NMNH calcium salt and crystal form and application thereof

Abstract

The invention relates to a calcium salt NMNH-Ca of reduced beta-nicotinamide mononucleotide, a preparation method and a crystal form of the calcium salt NMNH-Ca, and application of the calcium salt NMNH-Ca in acute kidney injury protection. Reduced beta-nicotinamide mononucleotide disodium salt is used as a raw material and reacts with calcium chloride in an aqueous solution, and the NMNH calcium salt NMNH-Ca is obtained through crystallization and purification. The crystal form A of the NMNH-Ca uses CuK alpha radiation, and an X-ray powder diffraction spectrum expressed by a 2 theta angle at least has diffraction peaks at 7.145 degrees + / -0.2 degrees, 12.964 degrees + / -0.2 degrees, 18.320 degrees + / -0.2 degrees and 25.756 degrees + / -0.2 degrees. The NMNH-Ca disclosed by the invention is convenient to prepare, good in stability, convenient to purify, store and transport, and suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a reduced β-nicotinamide mononucleotide calcium salt (NMNH-Ca) calcium salt, a preparation method, a crystal form and a use thereof. Background Art

[0002] NMNH, or reduced β-nicotinamide mononucleotide, is a natural compound present in cells and is a direct precursor of nicotinamide adenine dinucleotide (NAD+). It has a better NAD+ promoting effect than NMN and other biological functions such as increasing cellular antioxidant capacity, reducing fat accumulation, reducing inflammatory response and inhibiting tumor cell growth. It is a health promotion agent with significant commercial potential. NAD+ plays many important roles in cells, including being a key coenzyme in cellular energy metabolism, participating in DNA repair, cell signaling, and as an antioxidant in cells.

[0003]

[0004] NAD+ is a key molecule in cellular energy metabolism. It participates in the redox reaction in the cellular respiratory chain, helping cells convert food into energy. NAD+ participates in the repair process of DNA damage and helps maintain the stability of the genome. NAD+ also plays an important role in cell signaling, and it participates in regulating cell growth, differentiation, and apoptosis. NAD+ can act as an antioxidant to help cells resist oxidative stress.

[0005] As we age, the level of NAD+ in the human body gradually decreases, which is associated with a variety of aging-related physiological changes. Therefore, supplementing with NMN or NAD+ is thought to help increase the level of NAD+ in cells, thereby potentially fighting aging and some age-related diseases.

[0006] In recent years, the study of NMNH has become one of the hot topics in the health field, and its specific role and mechanism in human health and disease treatment still need more scientific research to verify.

[0007] Most NMNH products on the market are amorphous solids obtained by freeze-drying aqueous solutions, which are easy to absorb moisture, have poor fluidity, and are easy to degrade. The salt form of NMNH is mainly sodium salt. For example, the patent document with publication number CN115368423A reports a polymorphic form of reduced NMNH sodium salt and its preparation method and use. Experiments have shown that the thermal stability of this crystalline form is still poor and it is difficult to store for a long time.

[0008] Therefore, the art needs to develop new salt forms and crystal forms of NMNH to meet the needs of long-term storage and convenient transportation. Summary of the invention

[0009] The inventors have studied the salt form and crystal form (including monocrystalline and polymorphic forms) of NMNH, including sodium salt, potassium salt, calcium salt, magnesium salt, iron salt, zinc salt, etc. of NMNH. Through experimental comparison, it is found that the physicochemical properties of the calcium salt of NMNH (abbreviated as NMNH-Ca) are superior to those of the sodium salt (disodium salt, abbreviated as NMNH-2Na), and it also has new characteristics in physiological functions, such as being able to improve the condition of acute kidney injury. Therefore, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a reduced β-nicotinamide mononucleotide calcium salt (NMNH-Ca) as shown in formula I or a reduced β-nicotinamide mononucleotide calcium salt hydrate as shown in formula II,

[0011]

[0012] Wherein x refers to all possible values ​​between 1 and 9, including integers and non-integers. For example, x can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.

[0013] In one embodiment, the water content in the reduced β-nicotinamide mononucleotide calcium salt hydrate (II) is 5 to 30 wt %, preferably 20 to 25 wt %.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned reduced β-nicotinamide mononucleotide calcium salt (NMNH-Ca), comprising the following steps: using the reduced β-nicotinamide mononucleotide disodium salt (NMNH-2Na) shown in formula III as a raw material, reacting with calcium chloride in an aqueous solution, and obtaining the reduced β-nicotinamide mononucleotide calcium salt (I) by crystallization and purification.

[0015]

[0016] The reaction temperature of the above method is 10-40°C, preferably 20-25°C.

[0017] In the above method, during crystallization, it is preferred to add a small amount of Form A to the mother liquor as a seed crystal for induction, and it is more preferred to carry out the process at a relatively low temperature such as 5-10°C.

[0018] In a third aspect, the present invention provides a crystalline form of the reduced β-nicotinamide mononucleotide calcium salt hydrate, named Form A, which has an X-ray powder diffraction spectrum using CuKα radiation and expressed in 2θ angles at least at 7.145°±0.2°, 12.964°±0.2°, 18.320°±0.2° and 25.756°±0.2°; preferably, at least at 2θ values ​​of 7.145°±0.1°, 12.964°±0.1°, 18.320°±0.1° and 25.756°±0.1°; more preferably, at least at 2θ values ​​of about 7.145°, 12.964°, 18.320° and 25.756°.

[0019] Furthermore, the crystalline form also has diffraction peaks at 2θ values ​​of 9.752°±0.2°, 15.934°±0.2°, 21.352°±0.2° and 22.081°±0.2°; preferably, diffraction peaks are present at 2θ values ​​of 9.752°±0.1°, 15.934°±0.1°, 21.352°±0.1° and 22.081°±0.1°; more preferably, diffraction peaks are present at 2θ values ​​of approximately 9.752°, 15.934°, 21.352° and 22.081°.

[0020] Further, the crystalline form also has diffraction peaks at 2θ values ​​of 14.723°±0.2°, 16.965°±0.2°, 23.552°±0.2° and 28.665°±0.2°; preferably, diffraction peaks are present at 2θ values ​​of 14.723°±0.1°, 16.965°±0.1°, 23.552°±0.1° and 28.665°±0.1°; more preferably, diffraction peaks are present at 2θ values ​​of approximately 14.723°, 16.965°, 23.552° and 28.665°. Preferably, the XRPD pattern of the crystalline form A is substantially as follows Figure 1 shown.

[0021] Preferably, in DSC analysis, the crystalline form has an endothermic peak at about 81.56°C and an exothermic peak at about 238.06°C; preferably, the DSC spectrum of the crystalline form A is substantially as follows: Figure 4 shown.

[0022] Preferably, in TGA analysis, the crystalline form loses about 19.882% of weight at 50-200°C and about 38.385% of weight at 200-500°C; preferably, its TGA spectrum is substantially as follows Figure 5 shown.

[0023] It should be understood that when expressing numerical characteristics in this article, the terms "about", "approximately" or "around" mean that the number represented may have an error range or floating range of ±10%, ±8%, ±6%, ±4% or ±2%.

[0024] In a fourth aspect, the present invention provides uses of the reduced β-nicotinamide mononucleotide calcium salt (NMNH-Ca), reduced β-nicotinamide mononucleotide calcium salt hydrate and crystal form A as described above in the fields of medicine, food and beauty.

[0025] As a specific embodiment, the present invention provides a pharmaceutical composition, health food or cosmetic, which comprises the reduced β-nicotinamide mononucleotide calcium salt (NMNH-Ca) or reduced β-nicotinamide mononucleotide calcium salt hydrate as described above, or the crystalline form A as described above as an active ingredient.

[0026] The above-mentioned pharmaceutical composition, health food or cosmetics can be used for anti-aging, that is, treating and / or preventing age-related degenerative diseases; or the pharmaceutical composition is used to treat acute kidney injury.

[0027] Compared with the reduced β-nicotinamide mononucleotide sodium salt or its crystal form in the prior art, the NMNH-Ca and its crystal form A of the present invention have better overall moisture and heat stability, lower hygroscopicity, and are convenient for long-term storage and transportation. Animal experiments on mice have shown that NMNH-Ca crystal form A can also be used to protect the kidneys, improve the condition of acute kidney injury and improve the treatment prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the X-ray powder diffraction pattern (XPRD pattern) of NMNH-Ca crystal form A of the present invention.

[0029] Figure 2 This is the infrared spectrum (IR) of NMNH-Ca crystal form A of the present invention.

[0030] Figure 3 It is the mass spectrum (MS) of NMNH-Ca crystal form A of the present invention.

[0031] Figure 4 The differential scanning calorimetry diagram (DSC diagram) of the NMNH-Ca crystal form A of the present invention is shown in Figure 1. The abscissa is temperature (°C); the ordinate is heat flow (W / g).

[0032] Figure 5 This is a thermogravimetric analysis diagram (TGA diagram) of NMNH-Ca crystal form A of the present invention.

[0033] Figure 6 The NMR spectrum of the NMNH-Ca crystal form A of the present invention is ( 1 1H NMR).

[0034] Figure 7 The NMR carbon spectrum of NMNH-Ca crystal form A of the present invention is ( 13 CNMR).

[0035] Figure 8 The effects of NMN, NMNH-Ca, and NAM on plasma creatinine (Crea) and urea nitrogen (BUN) in acute renal injury induced by ischemia-reperfusion are shown. Asterisks in the statistical bar graphs indicate statistically different values ​​(Student's ttest, *, compared with the model control group (I / R), p < 0.05; **, compared with the model control group (I / R), p < 0.01; *** P<0.001). DETAILED DESCRIPTION

[0036] In order to solve the defects of NMNH solid such as easy moisture absorption, difficulty in long-term storage, and poor fluidity, the salt form and specific crystal form of NMNH are commonly used. Each metal salt form, such as sodium salt, potassium salt, calcium salt, magnesium salt, iron salt and zinc salt, has its own characteristics.

[0037] The calcium salt of NMNH, including its hydrate with a crystalline structure, has relatively high moisture and heat stability and low hygroscopicity. An unexpected discovery is that the calcium salt of NMNH, including crystalline form A, can be used for acute kidney injury and has a certain protective effect on acute kidney injury induced by renal ischemia-reperfusion.

[0038] In our study of mouse model, we investigated the improvement effect of NMNH-Ca on acute renal injury induced by renal ischemia-reperfusion in mice by oral pre-administration and compared it with β-nicotinamide mononucleotide (NMN) and nicotinamide (NAM).

[0039] During the experiment, normal C57BL / 6J mice (male) were used and divided into sham operation group (9 mice), ischemia reperfusion model group (13 mice), β-nicotinamide mononucleotide (NMN) 400mg / kg (13 mice), reduced β-nicotinamide mononucleotide calcium salt (NMNH-Ca) 400mg / kg (13 mice), nicotinamide (NAM) 400mg / kg (13 mice) according to body weight. Except for the NAM group, which was intraperitoneally injected (ip), the animals in other groups were orally administered (po), with a volume of 10mL / kg, 3 days and 1h before ischemia reperfusion. The surgical procedure was as follows: the back of the mouse was opened, and the right and left kidneys were clamped with artery clamps respectively, and the ischemia lasted for 22min; the mice in the sham operation group were not subjected to artery clamping. 24 hours after ischemia-reperfusion, the animals were anesthetized and plasma and kidneys were collected. The levels of plasma creatinine and urea nitrogen were measured by biochemical analyzer. The kidneys were weighed, some were frozen at -80℃, and some were fixed with 4% paraformaldehyde.

[0040] The results showed that NMN 400mg / kg significantly reduced the plasma creatinine and urea nitrogen levels 24h after ischemia-reperfusion; NMNH-Ca 400mg / kg significantly reduced the plasma creatinine and urea nitrogen levels 24h after ischemia-reperfusion; NAM 400mg / kg significantly reduced the plasma creatinine and urea nitrogen levels 24h after ischemia-reperfusion. That is, under this experimental condition, NMN 400mg / kg / day (po), NMNH-Ca 400mg / kg / day (po), NAM 400mg / kg / day (ip) pre-administered 3 days in advance all showed a certain protective effect on acute renal injury induced by renal ischemia-reperfusion.

[0041] Crystalline form A of NMNH-Ca (hereinafter referred to as crystal form A) belongs to NMNH-Ca hydrate. Hydrate is a special solvate. In the pharmaceutical industry, hydrates are worth discussing separately because of their particularity, whether in the synthesis of raw materials, drug preparations, drug storage, and drug activity evaluation.

[0042] An important indicator for characterizing the crystal form A of the present invention is the X-ray powder diffraction pattern (XPRD), such as Figure 1 As shown, the specific data are the diffraction peak position, i.e. the diffraction angle 2θ (°), the crystal plane spacing d The diffraction peak height (cts) and diffraction peak relative intensity (I / Imax) are summarized in Table 1.

[0043] Table 1. XPRD characteristics of NMNH calcium salt hydrate form A

[0044]

[0045]

[0046] The term "relative intensity" refers to the ratio of the intensity of other peaks to the intensity of the highest intensity peak among all diffraction peaks in an X-ray powder diffraction pattern when the intensity of the highest intensity peak is 100%.

[0047] In a preferred embodiment, the crystalline form A of the present invention has Figure 1 The X-ray powder diffraction spectrum shown in is essentially the same as the X-ray powder diffraction pattern.

[0048] The term "substantially the same" means that at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the peaks in the X-ray powder diffraction pattern appear in the exemplary X-ray powder diffraction spectrum given. Figure 1 middle.

[0049] It is well known to those skilled in the art that various factors may cause the difference between Form A and the above Table 1 and Figure 1There are measured data and / or differences between the X-ray powder diffraction analysis results shown in: (a) errors in sample preparation (e.g., sample height), (b) instrument errors, (c) calibration differences, (d) operator errors (including errors that occur when determining peak positions), and (e) the nature of the material (e.g., preferred orientation errors). Calibration errors and sample height errors often result in displacements of all peaks in the same direction. When a flat support is used, small differences in sample height will result in large displacements of XRPD peak positions. Systematic studies have shown that sample height differences of 1 mm can result in peak displacements of up to 1° of 2θ. These displacements can be identified from X-ray diffraction patterns and can be eliminated by compensating for the displacements (using the system calibration factor for all peak position values) or recalibrating the instrument. As described above, by applying the system calibration factor to make the peak positions consistent, measurement errors from different instruments can be corrected.

[0050] The present invention also analyzes the crystal form A by differential scanning calorimetry. When using differential scanning calorimetry for analysis, it is shown that there is an endothermic peak at 81.56°C in the DSC thermal analysis diagram, and the enthalpy change value is 516.3 J / g, which is caused by the endothermic absorption of water molecule evaporation; there is an exothermic peak at 238.06°C, and the enthalpy change value is -8866.3 J / g, which is formed by the decomposition and exothermic release of the crystal form A of the NMNH calcium salt. The DSC spectrum of the crystal form A is basically as follows Figure 4 It should be understood that similar to the situation with X-ray powder diffraction patterns, the values ​​quoted for differential scanning calorimetry techniques may vary and should not be interpreted as absolute values.

[0051] In addition, the present invention also analyzes the crystal form A by TGA thermogravimetric analysis technology. When using TGA technology for analysis, 50-200°C in the TGA spectrum is the first step, and the weight loss is 19.882%, which is caused by the loss of adsorbed water or crystal water. 200-500°C is the second step, and the weight loss is 38.385%, which is caused by the decomposition of the crystal form A of the NMNH calcium salt. The TGA spectrum of the crystal form A of the present invention is basically as follows Figure 5 It should be understood that, similar to the situation where there may be deviations in the values ​​of X-ray powder diffraction patterns, the values ​​quoted for TGA thermogravimetric analysis techniques cannot be interpreted as absolute values.

[0052] The method for preparing the crystal form A of the present invention is very simple, and reduced β-nicotinamide mononucleotide disodium salt (NMNH-2Na) and calcium chloride can be reacted in an aqueous solution at room temperature, and the crystal form A can be obtained after crystallization and purification.

[0053] During the crystallization process, it is preferred to add a small amount of Form A to the mother liquor as a seed crystal to induce the production of more Form A. It is more preferred to carry out the process at a lower temperature such as 5-10°C.

[0054] Based on the health and medical effects of NMNH-Ca, the anhydrous reduced β-nicotinamide mononucleotide calcium salt, or reduced β-nicotinamide mononucleotide calcium salt hydrate, or the crystalline form A as described above can be used as an anti-aging active ingredient or as an active ingredient for treating acute kidney injury for preparing pharmaceutical compositions, health foods or cosmetics.

[0055] It is easy for those skilled in the art to understand that the dosage form of the pharmaceutical composition, health food or cosmetics can be an oral preparation, an injection, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form or a cavity administration dosage form. Specific dosage forms include, but are not limited to, tablets, capsules, granules, aqueous solutions, enteric preparations, injections, slurries, emulsions, creams, foams, sprays, ointments, gels, lotions, pads, roll-on formulations, caplets, lozenges, lozenges, chewable tablets, gels, jelly agents, syrups, liquid solutions, suspensions, buccal films, sublingual films, oral adhesive films, powders, solid crystals, orally disintegrating tablets or pastes.

[0056] In one embodiment, the pharmaceutical composition, health food or cosmetic contains a therapeutically effective amount of Form A and further contains a pharmaceutically acceptable excipient or carrier, an excipient or carrier acceptable in health food, or an excipient or carrier acceptable in cosmetics.

[0057] The term "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (Tween), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0058] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and representative dosage forms include, but are not limited to, oral, injection (parenteral), external use such as transdermal absorption, and the like.

[0059] Oral solid dosage forms include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active ingredient NMNH-Ca, such as Form A, is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, such as microcrystalline cellulose, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrants (e) buffers, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.

[0060] Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient NMNH-Ca, such as Form A, can also be formed into microcapsules with one or more of the above-mentioned excipients.

[0061] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0062] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0063] In addition to the active ingredient NMNH-Ca, for example, Form A, the suspension may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

[0064] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0065] External dosage forms include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required when necessary.

[0066] The present invention is further described below by examples. It should be understood that these examples are only for illustrative purposes and are not intended to limit the present invention. Various changes or adjustments made by those skilled in the art based on the present invention should all fall within the scope of protection of the present invention.

[0067] Example

[0068] In the embodiments of the present invention, if there is no specific description on the experimental operation temperature, the temperature generally refers to room temperature (15-35° C.).

[0069] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, are all percentages by weight.

[0070] Reagents: The reactants used in the examples of the present invention are all chemically pure and can be used directly or after simple purification as needed; organic solvents are all analytically pure and used directly. Reagents were purchased from Aladdin Chemical Reagent Company.

[0071] X-ray powder diffraction:

[0072] X-ray powder diffraction analysis was performed on a Thermo Fisher Scientific powder X-ray diffractometer using CuKα radiation with a test power of 45 kV×40 mA, a scanning speed of 5° / min, a step width of 0.02°, and a θ~2θ continuous scan in the scanning range of 3~40° (2θ).

[0073] Infrared spectroscopy (IR) characterization:

[0074] Fourier transform infrared absorption spectrometer (FTIR), instrument model: Nicolet iS10, was used for testing after potassium bromide was ground and pressed into tablets.

[0075] Differential Scanning Calorimetry (DSC) Characterization:

[0076] The measurement was performed using a TA Q2000 / 2500 differential scanning calorimeter with nitrogen as the protective gas, a heating rate of 10°C / min, and a temperature gradually rising from 25°C to the set end point.

[0077] Thermogravimetric analysis (TGA):

[0078] The measurement was performed using a TA company's Q5000 / 5500 thermogravimetric analyzer with nitrogen as the protective gas, a heating rate of 10°C / min, and a temperature gradually rising from room temperature to a set end point.

[0079] NMR instrument models: Bruker AV-500 and Bruker AV-600.

[0080] Mass spectrometer (liquid chromatography-mass spectrometry (LC / MS)), model: Agilent 6120B, detector: DAD.

[0081] Example 1: Preparation of NMNH calcium salt form A

[0082]

[0083] Dissolve 100 g (0.23 mol, 1.0 eq, water content 11.8%) of reduced β-nicotinamide mononucleotide disodium salt in 500 mL of water, and stir to dissolve at about 20-25° C.; add dropwise a mixed solution of 29.2 g (0.26 mol, 1.1 eq) of calcium chloride and 200 mL of water, keep the temperature at 20-25° C. for 30 minutes, add seed crystals, continue stirring for 1 hour, cool the system to 5-10° C., stir for 30 minutes, filter, wash the filter cake with 100 mL of water and 100 mL of ethanol in turn, and dry in vacuum to obtain 108.0 g of NMNH monocalcium salt, which is form A. The moisture value measured by Karl Fischer method (KF) is 23.5%, the purity is 99.6%, and the molar yield is 95.2%.

[0084] The powder X-ray diffraction pattern of Form A of NMNH calcium salt is shown in Figure 1 , the absorption peak data are listed in Table 1.

[0085] The infrared spectrum of Form A of NMNH calcium salt can be found in Figure 2 The main absorption peaks are: 3333, 3227, 1690, 1657, 1536, 1428, 1237, 1071, 986, 819.

[0086] The mass spectrum of Form A of NMNH calcium salt is as follows Figure 3 In positive ion mode, the main peak is 375, which is the positive ion [M+H] of NMNH calcium salt. + , that is, C 11 H 16CaN2O8P + The molecular weight of the secondary peak is 337, which is the molecular weight of the positive ion without calcium ion [M-Ca+H] + , that is, C 11 H 18 N2O8P + The molecular weight of the negative ion is 337.08 (theoretical value: 337.08); in the negative ion mode, the main peak is 335, which is the molecular weight of the negative ion without calcium ion [M-Ca-H] - , that is, C 11 H 16 N2O8P - The molecular weight of the secondary peak is 671, which is the molecular weight of the two molecules of the above anion combined together [2M-2Ca-H] - , that is, C 22 H 33 N4O 16 P2 - Molecular weight (theoretical value: 671.14).

[0087] The DSC thermal analysis diagram of the crystal form A of NMNH calcium salt is shown in Figure 4 There is an endothermic peak at 81.56°C with an enthalpy change of 516.3 J / g, which is caused by the endothermic evaporation of water molecules; there is an exothermic peak at 238.06°C with an enthalpy change of -8866.3 J / g, which is formed by the decomposition and exothermic formation of the crystal form A of the NMNH calcium salt.

[0088] TGA thermogravimetric analysis of Form A of NMNH calcium salt, such as Figure 5 As shown, 50-200°C is the first step, with a weight loss of 19.882%, which is caused by the loss of adsorbed water or crystal water. 200-500°C is the second step, with a weight loss of 38.385%, which is caused by the decomposition of the crystal form A of the NMNH calcium salt.

[0089] The NMR hydrogen spectrum of the crystal form A of NMNH calcium salt is as follows: 1 H NMR(600MHz,Deuterium Oxide)δ7.10(d,J=7.2Hz,1H),6.22-6.06(m,1H),4.94(dq,J=8.0,4.0Hz,1H),4.81(t,J=7.8Hz,1H),4.26(dd,J=7.7 ,5.5Hz,1H),4.17(td,J=5.6,2.6Hz,1H),4.08-3.97(m,1H),3.78(dq,J=8.5,4.7,4.3Hz,2H),2.99(t,J=4.3Hz,2H). Such as Figure 6 shown.

[0090] The NMR carbon spectrum of the crystal form A of NMNH calcium salt is as follows: 13 C NMR (151MHz, Deuterium Oxide)δ138.19,125.16,105.33,100.76,94.91,83.20,83.14,70.76,70.68,63.95,57.42,21.96,16.77. Figure 7 shown.

[0091] Example 2: Preparation of NMNH calcium salt form A

[0092] Dissolve 200 g (0.41 mol, 1.0 eq, water content 22.4%) of reduced β-nicotinamide mononucleotide disodium salt in 1000 mL of water, and stir to dissolve at about 20-25° C.; add dropwise a mixed solution of 29.2 g (0.26 mol, 0.63 eq) of calcium chloride and 400 mL of water, keep the temperature at 20-25° C. for 30 minutes, add seed crystals, continue stirring for 1 hour, cool the system to 5-10° C., stir for 30 minutes, filter, wash the filter cake with 200 mL of water and 200 mL of ethanol in turn, and dry in vacuum to obtain 116.0 g of NMNH monocalcium salt. The moisture value measured by Karl Fischer method (KF) is 21.5%, the purity is 99.7%, and the molar yield is 59.6%.

[0093] The prepared NMNH calcium salt is the crystalline form A of NMNH calcium salt, and its spectral data is basically the same as that of the product in Example 1.

[0094] Example 3: Preparation of NMNH calcium salt crystal form A seed

[0095] 10.0 g (20 mmol, 1.0 eq, water content 23.5%) of NMNH calcium salt was added to 50 mL of water, heated to 40°C to dissolve, and then 20 mL of anhydrous ethanol was slowly added. The temperature was lowered to 5-10°C while stirring, and the temperature was maintained for crystallization for 24 hours. The filter cake was filtered and washed with cold anhydrous ethanol and vacuum dried to obtain 8.3 g of NMNH calcium salt form A product. The moisture content was 17.5% as measured by Karl Fischer method (KF), and the molar yield was 89%.

[0096] Example 4: Study on the improvement effect of oral pre-administration of NMNH-Ca on acute renal injury in mice

[0097] NMN, NMNH-Ca and NAM were added to 0.9% NaCl, shaken and mixed, and used immediately. 61 normal C57BL / 6J mice (male), 7-8 weeks old, were placed in the animal room and fed adaptively for 3 days. The dosage volume was 10 mL / kg, and the drug was administered orally (po) or intraperitoneally (ip), once a day.

[0098] The experiments were divided into the following groups:

[0099] 1. Sham operation group (Sham): 0.9% sodium chloride solution.

[0100] 2. Ischemia-reperfusion model group (I / R): 0.9% sodium chloride solution.

[0101] 3. NMN group: NMN 400 mg / kg.ip, dissolved in 0.9% NaCl solution.

[0102] 4. NMNH-Ca group: NMNH-Ca 400 mg / kg.ip, dissolved in 0.9% NaCl solution.

[0103] 5. NAM group: NAM 400 mg / kg.ip, dissolved in 0.9% NaCl solution.

[0104] After adaptive feeding, C57BL / 6J mice were divided into sham operation group (9 mice), ischemia reperfusion model group (13 mice), NMN 400mg / kg.po (13 mice), NMNH-Ca 400mg / kg.po (13 mice), and positive control NAM 400mg / kg.ip (13 mice) according to body weight. The administration method was: 3 days in advance, once a day, 1 hour before surgery, and no administration after surgery. The surgical procedure was as follows: the back of the mouse was opened, and the right and left kidneys were clamped with artery clamps respectively, and ischemia lasted for 22 minutes; the ischemia was observed, and the reperfusion time was calculated. The mice in the sham operation group were not clamped with artery clamps. The artery clamps were removed after 22 minutes, and the wounds were sutured. 24 hours after surgery, the animals were anesthetized and whole blood and kidneys were collected; the whole blood was centrifuged at 12000rpm for 2min, and the supernatant was taken to detect the levels of serum creatinine (Crea) and urea nitrogen (BUN) in the mice. The mice were killed and the kidneys were taken, some of which were frozen in a -80℃ refrigerator and some were fixed with 4% paraformaldehyde.

[0105] The plasma creatinine and urea levels were measured using creatinine and urea kits. Data are presented as mean ± standard error. One way ANOVA data were used for statistical analysis, and Fisher LSD method was used for multiple comparisons.

[0106] Blood was collected 24 hours after renal ischemia-reperfusion to detect plasma creatinine and urea nitrogen levels. The results showed that 24 hours after ischemia-reperfusion, the plasma creatinine and urea nitrogen levels in the model group (I / R) increased significantly; NMN, NMNH-Ca, and positive control NAM400mg / kg could significantly reduce the plasma creatinine (P=0.0029, P=0.0003, P<0.0001) and urea nitrogen levels (P=0.0062, P=0.0003, P<0.0001) of renal ischemia-reperfusion-induced renal injury 24 hours after ischemia-reperfusion, see Figure 8 , Table 2 and Table 3. This shows that NMN, NMNH-Ca and NAM can significantly improve acute kidney injury caused by ischemia-reperfusion.

[0107] Table 2 Effects of NMN, NMNH-Ca and NAM on plasma creatinine (Crea, μM) in mice with acute kidney injury induced by ischemia-reperfusion

[0108]

[0109] Note: *, compared with the model control group (I / R), p<0.05; **, compared with the model control group (I / R), p<0.01.

[0110] Table 3 Effects of NMN, NMNH-Ca and NAM on urea nitrogen (BUN, mM) in mice with acute renal injury induced by ischemia-reperfusion

[0111]

[0112]

[0113] Note: *, compared with the model control group (I / R), p<0.05; **, compared with the model control group (I / R), p<0.01.

[0114] The experimental results showed that NMN, NMNH-Ca and NAM were pre-administered for 3 days, once a day, 1 hour before surgery, and blood was collected 24 hours after surgery (i.e. 24 hours after reperfusion) to observe the protective effects of NMN, NMNH-Ca and NAM on ischemia-reperfusion renal injury. The results showed that NMN 400mg / kg.po, NMNH-Ca 400mg / kg.po, and positive control NAM 400mg / kg.ip could significantly reduce the plasma creatinine and urea nitrogen levels 24 hours after ischemia-reperfusion.

[0115] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention and should be included in the scope of the present invention.

Claims

1. A reduced β-nicotinamide mononucleotide calcium salt as shown in formula I or a reduced β-nicotinamide mononucleotide calcium salt hydrate as shown in formula II, Where x is a value between 1 and 9.

2. The reduced β-nicotinamide mononucleotide calcium salt hydrate (II) according to claim 1, characterized in that The water content is 5 to 30 wt %, preferably 20 to 25 wt %.

3. A method for preparing the reduced β-nicotinamide mononucleotide calcium salt according to claim 1, characterized in that: The steps include: The reduced β-nicotinamide mononucleotide disodium salt shown in formula III is used as a raw material, reacted with calcium chloride in an aqueous solution, and crystallized and purified to obtain the reduced β-nicotinamide mononucleotide calcium salt (I) 4. A crystalline form of the reduced β-nicotinamide mononucleotide calcium salt hydrate according to claim 1, named as crystalline form A, characterized in that: Its X-ray powder diffraction spectrum expressed in 2θ angles using CuKα radiation has diffraction peaks at least at 7.145°±0.2°, 12.964°±0.2°, 18.320°±0.2° and 25.756°±0.2°.

5. The crystal form according to claim 4, characterized in that: The crystal form also has diffraction peaks at 2θ values ​​of 9.752°±0.2°, 15.934°±0.2°, 21.352°±0.2° and 22.081°±0.2°.

6. The crystal form according to claim 5, characterized in that: The crystalline form also has diffraction peaks at 2θ values ​​of 14.723°±0.2°, 16.965°±0.2°, 23.552°±0.2° and 28.665°±0.2°; preferably, the XRPD spectrum of the crystalline form A is substantially as shown in FIG1 .

7. The crystal form according to claim 4, characterized in that: In the DSC analysis, the crystal form has an endothermic peak at 81.56°C and an exothermic peak at 238.06°C; preferably, the DSC spectrum of the crystal form A is substantially as shown in FIG. 4 .

8. The crystal form according to claim 4, characterized in that In TGA analysis, the crystalline form loses 19.882% of its weight in the 50-200° C. stage and loses 38.385% of its weight in the 200-500° C. stage; preferably, its TGA spectrum is substantially as shown in FIG. 5 .

9. A pharmaceutical composition, health food or cosmetic, characterized in that: The invention comprises the reduced β-nicotinamide mononucleotide calcium salt or the reduced β-nicotinamide mononucleotide calcium salt hydrate as claimed in claim 1 or 2, or the crystalline form A as claimed in any one of claims 4 to 8 as an active ingredient.

10. The pharmaceutical composition, health food or cosmetic according to claim 9, characterized in that: Used for anti-aging, i.e. treating and / or preventing age-related degenerative diseases; or the pharmaceutical composition is used for treating acute kidney injury.

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