Method for measuring concentration of 1-methylnicotinamide, kit for measuring concentration of 1-methylnicotinamide, reagent for measuring concentration of 1-methylnicotinamide, and screening method

Abstract

Disclosed is a method for measuring the concentration of 1-methylnicotinamide contained in a sample, the method including: a step (S1) for mixing the sample with a water-soluble pillar[6]arene to obtain a mixture; and a step (S2) for measuring the fluorescence intensity of the mixture. The water-soluble pillar[6]arene has an anionic group that is stronger than a carboxy group, or a salt thereof.

Description

Method for measuring 1-methylnicotinamide concentration, kit for measuring 1-methylnicotinamide concentration, reagent for measuring 1-methylnicotinamide concentration, and screening method

[0001] The present invention relates to a method for measuring 1-methylnicotinamide concentration, a kit for measuring 1-methylnicotinamide concentration, a reagent for measuring 1-methylnicotinamide concentration, and a screening method. This application claims priority to Japanese Patent Application No. 2024-089230, filed on May 31, 2024, the contents of which are incorporated herein by reference.

[0002] Conventionally, methods for quantifying 1-methylnicotinamide (1-MNA), which is obtained by metabolizing nicotinamide with NNMT (nicotinamide N-methyltransferase), in a sample include, for example, high performance liquid chromatography and liquid chromatography mass spectrometry.

[0003] Furthermore, Patent Document 1 discloses a method for measuring the concentration of 1-methylnicotinamide in a sample by mixing a water-soluble pillar[6]arene having a carboxy group with the sample containing 1-methylnicotinamide and measuring the fluorescence intensity.

[0004] In the method described in Patent Document 1, the concentration of 1-methylnicotinamide in a sample is measured from the fluorescence intensity, utilizing the fact that the fluorescence of the water-soluble pillar[6]arene is quenched when 1-methylnicotinamide enters the inside of the cyclic structure of the water-soluble pillar[6]arene. Therefore, the concentration of 1-methylnicotinamide can be easily measured without performing complicated procedures such as liquid chromatography mass spectrometry.

[0005] Japanese Patent Application Laid-Open No. 2022-065544

[0006] However, the method described in Patent Document 1, which uses a water-soluble pillar[6]arene having a carboxy group, has large variations in the measured fluorescence intensity and low sensitivity, leaving room for improvement in the measurement accuracy of the concentration of 1-methylnicotinamide.

[0007] In view of the above circumstances, one object of the present invention is to provide a method for measuring 1-methylnicotinamide concentration, a reagent for measuring 1-methylnicotinamide concentration, and a kit for measuring 1-methylnicotinamide concentration, which enable simple and accurate measurement of the concentration of 1-methylnicotinamide in a sample. Another object of the present invention is to provide a novel method for screening NNMT inhibitors.

[0008] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0009] [1] A method for measuring the concentration of 1-methylnicotinamide contained in a sample, comprising the steps of: mixing the sample with a water-soluble pillar[6]arene to obtain a mixture; and measuring the fluorescence intensity of the mixture, wherein the water-soluble pillar[6]arene has an anionic group or a salt thereof that is stronger than a carboxy group.

[0010] [2] The method for measuring 1-methylnicotinamide concentration according to claim 1, wherein the water-soluble pillar[6]arene has a cyclic structure in which six benzene rings are bonded via methylene groups.

[0011] [3] The method for measuring 1-methylnicotinamide concentration according to claim 2, wherein the water-soluble pillar[6]arene is a compound represented by the following general formula (1) or a salt thereof: (In the general formula (1), X is the anionic group.)

[0012] [4] The method for measuring 1-methylnicotinamide concentration according to any one of [1] to [3], wherein the anionic group is a sulfonic acid group or a phosphonic acid group.

[0013] [5] The method for measuring a 1-methylnicotinamide concentration according to any one of [1] to [4], wherein the sample is a biological sample.

[0014] [6.] The method for measuring the concentration of 1-methylnicotinamide according to [5], wherein the biological sample is urine.

[0015] [7] The method for measuring the concentration of 1-methylnicotinamide according to any one of [1] to [6.], further comprising a step of purifying the sample or the mixture prior to the step of measuring the fluorescence intensity.

[0016] [8] A kit for measuring the concentration of 1-methylnicotinamide, comprising a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof.

[0017] [9] The kit for measuring 1-methylnicotinamide concentration according to [8], wherein the water-soluble pillar[6]arene has a cyclic structure in which six benzene rings are bonded via methylene groups.

[0018]

[10] The kit for measuring 1-methylnicotinamide concentration according to [8] or [9], wherein the water-soluble pillar[6]arene is a compound represented by the following general formula (1) or a salt thereof:

[0019] (In the general formula (1), X is the anionic group.)

[0020]

[11] The kit for measuring 1-methylnicotinamide concentration according to any one of [8] to

[10] , wherein the anionic group is a sulfonic acid group or a phosphonic acid group.

[0021]

[12] The kit for measuring 1-methylnicotinamide concentration according to any one of [8] to

[11] , further comprising a column for adsorbing a hydrophobic compound.

[0022]

[13] A method for screening for an NNMT inhibitor, comprising the steps of: contacting nicotinamide methyltransferase (NNMT) with a candidate substance for an NNMT inhibitor in the presence of S-adenosylmethionine and nicotinamide to obtain a sample; obtaining a mixture of the sample obtained by the sample obtaining step and a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof; and measuring the fluorescence intensity of the mixture.

[0023]

[14] A method for screening for an NNMT activator, comprising the steps of: contacting nicotinamide methyltransferase (NNMT) with a candidate substance for an NNMT activator in the presence of S-adenosylmethionine and nicotinamide to obtain a sample; obtaining a mixture of the sample obtained by the sample obtaining step and a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof; and measuring the fluorescence intensity of the mixture.

[0024]

[15] A reagent for measuring the concentration of 1-methylnicotinamide, comprising a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof.

[0025]

[16] A measurement reagent for use in the method for measuring the concentration of 1-methylnicotinamide according to any one of [1] to [7], comprising a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof.

[0026] The present invention also has the following aspects.

[0027] [1A] A method for estimating changes in blood concentration of 1-methylnicotinamide over time, comprising a step of measuring the concentrations of 1-methylnicotinamide contained in multiple random urine samples from a mammal excreted at different times.

[0028] [2A] The method for estimating changes in blood concentration of 1-methylnicotinamide over time according to [1A], wherein the random urine sample is a random urine sample excreted from a mammal after ingesting at least one compound selected from the group consisting of nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide riboside, and tryptophan.

[0029] [3A] The method for estimating changes in blood concentration of 1-methylnicotinamide over time according to [1A] or [2A], further comprising a step of purifying the random urine sample prior to the step of measuring the concentration of 1-methylnicotinamide.

[0030] [4A] The method for estimating changes in blood concentration of 1-methylnicotinamide over time according to any one of [1A] to [3A], wherein the random urine sample is urine that has been stored at room temperature of 10 to 25°C after excretion, or urine that has been frozen and stored after excretion.

[0031] [5A] The method for estimating a change in blood concentration of 1-methylnicotinamide over time according to any one of [1A] to [4A], wherein the concentration of 1-methylnicotinamide is a concentration corrected by the value of the creatinine concentration of the random urine sample.

[0032] [6A] The method for estimating changes in blood concentration of 1-methylnicotinamide over time according to any one of [1A] to [5A], wherein the step of measuring the concentration of 1-methylnicotinamide includes the steps of mixing the random urine sample with a water-soluble pillar[6]arene to obtain a mixture, and measuring the fluorescence intensity of the mixture.

[0033] [7A] The method for estimating the change in blood concentration of 1-methylnicotinamide over time according to any one of [1A] to [6A], wherein the water-soluble pillar[6]arene has an anionic group stronger than a carboxy group or a salt thereof.

[0034] According to the present invention, it is possible to provide a method for measuring the concentration of 1-methylnicotinamide, which can simply and accurately measure the concentration of 1-methylnicotinamide in a sample, a reagent for measuring the concentration of 1-methylnicotinamide, and a kit for measuring the concentration of 1-methylnicotinamide. Furthermore, according to the present invention, it is possible to provide a novel method for screening for NNMT inhibitors.

[0035] 1 is a flow chart showing a method for measuring 1-methylnicotinamide concentration according to a preferred embodiment of the present invention.

[0023] FIG. 1 is a schematic longitudinal cross-sectional view showing an example of a column included in a kit for measuring 1-methylnicotinamide concentration.

[0024] FIG. 1 is a graph showing the fluorescence intensity at each concentration of 1-methylnicotinamide in an aqueous solution containing a water-soluble pillar[6]arene having a carboxy group and 1-methylnicotinamide.

[0025] FIG. 1 is a graph showing the fluorescence intensity at each concentration of 1-methylnicotinamide in an aqueous solution containing a water-soluble pillar[6]arene having a sulfonic acid group and 1-methylnicotinamide.

[0026] FIG. 1 is a graph showing the fluorescence intensity of a mixture of urine from a wild-type mouse, urine from an NNMT knockout mouse, and urine from a wild-type mouse that drank water containing nicotinamide with a water-soluble pillar[6]arene having a sulfonic acid group.

[0027] FIG. 1 is a graph showing the results of measurement of the concentrations of 1-methylnicotinamide in urine from a wild-type mouse, urine from an NNMT knockout mouse, and urine from a wild-type mouse that drank water containing nicotinamide, using a liquid chromatograph mass spectrometer. 1 is a graph showing the regression line between the fluorescence intensity of a mixture of mouse urine and a water-soluble pillar[6]arene having a sulfonic acid group and the concentration of 1-methylnicotinamide in the urine measured by liquid chromatography-mass spectrometry. 2 is a graph showing the regression line between the fluorescence intensity at a wavelength of 330 nm derived from the water-soluble pillar[6]arene having a carboxy group and the concentration of 1-methylnicotinamide in the urine of six humans mixed with 1-methylnicotinamide and a water-soluble pillar[6]arene having a carboxy group. 3 is a graph showing the regression line between the fluorescence intensity at a wavelength of 310 nm derived from the water-soluble pillar[6]arene having a sulfonic acid group and the concentration of 1-methylnicotinamide in the urine of six humans mixed with 1-methylnicotinamide and a water-soluble pillar[6]arene having a sulfonic acid group. 4 is a graph showing the fluorescence emission intensity in the wavelength range of 300 to 400 nm of all components contained in RPMI 1640 medium. 1 is a graph showing the fluorescence intensity of RPMI 1640 medium, Afr RPMI 1640 medium, FBS, and water-soluble pillar[6]arene (50 μM) having a sulfonic acid group.1 is a graph showing the results of measuring the concentration of 1-methylnicotinamide in the medium after 48 hours of culture of 293T cells, OE-NNMT cells, and OE-Y20A cells using a liquid chromatograph mass spectrometer. 2 is a graph showing the results of measuring the concentration of 1-methylnicotinamide in the medium after 48 hours of culture of 293T cells, OE-NNMT cells, and OE-Y20A cells using a water-soluble pillar[6]arene having a sulfonic acid group. 3 is a graph showing the results of measuring the concentration of 1-methylnicotinamide in the medium during 48 hours of culture of 293T cells and OE-NNMT cells with the addition of a water-soluble pillar[6]arene having a sulfonic acid group, and a regression curve. 4 is a graph showing the results of measuring the concentration of 1-methylnicotinamide in the medium during 48 hours of culture of 293T cells and OE-NNMT cells using a liquid chromatograph mass spectrometer, and a regression curve. 19A is a diagram showing the metabolic routes of various niacin-related substances to 1-methylnicotinamide. This is a graph showing the time course of 1-methylnicotinamide concentration in random urine after ingestion of nicotinamide. This is a graph showing the time course of 1-methylnicotinamide blood concentration after ingestion of nicotinamide. This is a graph showing the time course of 1-methylnicotinamide concentration in random urine after a subject ingestion of nicotinamide mononucleotide. This is a graph showing the time course of 1-methylnicotinamide blood concentration in random urine after ingestion of nicotinamide mononucleotide by the same subject as in FIG. 19A. This is a graph showing the time course of 1-methylnicotinamide concentration in random urine after ingestion of nicotinamide mononucleotide by another subject. This is a graph showing the time course of 1-methylnicotinamide blood concentration in random urine after ingestion of nicotinamide mononucleotide by the same subject as in FIG. 20A. This is a graph showing the time course of 1-methylnicotinamide concentration in random urine after ingestion of nicotinamide acid. This is a graph showing the time course of 1-methylnicotinamide blood concentration after ingestion of nicotinamide acid. 1 is a graph showing the concentration of 1-methylnicotinamide in random urine samples stored at room temperature of 10 to 25° C. for 3 days and the concentration of 1-methylnicotinamide in random urine samples stored at −20° C. for 3 days.26 shows the NMR spectrum of a water-soluble pillar[6]arene having a phosphonic acid group, the NMR spectrum of 1-methylnicotinamide, and the NMR spectrum when a water-soluble pillar[6]arene having a phosphonic acid group and 1-methylnicotinamide are mixed. This is a graph showing raw data obtained by isothermal titration calorimetry of a water-soluble pillar[6]arene having a phosphonic acid group. This is a graph showing a binding isotherm obtained from the raw data shown in FIG. 25. This is a diagram showing the binding constant Ka between a water-soluble pillar[6]arene having a phosphonic acid group, a water-soluble pillar[6]arene having a sulfonic acid group, or a water-soluble pillar[6]arene having a carboxylic acid group and 1-methylnicotinamide, calculated from the binding isotherm shown in FIG. 26. This is a graph showing the fluorescence intensity of a water-soluble pillar[6]arene having a sulfonic acid group or a water-soluble pillar[6]arene having a phosphonic acid group at various 1-methylnicotinamide concentrations. 1 is a graph showing the fluorescence intensity and regression line for each concentration of 1-methylnicotinamide in a mixed solution of 1-methylnicotinamide and a water-soluble pillar[6]arene having a sulfonic acid group. 2 is a graph showing the fluorescence intensity and regression line for each concentration of 1-methylnicotinamide in a mixed solution of 1-methylnicotinamide and a water-soluble pillar[6]arene having a phosphonic acid group.

[0036] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings as needed.

[0037] <<Method for Measuring (Evaluating) 1-Methylnicotinamide Concentration>> FIG. 1 is a flow chart showing a method for measuring 1-methylnicotinamide concentration according to a preferred embodiment of the present invention.

[0038] As shown in FIG. 1, the method for measuring the concentration of 1-methylnicotinamide includes a step S1 of mixing a sample with a water-soluble pillar[6]arene to obtain a mixture, and a step S2 of measuring the fluorescence intensity of the mixture.

[0039] <Step S1> In step S1, a water-soluble pillar[6]arene having an anionic group (anionic group) or a salt thereof that is stronger than a carboxyl group is mixed with a sample. The number of the anionic groups is not particularly limited, but is preferably two or more, and may be two.

[0040] In step S1, the water-soluble pillar[6]arene and the sample may be mixed in vitro (outside an animal's body) or in vivo, but mixing within the human body (e.g., mixing by administering the water-soluble pillar[6]arene to a human) is excluded. Therefore, mixing of the water-soluble pillar[6]arene and the sample may be performed in the body of a mammal other than a human, as described in detail below, or may be performed outside the human body.

[0041] (Water-soluble pillar[6]arene) Pillararene is a compound in which six-membered ring structures such as benzene ring structures or quinoid structures are linked by methylene chains at the 2- and 5-positions to form a cyclic structure. In other words, it is a cyclic compound in which specific repeating units are linked. Water-soluble pillar[6]arene is a water-soluble pillararene compound, and is a hexagonal cyclic compound in which six repeating units are linked.

[0042] Particularly preferred water-soluble pillar[6]arenes have a cyclic structure in which six benzene rings are bonded via methylene groups. Examples of water-soluble pillar[6]arenes having such a cyclic structure include compounds represented by the following general formula (1) or salts thereof (neutralized with a base), but are not limited to the compounds represented by general formula (1) or salts thereof. Therefore, the water-soluble pillar[6]arenes may have a cyclic structure including a quinoid structure.

[0043] (In the general formula (1), X is an anionic group stronger than a carboxy group.)

[0044] Examples of cations that neutralize X in the water-soluble pillar[6]arene include, but are not limited to, sodium ions, ammonium ions, or hydrogen ions.

[0045] X in the formula (1) may be any anionic group stronger than a carboxy group (carboxylate group), and the type thereof is not particularly limited. For example, it may be a sulfonic acid group as represented by the following formula (2), or a phosphonic acid group as represented by the following formula (3). These anionic groups may exist in water in both an ionized state and a salt state ionically bonded to a cation. An anionic group stronger than a carboxy group is a functional group that exhibits stronger anionicity (acidity) than a carboxy group, and is an anionic group that exhibits a higher degree of ionization than a carboxy group when added to water in a state in which the anionic group is ionically bonded to a hydrogen ion. In other words, a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof is an acid or a salt thereof that is stronger than a water-soluble pillar[6]arene having a carboxy group instead of the anionic group, and exhibits a higher degree of ionization than a water-soluble pillar[6]arene having a carboxy group instead of the anionic group. For example, a water-soluble pillar[6]arene having a sulfonic acid group as X is a stronger acid than a water-soluble pillar[6]arene having a total of 12 carboxy groups per molecule instead of X, and exhibits a higher degree of ionization than the water-soluble pillar[6]arene having 12 carboxy groups.

[0046]

[0047]

[0048] A water-soluble pillar[6]arene having, for example, a sulfonic acid group as X (having a total of 12 sulfonic acid groups) emits fluorescence in aqueous solution with a maximum emission wavelength of approximately 310 nm. The excitation wavelength at this time is preferably 276 nm. When 1-methylnicotinamide is inserted and bonded to the cyclic structure formed by the repeating units of the water-soluble pillar[6]arene to form a complex, the fluorescence is quenched.

[0049] Therefore, the concentration of 1-methylnicotinamide in the sample can be measured (evaluated) by checking the fluorescence intensity of the mixture of the sample and the water-soluble pillar[6]arene.

[0050] Here, since the X is a stronger anionic group than a carboxy group, the water-soluble pillar[6]arene can bond more strongly to 1-methylnicotinamide than, for example, a water-soluble pillar[6]arene having a carboxy group instead of X.

[0051] For example, in the case of a water-soluble pillar[6]arene having a carboxyl group instead of X, the association constant (binding constant) Ka with 1-methylnicotinamide is (8.05±0.96)×10 ３ M (mol / L) －１ Whereas, when X is a sulfonic acid group, the association constant Ka with 1-methylnicotinamide is (5.68±1.02)×10 ６ M －１ is.

[0052] In this way, the water-soluble pillar[6]arene having X strongly binds to 1-methylnicotinamide, thereby quenching the fluorescence of the water-soluble pillar[6]arene with high sensitivity, and the concentration of 1-methylnicotinamide can be measured with high accuracy.

[0053] (Sample) The sample to be measured, which is mixed with the water-soluble pillar[6]arene, may contain other components in addition to water and 1-methylnicotinamide, or may contain only water and 1-methylnicotinamide.

[0054] Examples of samples include biological samples, food, feed such as pet food, and pharmaceuticals, with biological samples being particularly preferred. However, the type of sample is not limited to these. By using a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof, 1-methylnicotinamide can be detected with high sensitivity, making it possible to subject a wide variety of samples to the measurement method of this embodiment. When the sample is solid, it is preferably dissolved in water to form an aqueous solution before mixing with the water-soluble pillar[6]arene, in order to facilitate measurement.

[0055] The biological sample is not particularly limited as long as it is derived from a living organism and may contain 1-methylnicotinamide. Examples of biological samples include blood (plasma, serum, whole blood), cerebrospinal fluid, lymph, urine, serous fluid, synovial fluid, aqueous humor, tears, saliva, culture media used for cell culture (culture supernatant, etc.), and dilutions thereof.

[0056] The biological sample is not particularly limited to the type of animal, as long as it is derived from a mammal. Therefore, for example, it may be derived from a human or a non-human mammal. Therefore, the biological sample may be, for example, mammalian urine or a diluted solution thereof. As will be described in detail in the Examples below, by using a water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group, the concentration of 1-methylnicotinamide can be measured with high accuracy by dilution without purification, even in urine samples with relatively high autofluorescence intensity.

[0057] The dilution ratio of the urine sample is not particularly limited, but may be, for example, 100 to 1500 times.

[0058] Examples of non-human mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, and sheep.

[0059] Examples of foods include breads, fermented foods, dried foods, paste products, frozen foods, retort foods, instant foods (instant noodles, dry foods), processed foods (processed fish products, processed livestock products), luxury foods such as confectionery, health foods (functional foods) such as supplements, foods for special dietary uses (foods for the sick, foods for children, foods for the elderly), foods with functional claims, foods for specified health uses, water, coffee, soft drinks, alcoholic beverages, tea, etc.

[0060] The sample has a fluorescence intensity at a wavelength of 300 to 400 nm, measured at room temperature (25°C) using a spectrofluorometer, a fluorescence microplate reader, or the like, which is substantially 0 within the detectable range, or is 3 times or less (more preferably 2 times or less, even more preferably 1 time or less, particularly preferably 1 / 10 or less, and most preferably 1 / 10) of the fluorescence intensity of 5 mL of an aqueous solution containing only the water-soluble pillar[6]arene represented by the above formula (2) at a concentration of 50 μM. ５ Preferably, the sample emits fluorescence with a fluorescence intensity of 1-methylnicotinamide (see below). By using such a sample, the concentration of 1-methylnicotinamide can be measured accurately.

[0061] (Mixing) The relative amount of the water-soluble pillar[6]arene to the sample used in the measurement method of this embodiment is preferably an amount such that the amount of bound water-soluble pillar[6]arene is in excess relative to the amount of 1-methylnicotinamide contained in the sample.

[0062] When the amount of 1-methylnicotinamide contained in a sample can be roughly predicted before measurement, the sample and the water-soluble pillar[6]arene can be mixed so that the amount of the water-soluble pillar[6]arene is sufficiently in excess of the amount of 1-methylnicotinamide.

[0063] If it is difficult to predict the amount of 1-methylnicotinamide contained in the sample before measurement, the sample and the water-soluble pillar[6]arene are mixed in tentative amounts, and when the % inhibition described below is calculated once, if it is close to 100%, the amount of the water-soluble pillar[6]arene is increased and the measurement is performed again, or if the % inhibition is close to 0%, the amount of the water-soluble pillar[6]arene is decreased and the measurement is performed again, and adjustments can be made as appropriate.

[0064] The mixing may involve adding the water-soluble pillar[6]arene to a sample, or adding the sample to the water-soluble pillar[6]arene. Stirring or the like may be performed during mixing. The water-soluble pillar[6]arene may be used as is or as a solution such as an aqueous solution. To facilitate fluorescence measurement, the resulting mixture is preferably prepared as a solution, more preferably as an aqueous solution. A solvent such as water may be added separately during mixing, if necessary.

[0065] The temperature at which mixing is carried out is not particularly limited, and mixing may be carried out under cooled conditions, room temperature, or heated conditions, but is preferably carried out under cooled conditions or room temperature. Specifically, mixing is preferably carried out at a temperature of 15°C to 30°C, more preferably 20°C to 25°C. The upper and lower limits of these numerical ranges can be combined arbitrarily. In the examples described below, mixing was carried out at room temperature in the range of 10°C to 25°C.

[0066] The method for measuring the concentration of 1-methylnicotinamide may further include a step of purifying the sample or the mixture of the sample and the water-soluble pillar[6]arene before step S1 or after step S1 (before step S2). By removing impurities in this step, the sensitivity and accuracy of the measurement can be improved.

[0067] Examples of methods for purifying a sample or mixture include, but are not limited to, subjecting the sample or mixture to a column that adsorbs impurities. Impurities to be removed from a sample or mixture include, for example, hydrophobic compounds such as lipids and tryptophan, and tyrosine. Removing fluorescent compounds such as tryptophan and lipids from a sample or mixture can improve measurement sensitivity and accuracy.

[0068] <Step S2> In step S2, the fluorescence intensity of the mixture obtained in step S1 is measured. For example, when a water-soluble pillar[6]arene having a sulfonic acid group as X is mixed with a sample, the fluorescence intensity at a wavelength of about 310 nm is measured.

[0069] The fluorescence intensity can be measured using, for example, a spectrofluorometer, but the measurement means is not particularly limited. When the number of samples is large or the amount of each sample is small, a multi-detection mode plate reader equipped with a fluorescence measurement function, such as Spark (Tecan), may be used.

[0070] The temperature at which the fluorescence intensity is measured is not particularly limited, and may be, for example, under cooled conditions, room temperature of 10 to 25°C, or heated conditions, but is preferably measured at 16 to 30°C, and more preferably at 20 to 25°C. The upper and lower limits of these numerical ranges can be combined arbitrarily. In the examples described below, measurements were performed at room temperatures ranging from 10 to 25°C.

[0071] The final evaluation method for the 1-methylnicotinamide concentration based on the thus measured fluorescence intensity is not particularly limited as long as it is based on the measurement result of the fluorescence intensity of a mixture of a sample and the water-soluble pillar[6]arene. For example, the following evaluation method can be mentioned.

[0072] One evaluation method involves first using an aqueous solution in which the content of 1-methylnicotinamide has been determined by a known analytical method such as liquid chromatography-mass spectrometry as a reference sample. Then, the fluorescence intensities of a mixture of the reference sample and the water-soluble pillar[6]arene and a mixture of the sample to be measured and the water-soluble pillar[6]arene are measured and compared. In this case, it is preferable that the amount of water-soluble pillar[6]arene mixed in the reference sample and the sample to be measured is the same.

[0073] It is preferable to prepare multiple reference samples with different concentrations of 1-methylnicotinamide. The fluorescence intensity of a mixture of each reference sample and the water-soluble pillar[6]arene is used as a marker (standard) of the 1-methylnicotinamide concentration, and by comparing it with the fluorescence intensity of a mixture of the sample to be measured and the water-soluble pillar[6]arene, a more accurate evaluation of the amount of 1-methylnicotinamide in the sample to be measured is possible. The type of reference sample is not particularly limited, but it is preferable that the components other than 1-methylnicotinamide are the same or similar to those of the sample to be measured.

[0074] Another evaluation method is to calculate the % inhibition described in JP 2022-065544 A using the following formula (I) and calculate it as an index of 1-methylnicotinamide concentration.

[0075] % inhibition=[1−(F / F0)]×100 (I) In formula (I), F0 is the fluorescence intensity of the water-soluble pillar[6]arene not bound to 1-methylnicotinamide, and F is the fluorescence intensity of the mixture.

[0076] When the sample is, for example, urine, the percent inhibition can be calculated using the above formula (I) by defining the fluorescence intensity of a mixture obtained by mixing a urine sample excreted from an Nnmt-deficient mouse substantially free of 1-methylnicotinamide with the water-soluble pillar[6]arene as F0 and the fluorescence intensity of a mixture of the urine sample and the water-soluble pillar[6]arene as F. In this embodiment, "substantially free of 1-methylnicotinamide" means that the concentration of 1-methylnicotinamide analyzed by a known method such as liquid chromatography-mass spectrometry is not detectable or is 50 nM or less.

[0077] Another evaluation method is to use the difference between the fluorescence intensity of a mixture of a sample and the water-soluble pillar[6]arene and the fluorescence intensity of the water-soluble pillar[6]arene not bound to 1-methylnicotinamide as an indicator of the 1-methylnicotinamide concentration. The fluorescence intensity of the water-soluble pillar[6]arene not bound to 1-methylnicotinamide may be the fluorescence intensity of a solution containing the water-soluble pillar[6]arene before mixing with the sample.

[0078] According to the method for measuring the concentration of 1-methylnicotinamide of the present embodiment described above in detail, the concentration of 1-methylnicotinamide can be measured simply, quickly, and accurately by mixing a sample with the water-soluble pillar[6]arene and measuring the fluorescence intensity of the resulting mixture.

[0079] <<Kit for Measuring 1-Methylnicotinamide Concentration>> The kit for measuring 1-methylnicotinamide concentration (measurement kit) of this embodiment includes a water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group.

[0080] The water-soluble pillar[6]arene may be any of the water-soluble pillar[6]arene described in detail in the above embodiment. The state of the water-soluble pillar[6]arene is not particularly limited, and may be, for example, dissolved in a solvent such as water, or may be in a dry or wet solid or powder state.

[0081] The kit for measuring 1-methylnicotinamide concentration includes a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof, and thus an operator of the kit can easily and accurately measure the concentration of 1-methylnicotinamide by the above-described measurement method.

[0082] 2 is a schematic longitudinal cross-sectional view showing an example of a column included in the kit for measuring 1-methylnicotinamide concentration. The kit for measuring 1-methylnicotinamide concentration may further include a column for adsorbing hydrophobic compounds and / or tyrosine. Examples of hydrophobic compounds include lipids and tryptophan. An example of a column for adsorbing hydrophobic compounds is column 100 including a carrier 10 that adsorbs hydrophobic compounds. Examples of the carrier 10 include, but are not limited to, a carrier including silica gel that adsorbs lipids and a carrier including a resin carrying hydrophobic residues.

[0083] The kit for measuring the concentration of 1-methylnicotinamide may include components, reagents, etc. other than the water-soluble pillar[6]arene and column.

[0084] <<Use of Kit for Measuring 1-Methylnicotinamide Concentration>> In another embodiment, the present invention provides use of a kit for measuring the concentration of 1-methylnicotinamide, which includes a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof. The measurement kit used in this embodiment is the same as the measurement kit described in detail in the embodiment of the measurement kit described above.

[0085] <<Use of Water-Soluble Pillar[6]arene Having an Anionic Group Stronger than a Carboxy Group or a Salt Thereof, for Producing a Kit for Measuring 1-Methylnicotinamide Concentration>> In another embodiment, the present invention provides use of a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof, for producing a kit for measuring 1-methylnicotinamide concentration. The water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof used in this embodiment can be the water-soluble pillar[6]arene described in detail in the embodiment of the method for measuring 1-methylnicotinamide concentration. The measurement kit produced using this embodiment is similar to the measurement kit described in detail in the embodiment of the measurement kit above.

[0086] The state of the water-soluble pillar[6]arene is not particularly limited, and may be, for example, dissolved in a solvent such as water, or may be in a dry or wet solid or powder state.

[0087] <<Reagent for Measuring 1-Methylnicotinamide Concentration>> The reagent for measuring 1-methylnicotinamide concentration of this embodiment contains a water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group.

[0088] The water-soluble pillar[6]arene may be the same as that described in detail in the embodiment of the method for measuring 1-methylnicotinamide concentration. The state of the water-soluble pillar[6]arene is not particularly limited, and may be, for example, dissolved in a solvent such as water, or in a dry or wet solid or powder state.

[0089] The reagent for measuring 1-methylnicotinamide concentration of this embodiment may be, for example, a reagent for measurement used in the measurement method described in detail in <<Method for measuring (evaluating) 1-methylnicotinamide concentration>>.

[0090] <<Use of a Reagent for Measuring 1-Methylnicotinamide Concentration>> In another embodiment, the present invention provides use of a reagent for measuring the concentration of 1-methylnicotinamide, which includes a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof. The measurement reagent used in this embodiment is the same as the measurement reagent described above.

[0091] <<Use of Water-Soluble Pillar[6]arene Having an Anionic Group Stronger than a Carboxy Group or a Salt Thereof, for Producing a Reagent for Measuring 1-Methylnicotinamide Concentration>> In another embodiment, the present invention provides use of a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof, for producing a reagent for measuring 1-methylnicotinamide concentration. The water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof used in this embodiment can be the water-soluble pillar[6]arene described in detail in the embodiment of the method for measuring 1-methylnicotinamide concentration.

[0092] The state of the water-soluble pillar[6]arene is not particularly limited, and may be, for example, dissolved in a solvent such as water, or may be in a dry or wet solid or powder state.

[0093] <<Method for screening for NNMT inhibitors>> The method for screening for NNMT inhibitors of the present embodiment includes the steps of contacting NNMT with a candidate substance for an NNMT inhibitor in the presence of S-adenosylmethionine and nicotinamide to obtain a sample, obtaining a mixture of the sample obtained in the sample obtaining step and a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof (mixing step), and measuring the fluorescence intensity of the mixture (fluorescence intensity measuring step).

[0094] An NNMT inhibitor is a substance that has the effect of inhibiting the production of 1-methylnicotinamide (transfer of a methyl group from S-adenosylmethionine to nicotinamide) by NNMT (nicotinamide methyltransferase), and the type of the inhibitor is not particularly limited, but examples thereof include proteins and low molecular weight compounds.

[0095] The water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group can be the water-soluble pillar[6]arene described in detail in the embodiment of the measurement method. By using the water-soluble pillar[6]arene, it is possible to simply and accurately screen the degree to which a candidate substance for an NNMT inhibitor inhibits NNMT.

[0096] <Sample Obtaining Step> In the sample obtaining step, NNMT is contacted with a candidate substance for an NNMT inhibitor in the presence of S-adenosylmethionine and nicotinamide to obtain a sample during or after the enzymatic reaction catalyzed by NNMT. The location where NNMT is contacted with the candidate substance for an NNMT inhibitor (i.e., the location where the sample is obtained) may be in vitro (outside an animal's body) or in vivo, but not within the human body. Therefore, the water-soluble pillar[6]arene and the sample may be mixed inside the body of a mammal other than a human, as described above. Examples of mixing inside the body of a mammal other than a human include administering the candidate substance to the mammal other than a human. When mixed inside the body of a mammal other than a human, the sample obtained from the body of the mammal other than a human in the sample obtaining step include, for example, blood (plasma, serum, whole blood), cerebrospinal fluid, lymph, urine, serous fluid, synovial fluid, aqueous humor, tears, saliva, or diluted solutions thereof.

[0097] When the step of obtaining a sample is carried out in vitro, for example, a method of mixing NNMT, nicotinamide, S-adenosylmethionine and a candidate substance for an NNMT inhibitor in a buffer solution or liquid medium and carrying out an enzymatic reaction to obtain a sample can be mentioned.

[0098] Examples of buffer solutions include, but are not limited to, phosphate buffer, phosphate buffered saline, acetate buffer, citrate buffer, Tris buffer, and HEPES buffer.

[0099] The step of obtaining a sample may involve, for example, contacting NNMT, nicotinamide, S-adenosylmethionine, and a candidate substance for an NNMT inhibitor in cultured cells expressing NNMT immersed in a liquid medium, and carrying out an enzymatic reaction.

[0100] For example, cancer tissue-derived cell lines are generally cultured in media containing inorganic salts, sugars, amino acids, and vitamins, such as RPMI 1640 medium or DMEM (Dulbecco's Modified Eagle's Medium). For long-term culture of mammalian cells, it is necessary to add 10% v / v FBS (Fetal Bovine Serum) to the medium.

[0101] However, both RPMI1640 medium and FBS emit strong autofluorescence in the range of 300 to 400 nm, which includes the wavelength of the fluorescence emitted by the water-soluble pillar[6]arene having a sulfonate group.

[0102] Therefore, when cells expressing NNMT are cultured using RPMI1640 medium or a medium containing FBS, there is a risk that the intensity of fluorescence derived from the water-soluble pillar[6]arene cannot be accurately measured in the fluorescence intensity measurement step.

[0103] When the present inventors analyzed the components of the medium, they confirmed that tyrosine, tryptophan, and p-aminobenzoic acid in particular emitted strong autofluorescence in the above wavelength range.

[0104] For these reasons, it is preferable to use a medium that does not contain tyrosine, tryptophan, or p-aminobenzoic acid as a medium for culturing NNMT-expressing cells. An example of such a medium is the Afr RPMI 1640 medium described in detail in the Examples.

[0105] On the other hand, NNMT is not limited to wild-type NNMT as long as it is an enzyme having the activity of transferring a methyl group from S-adenosylmethionine to nicotinamide, and therefore may be, for example, a recombinant NNMT (recombinant protein).

[0106] The pH of the enzymatic reaction by NNMT is not particularly limited, but may be, for example, pH 6-9 or pH 6-8.

[0107] The temperature for the enzymatic reaction by NNMT is not particularly limited, but may be, for example, room temperature of 10 to 25°C, 10 to 40°C, 20 to 35°C, or 30 to 40°C.

[0108] NNMT, nicotinamide, and S-adenosylmethionine may be added to cultured cells or administered to a mammal other than a human, or may be originally present in cultured cells or in the body of a mammal other than a human.

[0109] A sample purification step may be performed between the sample obtaining step and the mixing step, or between the mixing step and the fluorescence intensity measuring step. Examples of the purification method include, but are not limited to, subjecting the sample or the mixture to a column that adsorbs impurities.

[0110] Impurities to be removed from a sample or mixture include, for example, lipids, hydrophobic compounds such as tryptophan, and tyrosine. Removal of fluorescent compounds such as tryptophan and tyrosine and lipids from a sample or mixture can improve measurement sensitivity and accuracy.

[0111] <Mixing Step> In the mixing step, the sample obtained in the sample obtaining step is mixed with water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group. The water-soluble pillar[6]arene to be mixed with the sample may be dissolved in water, a buffer solution, or the like, or may be in a solid state. The mixture obtained in the mixing step may be stirred before measuring the fluorescence intensity.

[0112] [Step of Measuring Fluorescence Intensity] In the step of measuring fluorescence intensity in the screening method for an NNMT inhibitor, the fluorescence intensity of the mixture (mixture) is measured. The step of measuring fluorescence intensity can be performed in the same manner as step S2 in the method for measuring the concentration of 1-methylnicotinamide.

[0113] The specific method for selecting an NNMT inhibitor is not particularly limited, but for example, in the step of obtaining the above-mentioned sample, the greater the measured fluorescence intensity compared to a control group in which the candidate substance for the NNMT inhibitor has not been contacted with NNMT, the more excellent the NNMT inhibitor can be selected.

[0114] <<Method for screening NNMT activators>> The method for screening NNMT activators of the present embodiment includes the steps of contacting NNMT with a candidate substance for an NNMT activator in the presence of S-adenosylmethionine and nicotinamide to obtain a sample, obtaining a mixture of the sample obtained in the sample obtaining step and a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof (mixing step), and measuring the fluorescence intensity of the mixture (fluorescence intensity measuring step).

[0115] An NNMT activator is a substance that has the effect of activating (promoting) the production of 1-methylnicotinamide by NNMT (transfer of a methyl group from S-adenosylmethionine to nicotinamide), and the type of NNMT activator is not particularly limited, but examples thereof include proteins and low molecular weight compounds.

[0116] The screening method for NNMT activators is similar to the screening method for NNMT inhibitors described above, except that, for example, a candidate substance for an NNMT activator is used instead of a candidate substance for an NNMT inhibitor, and in the selection method, the smaller the fluorescence intensity measured in the fluorescence intensity measurement step compared to a control group in which the candidate substance for an NNMT activator has not been contacted with NNMT, the more excellent the NNMT activator that can be selected.

[0117] <<Other Embodiments>> In recent years, supplements containing niacin-related substances such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) in addition to nicotinamide (NAM) and nicotinic acid (NA) have been increasing. Ingestion of such niacin-related substances is expected to increase the concentration of nicotinamide adenine dinucleotide (NAD+) in the body, resulting in anti-aging effects such as suppressing cognitive decline and motor decline. After ingestion of a niacin-related substance, the blood concentration of 1-methylnicotinamide metabolized by NNMT is expected to increase.

[0118] On the other hand, the following literature describes that various cancer tissues increase the expression level of NNMT in the liver, resulting in an increase in the amount of 1-methylnicotinamide in the liver and the occurrence of various metabolic abnormalities.

[0119] Rin Mizuno et al., Remote solid cancers rewire hepatic nitrogen metabolism via host nicotinamide-N-methyltransferas. Nature Communications 13, 3346, 2022. Furthermore, it is known that NNMT expression levels increase and blood levels of 1-methylnicotinamide increase in diseases that cause inflammation in the liver, such as liver cirrhosis. Measuring blood levels of 1-methylnicotinamide may enable the early detection of various diseases.

[0120] As described above, it is expected that the concentration of 1-methylnicotinamide in the blood will increase after the ingestion of a niacin-related substance. Therefore, by measuring the blood concentration of 1-methylnicotinamide in patients who have taken a niacin-related substance for the treatment of various diseases, the effects of the ingestion of a niacin-related substance can be confirmed, and this may be useful for selecting the appropriate medication to be used for treatment.

[0121] Thus, there is a demand for measuring the blood concentration of 1-methylnicotinamide to confirm the effects of supplements, detect various diseases, etc. However, blood tests have the problem of imposing a great burden on the subject (or animal) from whom blood is taken.

[0122] In view of the above circumstances, in one embodiment, the present invention provides a method for estimating changes in blood concentration of 1-methylnicotinamide over time, which can reduce the burden on the subject or animal whose blood concentration is to be estimated.

[0123] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0124] A method for estimating changes in blood concentration of 1-methylnicotinamide over time according to a preferred embodiment of the present invention includes a step of measuring the concentrations of 1-methylnicotinamide contained in multiple random urine samples excreted at different times from a mammal.

[0125] The present inventors have surprisingly found that the concentration of 1-methylnicotinamide contained in a mammal's spot urine increases or decreases at the same time that the blood concentration of 1-methylnicotinamide increases or decreases (in other words, the manner in which the concentration of 1-methylnicotinamide changes in spot urine closely resembles the manner in which the blood concentration of 1-methylnicotinamide changes).

[0126] Therefore, by measuring the change in urinary 1-methylnicotinamide concentration over time using multiple random urine samples excreted at different times, it is possible to estimate the change in blood 1-methylnicotinamide concentration over time with high accuracy. It has not been known until now that the blood concentration of 1-methylnicotinamide fluctuates precisely at the same timing as the concentration of 1-methylnicotinamide in random urine. As will be shown in the Examples below, the coincidence of the timing of the peaks of the blood concentration of 1-methylnicotinamide and the concentration of 1-methylnicotinamide in urine is a result that could not be predicted based on conventional common technical knowledge. By using this method, it is possible to detect the presence of peaks and valleys caused by changes in blood 1-methylnicotinamide concentration, as well as patterns formed by these.

[0127] For example, the following literature describes that measurement of 1-methylnicotinamide concentrations in urine collected for five hours revealed that 1-methylnicotinamide concentrations were higher in patients with cirrhosis than in healthy individuals. However, it has not been known to date that measuring urinary 1-methylnicotinamide concentrations by collecting urine at short intervals, such as 2.5 hours, as in the Examples below, allows accurate estimation of the progression and peak of blood concentrations. R Cuomo et al., Nicotinamide methylation in patients with cirrhosis. J Hepatol, 20, 138-42, 1994.

[0128] The "blood concentration" whose change over time is estimated in this embodiment is a concept that includes the concentration of 1-methylnicotinamide in whole blood, the concentration of 1-methylnicotinamide in plasma, and the concentration of 1-methylnicotinamide in serum, and the change over time (change over time) of the 1-methylnicotinamide concentration in whole blood, plasma, or serum can be estimated based on the change over time of the 1-methylnicotinamide concentration in spot urine.

[0129] The mammal for which the time course of the blood concentration of 1-methylnicotinamide is to be estimated is not particularly limited, and may be, for example, a human or a mammal other than a human.

[0130] Examples of mammals other than humans include, but are not limited to, mice, rats, guinea pigs, rabbits, monkeys, pigs, cows, horses, dogs, and cats.

[0131] The multiple random urine samples excreted at different times are all random urine samples (in other words, spot urine samples) excreted (discharged) from the same individual (or the same person). In order to increase the accuracy of estimating the change in blood concentration of 1-methylnicotinamide over time, the multiple random urine samples are preferably urine samples excreted consecutively within an arbitrary period without skipping any urine samples (for example, four consecutive urine samples from the first to fourth urine samples on a certain day).

[0132] A random urine sample may be a spontaneous urination sample, a urine sample discharged from the bladder using a catheter or the like (catheterization), or a urine sample collected by pressing the abdomen of a mammal such as a mouse to encourage excretion.

[0133] The interval between the multiple random urine excretion times (in other words, the random urine collection times) is not particularly limited. For example, the interval may be within 0.5 to 2 hours, 1 to 2.5 hours, 2.5 to 5 hours, 5 to 10 hours, or 0.5 to 12 hours. It is expected that the interval between urination will be particularly large around the time of going to bed.

[0134] Examples of the lower limit of the interval between the excretion times of multiple random urine samples include, but are not limited to, 0.5 hours, 1 hour, 2 hours, 2.5 hours, 3 hours, 5 hours, 7 hours, 10 hours, 12 hours, and 15 hours.

[0135] Examples of upper limit values ​​for the interval between multiple random urine excretion times include, but are not limited to, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 11 hours, 15 hours, and 24 hours.

[0136] The above-mentioned lower limit and upper limit for the interval between the excretion times of a plurality of random urine samples can be combined arbitrarily.

[0137] The number of times the multiple random urine samples are excreted (in other words, the number of random urine samples collected) is not particularly limited, and may be, for example, two or more times in a 5-hour period, three or more times in a 5-hour period, two or more times in a 10-hour period, three or more times in a 10-hour period, four or more times in a 10-hour period, five or more times in a 10-hour period, two or more times in a 12-hour period, three or more times in a 12-hour period, four or more times in a 12-hour period, five or more times in a 12-hour period, or six or more times in a 12-hour period. The above 5, 10, and 12 hours may be 5, 10, and 12 hours, respectively, after a meal or after ingestion of a niacin-related substance, which will be described later.

[0138] During random urination, it is preferable to empty as much urine as possible from the bladder. This prevents a portion of the urine containing 1-methylnicotinamide from the bladder from being discharged from the bladder during the next random urination, thereby enabling more accurate estimation of changes in the blood concentration of 1-methylnicotinamide over time.

[0139] The temperature at which the excreted random urine sample is stored until the 1-methylnicotinamide concentration is measured is not particularly limited, and the sample may be stored at room temperature (10 to 25°C), in a refrigerator, or in a freezer. That is, the random urine sample may be urine that has been frozen and stored after excretion. The temperature at which frozen urine has been stored is, for example, -30 to -10°C, but is not limited thereto.

[0140] The random urine sample is preferably a random urine sample collected from a mammal after ingesting at least one substance (niacin-related substance) selected from the group consisting of nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide riboside, and tryptophan. Ingestion of one or more of these niacin-related substances is expected to increase blood and urinary 1-methylnicotinamide concentrations.

[0141] The time (period) from the time of ingestion of at least one substance selected from the above group to the time of urinary excretion is not particularly limited and can be set arbitrarily and appropriately. For example, the urine may be excreted 5 minutes or more, 30 minutes or more, 1 hour or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, or 24 hours or more after ingestion of the substance. For example, the urine may be excreted within 1 hour, 4 hours, 5 hours, 8 hours, 10 hours, 13 hours, 15 hours, 24 hours, 2 days, 5 days, or 1 week after ingestion of the substance. The lower and upper limits of these ranges can be combined arbitrarily.

[0142] Incidentally, random urine excreted before or during the ingestion of the above-mentioned substance may also be included in the measurement of concentration.

[0143] The method of ingesting the substance is not particularly limited, and may be, for example, oral ingestion of food or medicine containing the substance, or parenteral ingestion (administration) such as intravenous drip.

[0144] Examples of foods containing the above substances include, but are not limited to, breads, fermented foods, dried foods, paste products, frozen foods, retort foods, instant foods (instant noodles, dry foods), processed foods (processed fish products, processed livestock products), luxury foods such as confectionery, health foods (functional foods) such as supplements, foods for special dietary uses (foods for the sick, foods for children, foods for the elderly), foods with functional claims, foods for specified health uses, water, coffee, soft drinks, alcoholic beverages, tea, etc.

[0145] The method for measuring the concentration of 1-methylnicotinamide contained in multiple random urine samples is not particularly limited. The method for measuring the concentration of 1-methylnicotinamide may be, for example, a known method such as liquid chromatography-mass spectrometry, the measurement method described in JP 2022-065544 A, or the above-mentioned measurement method using a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof. That is, the above-mentioned step of measuring the concentration of 1-methylnicotinamide contained in random urine samples may include a step of mixing random urine samples excreted at different times with a water-soluble pillar[6]arene to obtain a mixture, and a step of measuring (evaluating) the fluorescence intensity of the mixture.

[0146] The concentration measured in the step of measuring the concentrations of 1-methylnicotinamide contained in multiple random urine samples is preferably a concentration corrected by the creatinine concentration values ​​of the random urine samples. More specifically, the 1-methylnicotinamide concentration measured in the step is preferably a concentration corrected by dividing by the creatinine concentration in the same random urine samples.

[0147] The concentration of 1-methylnicotinamide is affected by the amount of urine contained in the bladder, and therefore, the concentration of 1-methylnicotinamide can be measured with high accuracy by correcting the concentration by dividing it by the concentration of creatinine in the same random urine sample.

[0148] The blood concentration of 1-methylnicotinamide changes according to the concentration of 1-methylnicotinamide contained in random urine. Therefore, the change in the blood concentration of 1-methylnicotinamide over time can be estimated from the concentrations of 1-methylnicotinamide contained in multiple random urine samples excreted at different times, measured as described above.

[0149] According to the method for estimating the change in blood concentration of 1-methylnicotinamide over time according to the present embodiment, the change in blood concentration over time can be estimated from the concentration of 1-methylnicotinamide contained in a random urine sample, eliminating the need for blood testing of the mammal, thereby reducing the burden on the mammal (target person or target animal) whose blood concentration of 1-methylnicotinamide is to be estimated.

[0150] The method of estimating changes in blood concentration of 1-methylnicotinamide over time according to this embodiment may include other steps in addition to the step of measuring the concentrations of 1-methylnicotinamide contained in multiple random urine samples. For example, the method of estimating changes in blood concentration of 1-methylnicotinamide over time may further include a step of purifying the random urine samples prior to the step of measuring the concentrations of 1-methylnicotinamide contained in multiple random urine samples.

[0151] The compounds removed from the spot urine sample in this purification step are not particularly limited as long as they are compounds other than 1-methylnicotinamide, but examples include hydrophobic compounds such as lipids and tryptophan, as well as tyrosine. The above-mentioned column can be used, for example, to purify the spot urine sample. By removing hydrophobic compounds and the like from the spot urine sample before measuring the 1-methylnicotinamide concentration, the measurement sensitivity and accuracy of 1-methylnicotinamide contained in the spot urine sample can be improved.

[0152] The applications of the method for estimating the time-dependent change in blood concentration of 1-methylnicotinamide according to this embodiment are not particularly limited, and examples include evaluating the effects of administering a supplement containing a niacin-related substance, and evaluating the effects of calorie restriction and exercise. It is known that calorie restriction and exercise increase the blood concentration of 1-methylnicotinamide. The estimation method according to this embodiment may be a method for estimating the time-dependent change in blood concentration of 1-methylnicotinamide after a meal, or a method for estimating the time-dependent change in blood concentration of 1-methylnicotinamide after ingesting a niacin-related substance. The niacin-related substance may be, for example, at least one selected from the group consisting of nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide riboside, and tryptophan, as described above, or at least one selected from the group consisting of nicotinamide, nicotinic acid, and nicotinamide mononucleotide.

[0153] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0154] The present invention will be described below with reference to examples, but is not limited to these examples. All animal care and experiments were approved by the Kanazawa University Animal Care and Use Committee. Studies using human samples were approved by the Kanazawa University Hospital Institutional Review Board (CTRB).

[0155] <Methods> [Preparation of samples for liquid chromatography-mass spectrometry] (Acetonitrile containing internal standards) 180 μL each of d-1-methylnicotinamide, d-creatinine, and d-nicotinamide (10 μg / mL) as internal standards was added to 18 mL of acetonitrile to give a final concentration of 100 ng / mL. The method for preparing each compound as an internal standard is described below.

[0156] (Method for preparing D-isomer) Since the stock solutions of d-1-methylnicotinamide and d-nicotinamide were each 5 mg / mL, 98 μL of water was added to 2 μL of the stock solution to dilute 50 times, adjusting the concentration to 100 μg / mL.

[0157] Thereafter, 180 μL of water was added to 20 μL of the resulting 100 μg / mL solution to make it 10 μg / mL, and 180 μL of this solution was added to 18 mL of acetonitrile to make the final concentration 100 ng / mL.

[0158] (Method for adjusting creatinine) Creatinine was 1 mg / mL in stock solution, and 90 μL of water was added to 10 μL of the stock solution to dilute it 10 times, adjusting the concentration to 100 μg / mL.

[0159] Thereafter, 180 μL of water was added to 20 μL of the resulting 100 μg / mL solution to make it 10 μg / mL, and 180 μL of this solution was added to 18 mL of acetonitrile to make the final concentration 100 ng / mL.

[0160] (Human Blood Collection) Peripheral blood was collected from the finger using a Nipro LS lancet 25G 1.0 mm, and 100 to 200 μL of blood was collected using a heparin-containing capillary tube. The blood was then centrifuged at 1500 rpm for 15 minutes to collect the supernatant, which was then stored at −30°C.

[0161] (Collection of human urine) Urine was collected immediately after the above blood collection. The urine was centrifuged at 10,000 rpm for 1 minute, and the supernatant was collected and stored at -30°C.

[0162] (Method for diluting human urine) 1495 μL of PBS (Phosphate-Buffered Saline) was placed in an Eppendorf tube in advance. A frozen urine sample was thawed on ice, shaken and stirred using a vortex mixer, and then centrifuged at 4°C and 10,000 rpm for 5 minutes to remove the precipitate.

[0163] Thereafter, 5 μL of the supernatant was collected from the sample and added to the above-mentioned Eppendorf tube, and the mixture was thoroughly shaken and stirred using a vortex mixer (300-fold dilution, total 1500 μL).

[0164] (Method for diluting mouse urine) 1500 μL of PBS was placed in an Eppendorf tube in advance. A frozen urine sample was thawed on ice, shaken and stirred using a vortex mixer, and then centrifuged at 4° C. and 10,000 rpm for 5 minutes to remove the precipitate.

[0165] Thereafter, 1.5 μL of the supernatant was collected from the sample and added to the above-mentioned Eppendorf tube, and the mixture was thoroughly shaken and stirred using a vortex mixer (1000-fold dilution, total 1500 μL).

[0166] (Blood dilution method) 40 μL of PBS was placed in an Eppendorf tube in advance. A frozen blood sample was thawed on ice, shaken and stirred using a vortex mixer, and then centrifuged at 4°C and 10,000 rpm for 5 minutes to remove the precipitate (blood cell components). Then, 10 μL of the supernatant was collected from the sample and added to the Eppendorf tube, followed by thorough shaking and stirring using a vortex mixer (50-fold dilution, total 50 μL).

[0167] (Operations from sample dilution to storage) After diluting the urine or blood sample, 50 μL of each sample was added to an Eppendorf tube, and 150 μL of acetonitrile containing d-1-methylnicotinamide, d-creatinine, and d-nicotinamide as internal standards was added to make a total volume of 200 μL.

[0168] The mixture was then shaken and stirred using a vortex mixer, centrifuged at 10,000 rpm at 4°C for 5 minutes, and the supernatant was transferred to a vial using a pipette.The mixture was then stored at -20°C until the concentration of each component was measured.

[0169] (Method of diluting standards) 10 μL of 1 mg / mL nicotinamide, 10 μL of 1 mg / mL creatinine, and 10 μL of 1 mg / mL methylnicotinamide were each dissolved in 970 μL of PBS to give a concentration of 10 μg / mL.

[0170] Thereafter, the solution was diluted with PBS to prepare standards of 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL.

[0171] (Liquid Chromatography Mass Analysis) Using a liquid chromatograph mass spectrometer (model number: LCMS-8050) manufactured by Shimadzu Corporation, the concentrations of the target substances (1-methylnicotinamide, creatinine) in each sample were measured.

[0172] In the following experiments, "water-soluble pillar[6]arene having a sulfonic acid group" refers to the compound represented by the above formula (2). Also, "water-soluble pillar[6]arene having a carboxy group" refers to the compound represented by the above formula (1) in which X is a carboxy group.

[0173] [Experimental Example 1] <Binding Constant> Figure 3 is a graph showing the fluorescence intensity at various concentrations of 1-methylnicotinamide in a liquid medium containing a water-soluble pillar[6]arene having a carboxy group and 1-methylnicotinamide. Figure 4 is a graph showing the fluorescence intensity at various concentrations of 1-methylnicotinamide in a liquid medium containing a water-soluble pillar[6]arene having a sulfonic acid group and 1-methylnicotinamide. Afr RPMI 1640 medium, which will be described in detail later, was used as the liquid medium.

[0174] As shown in Figure 4, the fluorescence intensity of the water-soluble pillar[6]arene with sulfonic acid groups decreased significantly with increasing 1-methylnicotinamide concentration compared to the water-soluble pillar[6]arene with carboxyl groups. This result indicates that the water-soluble pillar[6]arene with sulfonic acid groups binds more strongly to 1-methylnicotinamide than the water-soluble pillar[6]arene with carboxyl groups.

[0175] Isothermal titration calorimetry (ITC) was performed, and the association constant (binding constant) Ka of the water-soluble pillar[6]arene having a carboxy group with 1-methylnicotinamide was found to be: Ka = (5.68 ± 1.02) × 10 ６ M －１

[0176] In contrast, the association constant (binding constant) Ka of the water-soluble pillar[6]arene having a sulfonic acid group with 1-methylnicotinamide was as follows: Ka = (8.05 ± 0.96) × 10 ３ M －１

[0177] Thus, the association constant K a of water-soluble pillar[6]arene with sulfonic acid groups is much larger (approximately 700 times) than that of water-soluble pillar[6]arene with carboxyl groups. Therefore, it was revealed that 1-methylnicotinamide can be detected with much higher sensitivity when water-soluble pillar[6]arene with sulfonic acid groups is used compared to when water-soluble pillar[6]arene with carboxyl groups is used.

[0178] Experimental Example 2 Measurement of 1-methylnicotinamide concentration in mouse urine In this experiment, water-soluble pillar[6]arene having a sulfonic acid group was added to urine from wild-type mice, urine from NNMT knockout mice, and urine from wild-type mice that had drunk water containing nicotinamide to a concentration of 50 μM, and then the fluorescence intensity was measured to determine the 1-methylnicotinamide concentration. Each urine was diluted 300-fold with PBS before use. Note that no purification of the urine samples was performed.

[0179] Figure 5 is a graph showing the fluorescence intensity of wild-type mouse urine, NNMT knockout mouse urine, and a mixture of the urine of wild-type mice that drank water containing nicotinamide and a water-soluble pillar[6]arene having a sulfonate group. Figure 6 is a graph showing the results of measurement, using a liquid chromatograph mass spectrometer, of the concentrations of 1-methylnicotinamide in the urine of wild-type (WT) mice, the urine of NNMT knockout mice, and the urine of wild-type mice that drank water containing nicotinamide. Figure 7 is a graph showing the regression line between the fluorescence intensity of a mixture of mouse urine and a water-soluble pillar[6]arene having a sulfonate group and the concentration of 1-methylnicotinamide in urine measured by a liquid chromatograph mass spectrometer.

[0180] The data shown in Figure 7 are for urine samples from wild-type mice, NNMT knockout mice, and wild-type mice that had been given nicotinamide water, which was a wild-type mouse that had been given water containing nicotinamide at a concentration of 2 mg / mL in its water bottle.

[0181] As shown in Figure 5, while fluorescence from substances other than the water-soluble pillar[6]arene having a sulfonic acid group was measured around 380 nm, fluorescence from the water-soluble pillar[6]arene having a sulfonic acid group was measured around 310 nm. This suggests that the use of the water-soluble pillar[6]arene having a sulfonic acid group allows the concentration of 1-methylnicotinamide to be measured without a sample purification process.

[0182] The measured fluorescence intensity was highest in the urine of NNMT knockout mice, followed by the urine of wild-type mice that had drunk water without nicotinamide, and lowest in the urine of wild-type mice that had drunk water containing nicotinamide.

[0183] As shown in Figure 6, measurements using a liquid chromatograph mass spectrometer revealed that the concentration of 1-methylnicotinamide was highest in the urine of wild-type mice that drank water containing nicotinamide, followed by the urine of wild-type mice that drank water without nicotinamide, and lowest in the urine of NNMT knockout mice. This result is similar to the measurement results shown in Figure 5 using water-soluble pillar[6]arene with a sulfonate group.

[0184] As shown in Figure 7, a negative correlation was observed between the fluorescence intensity in urine from mice mixed with water-soluble pillar[6]arene having sulfonic acid groups and the 1-methylnicotinamide concentration measured by liquid chromatography-mass spectrometry. This indicates that the fluorescence derived from water-soluble pillar[6]arene having sulfonic acid groups is quenched in a concentration-dependent manner by 1-methylnicotinamide.

[0185] From the above results, it was found that by using water-soluble pillar[6]arene having a sulfonic acid group, results similar to those of concentration measurement by liquid chromatography mass spectrometry could be obtained without a purification step.

[0186] Experimental Example 3: Measurement of 1-methylnicotinamide Concentration Added to Human Urine In this experiment, samples were prepared by adding different amounts of 1-methylnicotinamide to diluted human urine. Water-soluble pillar[6]arene having a carboxyl or sulfonic acid group was then mixed with the diluted urine to confirm the fluorescence intensity at the wavelength derived from the water-soluble pillar[6]arene. Specifically, the fluorescence intensity of the diluted urine before the addition of the water-soluble pillar[6]arene was measured as a background value, and then water-soluble pillar[6]arene having a carboxyl or sulfonic acid group was added. The fluorescence intensity at the wavelength derived from the water-soluble pillar[6]arene was measured again by subtracting the background value. The urine samples were not purified. Table 1 below shows the gender and age of the six people from whom urine was collected.

[0187] Figure 8 is a graph showing the regression line between the fluorescence intensity at a wavelength of 330 nm derived from water-soluble pillar[6]arene having a carboxy group and the 1-methylnicotinamide concentration in the urine of six people mixed with 1-methylnicotinamide and water-soluble pillar[6]arene having a carboxy group. Figure 9 is a graph showing the regression line between the fluorescence intensity at a wavelength of 310 nm derived from water-soluble pillar[6]arene having a sulfonic acid group and the 1-methylnicotinamide concentration in the urine of six people mixed with 1-methylnicotinamide and water-soluble pillar[6]arene having a sulfonic acid group.

[0188] The vertical axis of Fig. 8 shows the fluorescence intensity at 330 nm of mixtures prepared by mixing water-soluble pillar[6]arene having a carboxy group with urine samples from six people containing 1-methylnicotinamide at various concentrations, while the vertical axis of Fig. 9 shows the fluorescence intensity at 310 nm of mixtures prepared by mixing water-soluble pillar[6]arene having a sulfonic acid group with urine samples from six people containing 1-methylnicotinamide at various concentrations.

[0189] The horizontal axis in FIGS. 8 and 9 shows the concentration of 1-methylnicotinamide measured by liquid chromatography mass spectrometry.

[0190] As shown in Figure 8, when a water-soluble pillar[6]arene having a carboxy group was mixed with a human urine sample, the fluorescence intensity varied, and the regression line between the fluorescence intensity and the 1-methylnicotinamide concentration included a mixture of lines that extended almost horizontally, lines that sloped downward to the right, and lines that sloped downward to the left, indicating that the detection sensitivity of the 1-methylnicotinamide concentration was low.

[0191] In contrast, as shown in Figure 9, the regression line between the fluorescence intensity and 1-methylnicotinamide concentration when water-soluble pillar[6]arene having a sulfonic acid group was mixed showed a downward slope for all urine samples, with the fluorescence intensity decreasing as the 1-methylnicotinamide concentration increased. This confirms that 1-methylnicotinamide concentration can be detected (measured) with high sensitivity using water-soluble pillar[6]arene having a sulfonic acid group, regardless of the patient's background (age and gender). It also became clear that 1-methylnicotinamide concentration can be detected without purifying urine.

[0192] Experimental Example 4 Measurement of Autofluorescence of Each Component Contained in Culture Medium In this experiment, the fluorescence emission intensity of all components contained in RPMI1640 culture medium in the wavelength range of 300 to 400 nm was confirmed.

[0193] When measuring the fluorescence intensity, each component of the medium was dissolved individually in Hank's balanced salt solution (HBSS, Sigma-Aldrich), and the fluorescence intensity was measured using an excitation light with a wavelength of 265 nm.

[0194] Figure 10 is a graph showing the fluorescence emission intensity in the wavelength range of 300 to 400 nm for all components contained in RPMI 1640 medium. In Figure 10, the fluorescence intensity of each component is represented by a different color, with lighter colors indicating particularly strong fluorescence intensity. PABA is p-aminobenzoic acid.

[0195] As shown in FIG. 10, among all the components contained in the RPMI 1640 medium, tyrosine, tryptophan, and p-aminobenzoic acid in particular exhibited particularly strong fluorescence emission intensities.

[0196] Based on this result, an Afr (autofluorescence-reduced) RPMI 1640 medium was prepared which does not contain tyrosine, tryptophan, or p-aminobenzoic acid but contains all other components.

[0197] Figure 11 is a graph showing the fluorescence intensity of RPMI 1640 medium, Afr RPMI 1640 medium, FBS, and water-soluble pillar[6]arene (50 μM) having a sulfonic acid group. P6AS refers to the water-soluble pillar[6]arene of formula (2) having a sulfonic acid group. Original refers to the conventional RPMI 1640 medium.

[0198] 11, it was confirmed that the fluorescence intensity in the Afr RPMI 1640 medium was significantly lower than that in the conventional RPMI 1640 medium. Based on this result, the Afr RPMI 1640 medium was used in the experiments using cultured cells described in detail below.

[0199] [Experimental Example 5] <Measurement of Fluorescence Intensity of Medium Derived from Water-Soluble Pillar[6]arene Having a Sulfonic Acid Group> In this experiment, 293T cells overexpressing wild-type NNMT (OE-NNMT cells) were prepared and cultured in Afr RPMI1640 medium, and the concentration of 1-methylnicotinamide in the medium was measured using a water-soluble pillar[6]arene having a sulfonic acid group or a liquid chromatography mass spectrometer.

[0200] In addition, normal (Parental) 293T cells and cells (OE-Y20A cells) engineered with a gene encoding an enzyme that lacks 1-methylnicotinamide-producing activity by adding the amino acid mutation Y20A to NNMT were also cultured in Afr RPMI1640 medium, and the concentration of 1-methylnicotinamide in the medium was measured using a water-soluble pillar[6]arene having a sulfonate group or a liquid chromatography mass spectrometer.

[0201] To measure the concentration of 1-methylnicotinamide in the medium using a water-soluble pillar[6]arene having a sulfonic acid group, the water-soluble pillar[6]arene having a sulfonic acid group was directly added to Afr RPMI1640 medium, and the fluorescence intensity was measured.

[0202] FIG. 12 is a graph showing the results of measuring the concentration of 1-methylnicotinamide contained in the medium after 48 hours of culture of 293T cells, OE-NNMT cells, and OE-Y20A cells using a liquid chromatograph mass spectrometer.

[0203] Figure 13 is a graph showing the results of measuring the concentration of 1-methylnicotinamide in the medium after 48 hours of culture of 293T cells, OE-NNMT cells, and OE-Y20A cells using a water-soluble pillar[6]arene having a sulfonate group. Figure 13 also shows the fluorescence spectra of the water-soluble pillar[6]arene having a sulfonate group in the medium after 48 hours of culture of 293T cells, OE-NNMT cells, and OE-Y20A cells.

[0204] As shown in Figure 12, the concentration of 1-methylnicotinamide was measured using a liquid chromatography mass spectrometer. As a result, 1-methylnicotinamide was detected in the medium after culturing OE-NNMT cells, whereas 1-methylnicotinamide was not detected in the medium after culturing normal 293T cells or the medium after culturing OE-Y20A cells.

[0205] On the other hand, as shown in Figure 13, the fluorescence intensity of the medium after culturing OE-NNMT cells was significantly weaker than the fluorescence intensity of the medium after culturing normal 293T cells and the medium after culturing OE-Y20A cells, confirming that the medium after culturing OE-NNMT cells had a higher concentration of 1-methylnicotinamide.

[0206] These results demonstrate that the concentration of 1-methylnicotinamide can be measured using water-soluble pillar[6]arene with a sulfonic acid group, even when the sample is a cell culture medium.

[0207] Figure 14 is a graph showing the results of measuring the 1-methylnicotinamide concentration in the medium during 48-hour culture of 293T cells and OE-NNMT cells with the addition of a water-soluble pillar[6]arene having a sulfonate group, and the regression curve. Figure 15 is a graph showing the results of measuring the 1-methylnicotinamide concentration in the medium during 48-hour culture of 293T cells and OE-NNMT cells with a liquid chromatograph mass spectrometer, and the regression curve. The horizontal axis of Figures 14 and 15 shows the culture time.

[0208] In measuring the 1-methylnicotinamide concentration in the medium by adding a water-soluble pillar[6]arene having a sulfonic acid group, the % inhibition was calculated using the following formula (I) to determine the 1-methylnicotinamide concentration: % inhibition = [1 - (F / F0)] x 100 (I)

[0209] Here, F0 is the fluorescence intensity of the medium of 293T cells in which 1-methylnicotinamide was not detected, and F is the fluorescence intensity of the medium of OE-NNMT cells. The concentration of 1-methylnicotinamide was estimated from a linear standard curve showing the correspondence between % inhibition and 1-methylnicotinamide concentration, which was prepared by serially diluting 1-MNA with the medium in which 293T cells were cultured (see Figure 14 for measurement results).

[0210] As shown in Figures 14 and 15, the difference between the 1-methylnicotinamide concentration measured by adding water-soluble pillar[6]arene and the 1-methylnicotinamide concentration measured using a liquid chromatograph mass spectrometer was small, and the regression curves were very similar.

[0211] These results demonstrate that the concentration of 1-methylnicotinamide can be accurately measured using water-soluble pillar[6]arene with a sulfonic acid group, even when cell culture medium is used as a sample.

[0212] Experimental Example 6 Estimation of 1-methylnicotinamide Blood Concentration After Nicotinamide Ingestion In this experiment, the 1-methylnicotinamide concentration in random urine samples excreted at different times and the plasma concentration of 1-methylnicotinamide were measured from a subject who had ingested (administered) 250 mg of nicotinamide (Nam). The random urine and blood samples were collected almost simultaneously, with a time difference of less than 5 minutes. The random urine and blood samples were collected before nicotinamide ingestion (0 hours), and 2.5 hours, 5 hours, 10 hours, 24 hours, 34 hours, and 48 hours after ingestion.

[0213] Figure 16 is a diagram showing the metabolic pathways of various niacin-related substances to 1-methylnicotinamide. Figure 17 is a graph showing the time course of 1-methylnicotinamide concentrations in random urine after nicotinamide intake. Figure 18 is a graph showing the time course of 1-methylnicotinamide blood concentrations after nicotinamide intake.

[0214] The 1-methylnicotinamide concentration shown on the vertical axis of Fig. 17 is a value corrected by dividing the urinary 1-methylnicotinamide concentration value measured using a liquid chromatograph mass spectrometer by the urinary creatinine concentration value also measured using a liquid chromatograph mass spectrometer. The 1-methylnicotinamide concentration shown on the vertical axis of Fig. 18 is a concentration measured using a liquid chromatograph mass spectrometer.

[0215] 17 and 18, the blood concentration of 1-methylnicotinamide after nicotinamide ingestion fluctuated in sync with the concentration of 1-methylnicotinamide in random urine. In particular, the peaks of the 1-methylnicotinamide concentration in random urine and the blood concentration of 1-methylnicotinamide both coincided 2.5 hours after nicotinamide ingestion.

[0216] These results demonstrate that the time course of changes in blood concentration of 1-methylnicotinamide can be estimated with high accuracy based on the changes in 1-methylnicotinamide concentration in multiple random urine samples excreted at different times.

[0217] Experimental Example 7 Estimation of Blood Concentration of 1-methylnicotinamide After Ingestion of Nicotinamide Mononucleotide In this experiment, the 1-methylnicotinamide concentration in random urine samples excreted at different times and the blood (plasma) concentration of 1-methylnicotinamide were measured from a subject who had ingested (administered) 250 mg of nicotinamide mononucleotide (NMN). Random urine and blood samples were collected before (0 hour), and 2.5 hours, 5 hours, 10 hours, 24 hours, 34 hours, and 48 hours after ingestion of nicotinamide mononucleotide.

[0218] Figure 19A is a graph showing the time course of 1-methylnicotinamide concentration in random urine after a subject ingested nicotinamide mononucleotide. Figure 19B is a graph showing the time course of 1-methylnicotinamide concentration in blood after the same subject as in Figure 19A ingested nicotinamide mononucleotide. The random urine sample shown in Figure 19A and the blood sample shown in Figure 19B were collected almost simultaneously, with a time difference of less than 5 minutes.

[0219] Figure 20A is a graph showing the time course of 1-methylnicotinamide concentration in random urine from a different subject after taking nicotinamide mononucleotide. Figure 20B is a graph showing the time course of 1-methylnicotinamide concentration in blood from the same subject as in Figure 20A after taking nicotinamide mononucleotide. The random urine sample shown in Figure 20A and the blood sample shown in Figure 20B were collected almost simultaneously, with a time difference of within 5 minutes.

[0220] The 1-methylnicotinamide concentrations shown on the vertical axes of Figures 19A and 20A are values ​​corrected by dividing the urinary 1-methylnicotinamide concentration value measured using a liquid chromatograph mass spectrometer by the urinary creatinine concentration value also measured using a liquid chromatograph mass spectrometer. The 1-methylnicotinamide concentrations shown on the vertical axes of Figures 19B and 20B are concentrations measured using a liquid chromatograph mass spectrometer.

[0221] As shown in Figures 19A and 19B and Figures 20A and 20B, it was confirmed that even after ingestion of nicotinamide mononucleotide, the blood concentration of 1-methylnicotinamide fluctuated in sync with the concentration of 1-methylnicotinamide in random urine. In particular, the peaks of the 1-methylnicotinamide concentration in the random urine of the subjects shown in Figures 19A and 19B and the peaks of the 1-methylnicotinamide blood concentration both coincided 5 hours after ingestion of nicotinamide mononucleotide. Furthermore, the peaks of the 1-methylnicotinamide concentration in the random urine of the subjects shown in Figures 20A and 20B and the peaks of the 1-methylnicotinamide blood concentration both coincided 24 hours after ingestion of nicotinamide mononucleotide. This accuracy far exceeds expectations based on conventional technical common sense.

[0222] [Experimental Example 8] <Estimation of blood concentration of 1-methylnicotinamide after ingestion of nicotinic acid> In this experiment, the 1-methylnicotinamide concentration in random urine samples excreted at different times and the blood (plasma) concentration of 1-methylnicotinamide were measured from a subject who had ingested (administered) 50 mg of nicotinic acid. The random urine sample and the blood sample were collected almost simultaneously, with a time difference of within 5 minutes. The random urine and blood samples were collected before nicotinic acid ingestion (0 hour), and 5 hours, 10 hours, and 24 hours after ingestion.

[0223] Figure 21 is a graph showing the time course of 1-methylnicotinamide concentration in random urine after ingestion of nicotinic acid. Figure 22 is a graph showing the time course of 1-methylnicotinamide concentration in blood after ingestion of nicotinic acid.

[0224] The 1-methylnicotinamide concentration shown on the vertical axis of Figure 21 is a value corrected by dividing the urinary 1-methylnicotinamide concentration value measured using a liquid chromatograph mass spectrometer by the urinary creatinine concentration value also measured using a liquid chromatograph mass spectrometer. The 1-methylnicotinamide concentration shown on the vertical axis of Figure 22 is a concentration measured using a liquid chromatograph mass spectrometer.

[0225] 21 and 22, the blood concentration of 1-methylnicotinamide after nicotinic acid intake fluctuated in sync with the concentration of 1-methylnicotinamide in random urine. In particular, the peaks of the 1-methylnicotinamide concentration in random urine and the blood concentration of 1-methylnicotinamide both coincided 5 hours after nicotinic acid intake.

[0226] The above results suggest that, regardless of the type of ingested substance, it is possible to predict with high accuracy the change in blood concentration of 1-methylnicotinamide over time based on the change in 1-methylnicotinamide concentration in multiple random urine samples excreted at different times.

[0227] Experimental Example 9 Confirmation of the effect on 1-methylnicotinamide concentration of random urine samples stored at room temperature (10 to 25°C) for 3 days In this experiment, it was confirmed whether there was a difference in 1-methylnicotinamide concentration between random urine samples stored at room temperature (10 to 25°C) for 3 days and random urine samples stored at -30°C for 3 days. The number of samples used was n=4. The samples used were Sample 1, which showed a high 1-methylnicotinamide concentration, Samples 3 and 4, which showed low values, and Sample 2, which showed an intermediate value.

[0228] FIG. 23 is a graph showing the concentration of 1-methylnicotinamide in random urine samples stored at room temperature of 10 to 25° C. for 3 days and the concentration of 1-methylnicotinamide in random urine samples stored at −20° C. for 3 days.

[0229] 23, there was a slight variation in the 1-methylnicotinamide concentration in sample 1 between storage at room temperature of 10 to 25° C. and storage at −20° C., but there was almost no difference in the 1-methylnicotinamide concentration in samples 2 to 4. This demonstrates that even when random urine samples were stored at room temperature of 10 to 25° C. for 3 days, there was essentially no difference in the 1-methylnicotinamide concentration between before and after storage, and that the 1-methylnicotinamide concentration was stable even at room temperature.

[0230] Experimental Example 10 In this experiment, an NMR spectrum was obtained by nuclear magnetic resonance when a water-soluble pillar[6]arene having a phosphonic acid group was mixed with 1-methylnicotinamide.

[0231] Figure 24 shows the NMR spectrum of a water-soluble pillar[6]arene having a phosphonic acid group, the NMR spectrum of 1-methylnicotinamide, and the NMR spectrum of a mixture of a water-soluble pillar[6]arene having a phosphonic acid group and 1-methylnicotinamide. ２ 0) and the peaks obtained when a water-soluble pillar[6]arene having a phosphonate group was soaked in deuterium oxide (DO) at a concentration of 5 mM.

[0232] As shown in Figure 24, when a water-soluble pillar[6]arene having a phosphonic acid group (the compound represented by the above formula (3), P6AP in the figure) was mixed with 1-methylnicotinamide, the proton signal was broadened. This result confirmed the complexation of the water-soluble pillar[6]arene having a phosphonic acid group with 1-methylnicotinamide.

[0233] Experimental Example 11 In this experiment, the strength of the bond between a water-soluble pillar[6]arene having a phosphonic acid group and 1-methylnicotinamide was confirmed by isothermal titration calorimetry.

[0234] Figure 25 is a graph showing raw data obtained by isothermal titration calorimetry of a water-soluble pillar[6]arene having a phosphonate group. Figure 26 is a graph showing a binding isotherm obtained from the raw data shown in Figure 25. Figure 25 also shows the titration results of a water-soluble pillar[6]arene having a phosphonate group at a concentration of 0.08 mM and 1-methylnicotinamide at a concentration of 0.25 mM.

[0235] FIG. 27 is a diagram showing the binding constants K a of water-soluble pillar[6]arene having a phosphonic acid group, water-soluble pillar[6]arene having a sulfonic acid group, or water-soluble pillar[6]arene having a carboxylic acid group with 1-methylnicotinamide, calculated from the binding isotherms shown in FIG. 26 .

[0236] As shown in Figure 27, the binding constant K a of the water-soluble pillar[6]arene having a phosphonic acid group as X with 1-methylnicotinamide was approximately 1000 times that of the water-soluble pillar[6]arene having a carboxylic acid group instead of X, and approximately 1.4 times that of the water-soluble pillar[6]arene having a sulfonic acid group as X.

[0237] These results demonstrate that water-soluble pillar[6]arenes bearing phosphonic acid groups bind to 1-methylnicotinamide as strongly as or more strongly than water-soluble pillar[6]arenes bearing sulfonic acid groups.

[0238] [Experimental Example 12] In this experiment, the detection limits of 1-methylnicotinamide concentration using water-soluble pillar[6]arene having a phosphonic acid group and water-soluble pillar[6]arene having a sulfonic acid group were confirmed.

[0239] FIG. 28 is a graph showing the fluorescence intensity of water-soluble pillar[6]arene having a sulfonic acid group or water-soluble pillar[6]arene having a phosphonic acid group at various concentrations of 1-methylnicotinamide.

[0240] 28 shows the average fluorescence intensity (n=5) of aqueous solutions containing water-soluble pillar[6]arene having a sulfonic acid group or water-soluble pillar[6]arene having a phosphonic acid group and 1-methylnicotinamide at various 1-methylnicotinamide concentrations. For example, 1 eq (equivalent) is a mixture of 5 μM water-soluble pillar[6]arene and 5 μM 1-methylnicotinamide, and 0.2 eq is a mixture of 5 μM water-soluble pillar[6]arene and 1 μM 1-methylnicotinamide.

[0241] As shown in FIG. 28, in the case of water-soluble pillar[6]arene having a phosphonate group, even 0.05 eq (0.25 μM) of 1-methylnicotinamide could be detected.

[0242] Figure 29 is a graph showing the fluorescence intensity and regression line for each concentration of 1-methylnicotinamide in a mixture of 1-methylnicotinamide and a water-soluble pillar[6]arene having a sulfonic acid group. Figure 30 is a graph showing the fluorescence intensity and regression line for each concentration of 1-methylnicotinamide in a mixture of 1-methylnicotinamide and a water-soluble pillar[6]arene having a phosphonic acid group.

[0243] The fluorescence intensity ratio F / F on the vertical axis in FIGS. 29 and 30 ０ The "F" in the above table means the fluorescence intensity when 1-methylnicotinamide is mixed with a water-soluble pillar[6]arene having a sulfonic acid group or a water-soluble pillar[6]arene having a phosphonic acid group at each concentration. ０ " means the fluorescence intensity of an aqueous solution containing only water-soluble pillar[6]arene having a sulfonic acid group or water-soluble pillar[6]arene having a phosphonic acid group.

[0244] As shown in Figures 29 and 30, the fluorescence intensity ratio decreased steadily with increasing 1-methylnicotinamide concentration. This result suggests that the 1-methylnicotinamide concentration can be accurately measured using either water-soluble pillar[6]arene with sulfonic acid groups or water-soluble pillar[6]arene with phosphonic acid groups.

[0245] Furthermore, the detection limit for water-soluble pillar[6]arene containing sulfonic acid groups was 2.84 × 10 －７ M, whereas the detection limit of water-soluble pillar[6]arene with phosphonic acid groups is 1.42 × 10 －７ M, which was equivalent to or slightly lower than the sulfonic acid group.

[0246] According to the present invention, a water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group strongly binds to 1-methylnicotinamide, and therefore the concentration of 1-methylnicotinamide can be measured simply and accurately using the water-soluble pillar[6]arene, which is industrially applicable. Furthermore, by using a water-soluble pillar[6]arene having an anionic group or a salt thereof that is stronger than a carboxy group, it is possible to simply and accurately screen candidate substances for NNMT inhibitors to determine the extent to which they inhibit NNMT, which is industrially applicable. Furthermore, according to the present invention, the time course of 1-methylnicotinamide concentration in random urine coincides with the time course of 1-methylnicotinamide blood concentration, and therefore, by measuring the 1-methylnicotinamide concentration in random urine contained in multiple random urine samples excreted at different times, it is possible to accurately estimate the time course of 1-methylnicotinamide blood concentration, which is industrially applicable.

[0247] 100... column, 10... carrier

Claims

1. A method for measuring the concentration of 1-methylnicotinamide contained in a sample, comprising the steps of: mixing the sample with a water-soluble pillar[6]arene to obtain a mixture; and measuring the fluorescence intensity of the mixture, wherein the water-soluble pillar[6]arene has an anionic group or a salt thereof that is stronger than a carboxy group.

2. The method for measuring 1-methylnicotinamide concentration according to claim 1, wherein the water-soluble pillar[6]arene has a cyclic structure in which six benzene rings are bonded via methylene groups.

3. The method for measuring 1-methylnicotinamide concentration according to claim 2, wherein the water-soluble pillar[6]arene is a compound represented by the following general formula (1) or a salt thereof: (In the general formula (1), X is the anionic group.) 4. A method for measuring 1-methylnicotinamide concentration according to any one of claims 1 to 3, wherein the anionic group is a sulfonic acid group or a phosphonic acid group.

5. A method for measuring 1-methylnicotinamide concentration according to any one of claims 1 to 3, wherein the sample is a biological sample.

6. The method for measuring 1-methylnicotinamide concentration according to claim 5, wherein the biological sample is urine.

7. A method for measuring 1-methylnicotinamide concentration according to any one of claims 1 to 3, further comprising a step of purifying the sample or the mixture prior to the step of measuring the fluorescence intensity.

8. A kit for measuring the concentration of 1-methylnicotinamide, comprising a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof.

9. The kit for measuring 1-methylnicotinamide concentration according to claim 8, wherein the water-soluble pillar[6]arene has a cyclic structure in which six benzene rings are bonded via methylene groups.

10. The kit for measuring 1-methylnicotinamide concentration according to claim 9, wherein the water-soluble pillar[6]arene is a compound represented by the following general formula (1) or a salt thereof: (In the general formula (1), X is the anionic group.) 11. The kit for measuring 1-methylnicotinamide concentration according to any one of claims 8 to 10, wherein the anionic group is a sulfonic acid group or a phosphonic acid group.

12. A kit for measuring the concentration of 1-methylnicotinamide according to any one of claims 8 to 10, further comprising a column for adsorbing hydrophobic compounds.

13. A method for screening for an NNMT inhibitor, comprising the steps of: contacting nicotinamide methyltransferase (NNMT) with a candidate substance for an NNMT inhibitor in the presence of S-adenosylmethionine and nicotinamide to obtain a sample; obtaining a mixture of the sample obtained in the sample obtaining step and a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof; and measuring the fluorescence intensity of the mixture.

14. A method for screening for an NNMT activator, comprising the steps of: contacting nicotinamide methyltransferase (NNMT) with a candidate substance for an NNMT activator in the presence of S-adenosylmethionine and nicotinamide to obtain a sample; obtaining a mixture of the sample obtained in the sample obtaining step and a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof; and measuring the fluorescence intensity of the mixture.

15. A reagent for measuring the concentration of 1-methylnicotinamide, comprising a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof.

16. A measuring reagent for use in the method for measuring the concentration of 1-methylnicotinamide according to any one of claims 1 to 3, comprising a water-soluble pillar[6]arene having an anionic group stronger than a carboxy group or a salt thereof.

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