potently Enhances NAD +, Suppresses Glycolysis, TCA Cycle and Cell Growth, https: / / doi.org / 10.1101 / 2020.11.03.366427" was used. In the present invention, the following method was used. The HPLC system used is Prominence (Shimadzu Corporation). Dissolve the β-NMNH sample in 200 mM NaHCO3 buffer (pH 10.0) to a concentration of 1 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-NMNH 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 0.15 mL / min, and the absorbance is measured at 340 nm.

[0022] <Molar absorption coefficient at a wavelength of 340 nm> The molar absorption coefficient of the β-NMNH compound according to the present invention at a wavelength of 340 nm is preferably a predetermined value representing β-NMNH. The molar absorption coefficient of the β-NMNH compound at a wavelength of 340 nm is not particularly limited and can be appropriately selected, but is preferably 5,500 [L / (mol cm)] or more, and more preferably 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less.

[0023] The molar absorption coefficient at a wavelength of 340 nm is calculated by the following (Equation 2). For the measurement, a sample with a clear molar concentration of NMNH is prepared, and the absorbance at a wavelength of 340 nm of the sample appropriately diluted to a concentration suitable for the detector is measured in a cell with a known optical path length. -(Formula 2)- Molar absorption coefficient at a wavelength of 340 nm [L / (mol cm)] = absorbance at a wavelength of 340 nm / [molar concentration of NMNH in the measurement sample (mol / L) × cell path length (cm)]

[0024] Here, the method for clarifying the molar concentration of NMNH in the measurement sample is not particularly limited and can be appropriately selected as long as it does not utilize a method that applies the existing molar absorption coefficient of NMNH, and examples of such methods include a method of converting the NMNH molecular weight using a sample with a known NMNH content based on weight, quantitative NMR, and a method of quantitatively converting NMNH into a substance with a known molecular absorption coefficient and measuring it. In the present invention, the molar concentration was clarified by the following method. The moisture content (%) per powder weight is calculated using the Karl Fischer method. The sodium content (%) per powder weight is calculated by atomic absorption spectrometry. Calculate HPLC purity. Weigh out approximately 0.3 g of NMNH powder and record the powder weight. Dissolve in 200 mM NaHCO3 buffer (pH 10.0) and make up to 50 mL. Calculate the NMNH molar concentration in this sample using the following (Equation 3). For the molecular weight of NMNH (g / mol), use the molecular weight of anhydrous, salt-free NMNH, 336.2 g / mol. -(Formula 3)- Molar concentration of NMNH in sample (mol / L) = [A × {(100-BC) / 100} × {D / 100}] / 0.05(L) / E In the above formula, A represents the powder weight (g), B represents the water content (%), C represents the sodium content (%), D represents the HPLC purity (%), and E represents the molecular weight of NMNH (g / mol).

[0025] The method for measuring the absorbance of the measurement sample at a wavelength of 340 nm is not particularly limited and can be appropriately selected as long as the operation is performed under conditions under which the β-NMNH compound in the measurement sample is stable and the measurement is performed under conditions suitable for detection. In the present invention, the following method was used. A sample with a clear molar concentration of NMNH is diluted with 200 mM NaHCO3 buffer (pH 10.0) to approximately 0.2 mmol / L NMNH to prepare a measurement sample with a known molar concentration. The absorbance of the measurement sample at a wavelength of 340 nm is measured using a spectrophotometer UV-1800 (Shimadzu Corporation). The molar extinction coefficient at a wavelength of 340 nm is calculated using the absorbance and molar concentration of the measurement sample according to the above formula (2).

[0026] <Catalytic activity as a coenzyme when converted to β-NADH> The catalytic activity of the β-NMNH compound of the present invention against lactate dehydrogenase, which converts pyruvate to lactate when converted to β-NADH by Nmnat, is not particularly limited as long as it is 85% or more and can be appropriately selected, but is preferably 90% or more. In the present invention, the value of "catalytic activity of lactate dehydrogenase that converts pyruvate to lactic acid" refers to the relative activity value (%) when the activity of the standard substance β-NADH is taken as 100%. In the present invention, the "standard substance of β-NADH" refers to NADH that satisfies the reagent specifications in the "Recommended Method for Measurement of Enzyme Activity in Human Serum" of the Japan Society of Clinical Chemistry.

[0027] In the present invention, the catalytic activity of a β-NMNH compound with lactate dehydrogenase that converts pyruvate to lactate when the compound is converted to β-NADH by Nmnat means a value obtained by measurement as described below.

[0028] -Conversion synthesis of β-NADH from β-NMNH compounds- First, the β-NMNH compound is converted to β-NADH by Nmnat.

[0029] The enzymatic properties of Nmnat (EC 2.7.7.1) are described in https: / / www.brenda-enzymes.org / enzyme.php?ecno=2.7.7.1&Suchword=&reference=&UniProtAcc=&organism%5B%5D=. For example, human Nmnat is known to utilize NMNH as a substrate (Biochemistry 2007, 46, 16, 4912-4922). The Nmnat is preferably a heat-stable Nmnat (Thermococcus kodakaraensis TK0067 gene, see SEQ ID NO: 3 for the amino acid sequence). The heat-stable Nmnat may be expressed in Escherichia coli or the like.

[0030] The reaction using Nmnat can be carried out by the method described in the Examples section below. As shown in the Examples section below, by using Nmnat, β-NADH can be synthesized from β-NMNH compound.

[0031] -Catalytic activity- The catalytic activity of β-NADH converted by Nmnat with lactate dehydrogenase, which converts pyruvate to lactate, can be measured as follows. --Nmnat conversion mixture-- · Equivalent to 3mM NADH 100mM NaHCO3 buffer (pH 8.5) 20mM ATP 5mM MgCl2 --R1 test solution-- Dilute 0.15 mL of the Nmnat conversion mixture to 0.15 mM NADH equivalent with 4.85 mL of 0.1 M sodium phosphate buffer (pH 7.0) containing 0.02% NaN3. As a control (standard substance), an R1 test solution using β-NADH powder (manufactured by Oriental Yeast Co., Ltd.) is prepared using the same procedure. --R3 test solution-- Dissolve sodium pyruvate in purified water to a concentration of 5 mM. --Enzyme solution-- Prepare an enzyme solution so that the final concentration of lactate dehydrogenase (hereinafter sometimes referred to as "LDH") in the reaction solution is 5 U / mL. The diluent is 0.1 M potassium phosphate buffer (pH 7.0) containing 0.5% BSA and 0.02% NaN3. 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 according to the IFCC is used.

[0032] --Activity measurement-- Coenzyme activity was measured using a Hitachi 7170 automatic analyzer (Hitachi High-Tech Corp.). The measurement parameters were as follows: [Measurement parameters] · Measurement temperature ··· 37℃ · Measurement wavelength (secondary / main) ··· 405nm / 340nm Measurement mode 2 point rate

[10]

[18]

[20] [0][0] Sample volume (volume of enzyme solution) 4μL R1 test solution volume: 160 μL (equivalent to 0.15 mM NADH) R3 test solution volume: 40μL Start-up calibration: Yes (purified water)

[0033] [Measurement procedure] 4 μL of enzyme solution and 160 μL of R1 test solution are mixed and incubated at 37°C for 4.5 minutes (photometric points 1-16), after which 40 μL of R3 test solution is added to start the reaction (photometric point 17). Measurements are performed for 5 minutes after the start of the reaction, and the absorbance of the measurement sample at the same photometric points from 0.3-0.9 minutes (photometric points 18-20) from the start of the reaction is subtracted from the absorbance of water (blank) at the same photometric points to calculate the change in absorbance per minute (ΔmAbs / min).

[0034] In the present invention, LDH is a dehydrogenase also known as L-lactate dehydrogenase (EC 1.1.1.27). The lactate dehydrogenase is preferably mammalian heart-derived lactate dehydrogenase, more preferably porcine lactate dehydrogenase (LDH) 1 having the amino acid sequence of SEQ ID NO: 2, but other mammalian lactate dehydrogenases, such as skeletal muscle-derived lactate dehydrogenase (LDH) 5 consisting of a tetramer of subunits having the amino acid sequence of SEQ ID NO: 1, human lactate dehydrogenase (LDH) 1, and human lactate dehydrogenase (LDH) 5, can also be used. LDH catalyzes a reaction using pyruvate as a substrate to convert it to lactate, and at this time NADH is converted to NAD.

[0035] It is preferable that the β-NMNH compound according to the present invention does not substantially contain 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-NMNH compound is not detected by the above-mentioned HPLC analysis. Note that this does not exclude an embodiment in which NAD is contained in a preparation or food or drink containing the β-NMNH compound according to the present invention.

[0036] The β-NMNH compound of the present invention not only has a high purity as measured by HPLC, but also exhibits high catalytic activity as a coenzyme when converted to β-NADH. Therefore, it has high bioavailability compared to conventional β-NMNH products (drug substances), and is of high quality as β-NMNH 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-NMNH 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 high effects.

[0037] (Method of evaluating the quality of compounds) The method for evaluating the quality of a compound of the present invention is a method for evaluating the quality of a β-NADH precursor compound consisting of β-NMNH 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.

[0038] <Evaluation process> The evaluation step in the quality evaluation method of the present invention is a step of evaluating the quality of a β-NADH precursor compound consisting of the β-NMNH or a pharmacologically acceptable salt thereof, using as indicators a purity of 95% or more as measured by HPLC and a catalytic activity of 85% or more for lactate dehydrogenase, which converts pyruvate to lactate when converted to β-NADH by Nmnat. 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 catalytic activity of lactate dehydrogenase that converts pyruvic acid to lactic acid is not particularly limited and may be appropriately selected as long as it is 85% or more, but is preferably 90% or more. In the evaluation step, it is preferable that the molar absorption coefficient at a wavelength of 340 nm is a predetermined value indicating β-NMNH as an index. The molar absorption coefficient at a wavelength of 340 nm as the index is not particularly limited and can be appropriately selected, but is preferably 5,500 [L / (mol cm)] or more, and more preferably 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less.

[0039] -evaluation- In the evaluation step in the quality evaluation method of the present invention, when the β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof to be evaluated has a purity of 95% or more as measured by HPLC, and when converted to β-NADH by Nmnat, the catalytic activity of the lactate dehydrogenase that converts pyruvate to lactate is 85% or more, the quality of the evaluation object is evaluated as good. More specifically, when the purity is 95% or more as measured by HPLC, and when converted to β-NADH by Nmnat, the catalytic activity of the lactate dehydrogenase that converts pyruvate to lactate is 85% or more, the evaluation object is evaluated as a compound consisting of β-NMNH or a pharmacologically acceptable salt thereof with high bioavailability. In addition, when the molar absorption coefficient at a wavelength of 340 nm is a predetermined value indicating β-NMNH, the quality of the evaluation object is evaluated as good when the predetermined value is used as the index. More specifically, when it is the above-mentioned predetermined value, the evaluation subject is evaluated as being a compound consisting of β-NMNH or a pharmacologically acceptable salt thereof having high bioavailability. The purity as measured by the HPLC, the catalytic activity for lactate dehydrogenase that converts pyruvate to lactate when converted to β-NADH by Nmnat, and the molar absorption coefficient at a wavelength of 340 nm may be measured when carrying out the quality evaluation method of the compound of the present invention, or may be measured separately.

[0040] <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.

[0041] -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 when measured by HPLC of a β-NADH precursor compound composed of β-NMNH to be evaluated or a pharmacologically acceptable salt thereof, and the catalytic activity against lactate dehydrogenase that converts pyruvic acid to lactic acid when converted to β-NADH by Nmnat. In the said measurement step, you may measure the molar extinction coefficient at a wavelength of 340 nm further as needed. The purity when measured by the said HPLC, the catalytic activity against lactate dehydrogenase that converts pyruvic acid to lactic acid when converted to β-NADH by Nmnat, and the molar extinction coefficient at a wavelength of 340 nm can be measured in the same manner as those described in the items of <HPLC purity>, <Catalytic activity as a coenzyme when converted to β-NADH>, and <Molar extinction coefficient at a wavelength of 340 nm> of the above-mentioned (compound).

[0042] (Method for determining the enzymatic reactivity of a compound) The method for determining the enzymatic reactivity of the compound of the present invention is a method for determining the enzymatic reactivity of a β-NADH precursor compound composed of β-NMNH or a pharmacologically acceptable salt thereof, and includes at least a determination step, and further includes other steps as needed.

[0043] <Determination step> The determination step in the method for determining the enzymatic reactivity of the compound of the present invention is a step of determining the reactivity of the β-NADH precursor compound composed of β-NMNH or a pharmacologically acceptable salt thereof against lactate dehydrogenase, using as an index that the purity when measured by HPLC is 95% or more and the catalytic activity against lactate dehydrogenase that converts pyruvic acid to lactic acid when converted to β-NADH by Nmnat is 85% 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 catalytic activity of lactate dehydrogenase that converts pyruvic acid to lactic acid is not particularly limited and may be appropriately selected as long as it is 85% or more, but is preferably 90% or more. In the determination step, it is preferable that the molar absorption coefficient at a wavelength of 340 nm is a predetermined value indicating β-NMNH as an index. The molar absorption coefficient at a wavelength of 340 nm as the index is not particularly limited and can be appropriately selected, but is preferably 5,500 [L / (mol cm)] or more, and more preferably 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less.

[0044] -judgement- In the determination step in the method for determining the enzymatic reactivity of a compound of the present invention, when the β-NADH precursor compound to be determined, which is composed of β-NMNH or a pharmacologically acceptable salt thereof, has a purity of 95% or more as measured by HPLC, and when converted to β-NADH by Nmnat, has a catalytic activity of 85% or more against lactate dehydrogenase that converts pyruvate to lactate, the enzymatic reactivity of the compound to be determined is high. More specifically, when the purity is 95% or more as measured by HPLC, and when converted to β-NADH by Nmnat, has a catalytic activity of 85% or more against lactate dehydrogenase that converts pyruvate to lactate, the compound to be determined is determined to be a reduced β-nicotinamide mononucleotide or a reduced β-nicotinamide adenine dinucleotide precursor compound composed of a pharmacologically acceptable salt thereof that has a high reactivity against lactate dehydrogenase. In addition, when the molar extinction coefficient at a wavelength of 340 nm is a predetermined value indicating β-NMNH as the index, the compound to be determined is evaluated to have a high enzymatic reactivity when it is the predetermined value. More specifically, if the value is the specified value, the subject is evaluated as a reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, which has high reactivity to lactate dehydrogenase. The purity as measured by the HPLC, the catalytic activity for lactate dehydrogenase that converts pyruvate to lactate when converted to β-NADH by Nmnat, and the molar absorption coefficient at a wavelength of 340 nm may be measured when carrying out the method for determining the enzymatic reactivity of the compound of the present invention, or may be measured separately.

[0045] <Other processes> Other steps in the method for determining the enzymatic reactivity of a compound of the present invention are not particularly limited and can be appropriately selected as long as they do not impair the effects of the present invention. For example, a measurement step can be mentioned.

[0046] -Measurement process- In the measurement step in the method for determining the enzymatic reactivity of the compound of the present invention, the purity when measured by HPLC of a β-NADH precursor compound consisting of β-NMNH to be evaluated or a pharmacologically acceptable salt thereof, and when converted to β-NADH by Nmnat, the catalytic activity against lactate dehydrogenase that converts pyruvic acid to lactic acid is measured. In the said measurement step, you may measure the molar extinction coefficient at a wavelength of 340 nm further as needed. The purity when measured by the said HPLC, the catalytic activity against lactate dehydrogenase that converts pyruvic acid to lactic acid when converted to β-NADH by Nmnat, and the molar extinction coefficient at a wavelength of 340 nm can be measured in the same manner as described in the items of <HPLC purity> of the above-mentioned (compound), <catalytic activity as a coenzyme when converted to β-NADH>, and <molar extinction coefficient at a wavelength of 340 nm>.

[0047] 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 a β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof, and thereby provide a high-quality β-NADH precursor compound consisting of β-NMNH or a pharmacologically acceptable salt thereof.

[0048] Furthermore, the present invention also includes a method for producing a high-quality β-NADH precursor compound consisting of β-NMNH 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.

Example

[0049] Hereinafter, the present invention will be described by showing Examples, Comparative Examples, and Test Examples, but the present invention is not limited to these at all.

[0050] (Example 1: Preparation of β-NMNH compound powder) Commercially available NADH powder (manufactured by Oriental Yeast Co., Ltd.) was used as the starting material, and β-NMNH was produced using a recombinant NADH pyrophosphatase (SEQ ID NO: 4) prepared in-house. The specific reaction composition and conditions are as follows. Reaction temperature: 37℃ pH: pH8.0~9.0 (0.1M NaHCO3-NaOH buffer) Substrate NADH concentration: 20g powder per liter NADH pyrophosphatase added: 4,000U / L

[0051] <Monitoring the production of β-NMNH and terminating the reaction> The production of β-NMNH was monitored by reversed-phase chromatography using an ODS80Ts column (Tosoh Corporation). Under the following HPLC operating conditions, β-NMNH was eluted at the 7th minute, and NADH at the 60th minute. When the NADH peak completely disappeared, the reaction was stopped by removing the added enzyme using an ultrafiltration membrane with a molecular weight cutoff of 10,000. β-NMNH was collected in the external solution during the ultrafiltration process. -HPLC conditions- C18 reversed phase column size: TSKgel ODS80Ts (inner diameter 2mm, length 15cm, particle size 5μm) · Flow rate: 0.15mL / min Measurement wavelength: 340nm and 260nm · Elution conditions: A solution) 50mM Tris-Acetate buffer (pH7.5) B) Solution A above containing 15% methanol Gradient program: 0-20 minutes; A liquid 100% - B liquid 0% 20-60 min; A / B gradient 60-80 minutes; Part A 0% - Part B 100%

[0052] <Purification from β-NMNH mixture> β-NMNH was purified by column chromatography using ion exchange resin. HPA25 (manufactured by Mitsubishi Chemical Corporation) resin was charged at a ratio of 10 to 20 mL of the reaction mixture per 1 mL, and after thorough washing with equilibration buffer, gradient elution was performed with 0 to 0.2 M NaCl (pH 9.0). β-NMNH was collected at the elution position of 50 to 100 mM NaCl. -HPA25 resin chromatography conditions- Operating temperature: Refrigerated Equilibration buffer: 10mM NaHCO3-NaOH (pH 9.0) Elution buffer: The above NaHCO3-NaOH buffer containing 0.2M NaCl Elution procedure: Gradient

[0053] After collecting the purified main fraction, excess NaCl was removed by electrodialysis, and the primary concentration was performed by freeze-drying. The resulting yellowish powder was redissolved in a small amount of purified water, and the final powder was adjusted to 75% by weight as β-NMNH (anhydrous and salt-free), and the final vial freeze-drying was performed. Approximately 2 g of β-NMNH compound powder was obtained from 20 g of NADH powder.

[0054] (Comparative Example 1: Preparation of β-NMNH compound powder by chemical treatment) As a chemical agent to reduce β-NMN, sodium dithionite (Na2S2O4) was used in accordance with the reference (ChemRxiv, 2019, NADH in Solution: A Single State, https: / / chemrxiv.org / articles / preprint / NADH_in_Solution_A_Single_State / 8208407 / 1) to prepare the β-NMNH compound (hereinafter sometimes referred to as "1,4-dihydroNMN"). After confirming the increase in absorbance at 340 nm under the chemical reaction conditions below, we attempted to separate and purify 1,4-dihydroNMN in the same manner as described in Example 1. <Composition of chemical reaction mixture (per liter)> β-NMN powder (manufactured by Oriental Yeast Co., Ltd.) 5g Sodium dithionite powder 5g 35g NaHCO 10g Na2CO3

[0055] Specifically, β-NMN powder was dissolved in the above-mentioned alkaline solution of sodium dithionite and allowed to stand at room temperature for 30 minutes or more while shielded from light. The reduced solution, which showed a stable increase in absorbance at 340 nm and was pale yellow in color, was subjected to column chromatography using HPA25 ion exchange resin to isolate 1,4-dihydroNMN.

[0056] (Test Example 1: Measurement of HPLC Purity) The HPLC purity of the β-NMNH compounds produced in Example 1 and Comparative Example 1 was measured by the following method. The results are shown in Table 1 below. The measurement results are the average value of three measurements.

[0057] [HPLC Purity Measurement] The HPLC system used was Prominence (Shimadzu Corporation). The β-NMNH sample was dissolved in 200 mM NaHCO3 buffer (pH 10.0) to a concentration of 1 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). The column-adsorbed β-NMNH fraction was separated as follows: As the eluent, 50 mM Tris-acetate (pH 7.5) / methanol was used, and elution and separation were performed with a concentration gradient of 0-15% methanol adjusted to a flow rate of 0.15 mL / min, and absorbance was measured at 340 nm.

[0058] [Table 1]

[0059] In addition, in both of the β-NMNH compounds produced in Example 1 and Comparative Example 1, no NAD peak was observed by HPLC, and they were found to contain substantially no NAD.

[0060] (Test Example 2: Calculation of molar extinction coefficient) The molar absorption coefficients at a wavelength of 340 nm were calculated by the following method for the three lots of β-NMNH compound produced by the method of Example 1. The results are shown in Table 2 below.

[0061] [Calculation of Molar Absorption Coefficient] A sample with a clear molar concentration of NMNH was prepared, and the absorbance of the sample appropriately diluted to a concentration suitable for the detector at a wavelength of 340 nm was measured in a cell with a known path length, and the molar absorption coefficient at a wavelength of 340 nm was calculated using the above-mentioned (Equation 2). The samples for clarifying the molar concentration of NMNH were prepared as follows. The moisture content (%) per powder weight was calculated using the Karl Fischer method. The sodium content (%) per powder weight was calculated by atomic absorption spectrometry. - HPLC purity was calculated. Approximately 0.3 g of NMNH powder was weighed out and the powder weight was recorded. It was dissolved in 200 mM NaHCO3 buffer (pH 10.0) and made up to 50 mL. The molar concentration of NMNH in this sample was calculated using the above formula (3). The absorbance of the measurement sample at a wavelength of 340 nm was measured as follows. A sample with a clear molar concentration of NMNH was diluted with 200 mM NaHCO3 buffer (pH 10.0) to approximately 0.2 mmol / L NMNH to prepare a measurement sample with a known molar concentration. The absorbance of the measurement sample at a wavelength of 340 nm was measured using a spectrophotometer UV-1800 (Shimadzu Corporation). The molar absorption coefficient at a wavelength of 340 nm was calculated from the absorbance and molar concentration of the measurement sample using the above-mentioned (Equation 2).

[0062] [Table 2]

[0063] (Test Example 3) <Test Example 3-1: Conversion and synthesis of β-NADH from β-NMNH compound> The β-NMNH compounds produced in Example 1 and Comparative Example 1 were reacted with Nmnat to perform conversion and synthesis of β-NADH in the presence of ATP. In this test example, the Nmnat used was a heat-stable Nmnat expressed in Escherichia coli (Thermococcus kodakaraensis TK0067 gene; see SEQ ID NO: 3 for the amino acid sequence), which recognizes and acts on both β-NMNH and its chemically reduced derivative, 1,4-dihydroNMN, as substrates.

[0064] The composition of the reaction solution for the Nmnat conversion reaction is as follows. 100mM NaHCO3 buffer (pH 8.5) 5mM MgCl2 20mM ATP · 50U / L heat resistance Nmnat The activity unit (1U) of Nmnat was defined as the catalytic amount that produces 1 μmole of NAD from β-NMN per minute in the presence of ATP. 3 mM β-NMNH (Example 1) or 1,4-dihydroNMN (Comparative Example 1) The molar concentrations of β-NMNH and 1,4-dihydroNMN were determined using a molar absorption coefficient of 6,500 [L / (mol cm)] at a wavelength of 340 nm (the value reported in the reference literature described in Comparative Example 1).

[0065] The state of the Nmnat conversion reaction after standing overnight at 37°C was confirmed by comparing the HPLC elution patterns. The HPLC conditions were as follows. -HPLC conditions- The HPLC system used was Prominence (Shimadzu Corporation). The sample was dissolved in 200 mM NaHCO3 buffer (pH 10.0) to a total of approximately 1 mM equivalent to NMNH and NADH, 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). The column-adsorbed β-NMNH fraction, NADH, or NADH derivatives were separated as follows: The eluent was 50 mM Tris-acetate (pH 7.5) / methanol, and the concentration gradient was 0-15% methanol. The flow rate was adjusted to 0.15 mL / min. The absorbance was measured at 340 nm. The β-NMNH fraction was eluted at 7 min, and NADH or NADH derivatives at 60 min.

[0066] The HPLC elution pattern of β-NMNH in Example 1 before the Nmnat conversion reaction is shown in Figure 1A, and the HPLC elution pattern after the Nmnat conversion reaction is shown in Figure 1B. The HPLC elution pattern of 1,4-dihydroNMN in Comparative Example 1 before the Nmnat conversion reaction is shown in Figure 2A, and the HPLC elution pattern after the Nmnat conversion reaction is shown in Figure 2B. As shown in Figures 1A-2B, the elution retention times of the NADH precursor and the converted NADH and its derivatives were the same between the two. In addition, in both cases, the NADH precursor was converted to NADH or its derivatives at almost 100%, and it was confirmed that there was no difference between the two in the Nmnat conversion reaction.

[0067] <Test Example 3-2: Comparison of NADH precursor ability from the viewpoint of coenzyme activity responsiveness to LDH> -Conversion synthesis of β-NADH from β-NMNH compounds- In the same manner as in Test Example 3-1, the β-NMNH compounds produced in Example 1 and Comparative Example 1 were converted to β-NADH. The reaction was terminated by ultrafiltration with a molecular weight cutoff of 10,000.

[0068] -Evaluation of coenzyme activity response to LDH- The filtrate (hereinafter sometimes referred to as Nmnat conversion mixture) was used as is to examine the response of the converted NADH and NADH derivatives to LDH activity as described below.

[0069] [Evaluation] -Nmnat conversion mixture- · Equivalent to 3mM NADH 100mM NaHCO3 buffer (pH 8.5) 20mM ATP 5mM MgCl2 -R1 test liquid- 0.15 mL of the Nmnat conversion mixture was diluted with 4.85 mL of 0.1 M sodium phosphate buffer (pH 7.0) containing 0.02% NaN3 to the equivalent of 0.15 mM NADH. As a control (standard substance), an R1 test solution using β-NADH powder (manufactured by Oriental Yeast Co., Ltd.) was prepared using the same procedure. -R3 test liquid- Sodium pyruvate was dissolved in pure water to a concentration of 5 mM. -Enzyme solution- The enzyme solution was prepared so that the final concentration of LDH in the reaction mixture was 0, 0.5, 1, 2, 5, or 10 U / mL. The dilution solution was 0.1 M potassium phosphate buffer (pH 7.0) containing 0.5% BSA and 0.02% NaN3. As the LDH, recombinant LDH (derived from pig heart, SEQ ID NO: 2) or recombinant LDH (derived from rabbit skeletal muscle, SEQ ID NO: 1) was used.

[0070] -Activity measurement- Coenzyme activity was measured using a Hitachi 7170 automatic analyzer (Hitachi High-Tech Corp.). The measurement parameters are as follows: --Measurement parameters-- · Measurement temperature ··· 37℃ · Measurement wavelength (secondary / main) ··· 405nm / 340nm Measurement mode 2 point rate

[10]

[18]

[20] [0][0] Sample volume (volume of enzyme solution) 4μL R1 test solution volume: 160 μL (equivalent to 0.15 mM NADH) R3 test solution volume: 40μL Start-up calibration: Yes (purified water)

[0071] --Measurement procedure-- 4 μL of the enzyme solution and 160 μL of the R1 test solution were mixed and incubated at 37°C for 4.5 minutes (photometric points 1-16), after which 40 μL of the R3 test solution 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 0.3-0.9 minutes (photometric points 18-20) from the start of the reaction was subtracted from the absorbance of water (blank) at the same photometric points to calculate the change in absorbance per minute (ΔmAbs / min).

[0072] The relative activity value (%) was calculated for each case when the activity value of the β-NADH standard substance was set to 100% when an enzyme solution prepared to have a final concentration in the reaction solution of 5 U / mL was used, and the results are shown in Figure 3A (when pig heart-derived LDH was used) and 3B (when rabbit skeletal muscle-derived LDH was used) (○: control, ●: Example 1, △: Comparative Example 1). Furthermore, the relative activity values ​​when an enzyme solution prepared to have a final concentration in the reaction solution of 5 U / mL was used and when β-NADH converted from the β-NMNH compound produced in Example 1 was used and when β-NADH converted from 1,4-dihydroNMN produced in Comparative Example 1 was used are shown in Table 3. The measurement results are the average of measurements performed three times.

[0073] [Table 3]

[0074] As shown in FIGS. 3A and 3B and Table 3, the β-NMNH compound produced in Example 1 of the present invention exhibited higher catalytic activity as a coenzyme when converted to β-NADH than 1,4-dihydroNMN produced in Comparative Example 1, and was confirmed to have excellent NADH precursor activity.

[0075] (Test Example 4: Effect of β-NMNH compound on Parkinson's disease cell model based on rotenone-induced cell death) The biological activities of the β-NMNH compounds prepared in Example 1 and Comparative Example 1 were evaluated as follows.

[0076] Starting from human neural stem cells (SH-SY5Y), we induced differentiation of human neural cells with retinoic acid and phorbol 12-myristate-13 acetate, and determined the degree of inhibition of rotenone-induced cell death using the amount of intracellular ATP as an index. The procedure is shown below.

[0077] <Day 0> 10 viable cells per well 4 SH-SY5Y cells were seeded onto a 96-well plate so that each well became 1 cell.

[0078] <Day 1> The medium was replaced with medium supplemented with 10 μM retinoic acid.

[0079] <Day 4> Phorbol 12-myristate-13 acetate was added to the medium to a final concentration of 80 μM to induce differentiation.

[0080] <Day 7> The medium was replaced with a medium containing 100 μM rotenone to induce cell death. At this time, β-NMN (oxidized NMN, control) or the β-NMNH compound produced in Example 1 or Comparative Example 1 was added to the medium at various concentrations (0, 5, 50, or 500 μM).

[0081] <Day 9> The culture supernatant was removed, and the amount of ATP in the adherent cells was measured using a commercially available cell ATP measurement reagent (Toyo B-Net, CAT NO. CA2-100). It was previously confirmed from a calibration curve using an ATP standard solution that the amount of ATP in the cells was directly proportional to the amount of luminescence obtained using a luminescence plate reader based on the above measurement reagent.

[0082] The medium composition used in this test is as follows. For cell proliferation F12 / E-MEM + 1% NEAA (non-essential amino acids) + 15% FBS (fetal bovine serum) For differentiation induction and rotenone induction assays F12 / E-MEM + 1% NEAA + 1% N2 + 1mg / mL HSA (human serum albumin)

[0083] The results are shown in Figure 4. In Figure 4, the average of the measured values ​​(n = 3) is shown by a vertical bar, and the SD (standard deviation) is shown by an error bar. As shown in Figure 4, it was confirmed that the use of the β-NMNH compound of Example 1 showed a higher degree of inhibition of rotenone-induced cell death than the β-NMNH compound of Comparative Example 1. Furthermore, when added at 500 μM, the β-NMNH compound of Example 1 gave better results than the control oxidized β-NMN.

Claims

1. A composition comprising a reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, characterized in that the purity of the reduced β-nicotinamide adenine dinucleotide precursor compound when the composition is measured by HPLC is 95% or more and less than 100%, the molar absorption coefficient of the reduced β-nicotinamide adenine dinucleotide precursor compound at a wavelength of 340 nm is 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less, and when the composition is treated with nicotinamide mononucleotide adenine transferase (Nmnat) to convert the reduced β-nicotinamide adenine dinucleotide precursor compound to reduced β-nicotinamide adenine dinucleotide (β-NADH), the catalytic activity of lactate dehydrogenase, which converts pyruvate to lactic acid, is 85% or more.

2. The composition described in claim 1, wherein the purity of the reduced β-nicotinamide adenine dinucleotide precursor compound is 98% or more but less than 100% when the composition is measured by HPLC, and the catalytic activity of the composition against lactate dehydrogenase is 90% or more.

3. The composition described in claim 1 or 2, wherein the lactate dehydrogenase is lactate dehydrogenase derived from mammalian heart.

4. The composition described in claim 3, wherein the lactate dehydrogenase has the amino acid sequence of SEQ ID NO:

2.

5. A composition described in any of claims 1 to 4, wherein the Nmnat is a heat-resistant Nmnat (Thermococcus kodakaraensis TK0067 gene).

6. A composition described in 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 reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, comprising: The method comprises: evaluating the quality of the composition using as indicators the purity of the reduced β-nicotinamide adenine dinucleotide precursor compound being 95% or more when the composition is measured by HPLC, the molar absorption coefficient of the reduced β-nicotinamide adenine dinucleotide precursor compound being 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less at a wavelength of 340 nm, and the catalytic activity of lactate dehydrogenase, which converts pyruvate to lactic acid, being 85% or more when the composition is treated with nicotinamide mononucleotide adenine dinucleotide adenine dinucleotide (Nmnat) to convert the reduced β-nicotinamide adenine dinucleotide precursor compound to reduced β-nicotinamide adenine dinucleotide (β-NADH).

8. A method for determining the enzymatic reactivity of a composition containing a reduced β-nicotinamide adenine dinucleotide precursor compound consisting of reduced β-nicotinamide mononucleotide or a pharmacologically acceptable salt thereof, comprising: The method comprises: determining the reactivity of the composition with lactate dehydrogenase using as an index the purity of the reduced β-nicotinamide adenine dinucleotide precursor compound is 95% or more when the composition is measured by HPLC; the molar absorption coefficient of the reduced β-nicotinamide adenine dinucleotide precursor compound is 6,000 [L / (mol cm)] or more and 7,000 [L / (mol cm)] or less at a wavelength of 340 nm; and when the composition is treated with nicotinamide mononucleotide adenylyltransferase (Nmnat) to convert the reduced β-nicotinamide adenine dinucleotide precursor compound to reduced β-nicotinamide adenine dinucleotide (β-NADH), the catalytic activity of the reduced β-nicotinamide adenine dinucleotide precursor compound with lactate dehydrogenase, which converts pyruvate to lactate, is 85% or more.

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