A method of preparing a nicotinamide mononucleotide co-crystal

Abstract

The present application aims to solve the poor flowability of existing nicotinamide mononucleotide (NMN) single material flow, which leads to poor flowability, uneven mixing and poor content uniformity during preparation of NMN preparation product, and provides a safe and good flowability nicotinamide mononucleotide-proline co-crystal preparation method, the co-crystal uses Cu-Ka radiation, and the X-ray powder diffraction expressed by 2θ angle has diffraction peaks at 5.5±0.2° and 16.8±0.2°, the co-crystal has good bulk density and flowability, can realize direct tabletting or filling capsule operation of powder, the product weight difference meets the requirements of pharmacopoeia, and the operation is simple, more suitable for commercial production, at the same time, the natural amino acid-proline is used as a co-crystal former, which is safer and more reliable than the existing technology disclosed isoniazid (WHO is listed as a class 3 carcinogen).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compound crystal, in particular to a method for preparing nicotinamide mononucleotide co-crystal. BACKGROUND

[0002] Beta nicotinamide mononucleotide (NMN) is a direct precursor of coenzyme I (NAD+), as an important intermediate in the NAD rescue synthesis pathway in biological cells, which directly affects the concentration of NAD in biological cells. Coenzyme I is an important coenzyme in human oxidation-reduction reactions, and is involved in thousands of physiological reactions. Studies have shown that the content of coenzyme I in the human body decreases with age. Supplementing NMN in vitro is the most ideal way to effectively improve the level of coenzyme I in the body, which can prevent cardiovascular diseases, delay aging and prevent Alzheimer's disease, etc.

[0003] Due to the poor stability of NMN, the product prepared directly using amorphous powder is prone to degradation and inactivation in the production, storage and transportation links.

[0004] Patent CN108697722A discloses two crystal forms of NMN. It is found that the crystalline form can improve the stability of the product, but when used in oral dosage forms, there are still problems such as poor flowability, difficult operation, and uneven content. How to improve the performance indicators of NMN dosage forms and improve the stability is a problem to be solved.

[0005] Patent CN113292619B discloses a co-crystal of NMN and isoniazid, which can improve the problem of poor flowability. However, isoniazid is a highly toxic drug for treating tuberculosis, and was listed as a class 3 carcinogen (with carcinogenic potential) by the World Health Organization (WHO) in 2017. When used in healthy people, there is a safety hazard. If it is only to solve the problem of poor flowability of the product, a safe alternative substance needs to be selected. SUMMARY

[0006] The purpose of the present application is to provide a method for preparing nicotinamide mononucleotide co-crystal, which solves the problems of uneven content of NMN preparation product caused by poor flowability of NMN, and the safety hazard caused by using toxic reagent isoniazid as co-crystal former in the prior art. By forming a co-crystal of NMN and proline, the flowability and stability of the material are improved.

[0007] The above technical purpose of the present application is achieved by the following technical scheme: a method for preparing nicotinamide mononucleotide co-crystal, taking nicotinamide mononucleotide as an active ingredient and proline as a co-crystal former, and using a solution synthesis method to mix and crystallize the nicotinamide mononucleotide and the proline in an organic solvent and water mixture system.

[0008] By adopting the technical scheme, proline is a natural amino acid, and its in-vivo metabolite 4-hydroxyproline is an important component of animal collagen. Therefore, as a co-crystal former, it is safer and more reliable than the existing patent disclosed isoniazid (CN113292619B), and is more suitable for long-term use by healthy people. The drug co-crystal is a solid crystal formed by the combination of active pharmaceutical ingredients (API) and co-crystal formers (CCF) through hydrogen bonds or other non-covalent bonds. The crystalline form guidelines in the Chinese Pharmacopoeia 2020 edition include co-crystal drugs in the category of crystalline substances. Co-crystal drugs can improve the mechanical processing performance, solubility, permeability, bioavailability, stability, and other properties of the raw drug. The type of CCF affects the properties of the drug co-crystal. When selecting a suitable CCF, not only the strong intermolecular interaction between the CCF and the API should be considered, but also the safety, non-toxicity, and no side effects of the CCF must be ensured. The common methods for preparing co-crystals can be divided into two categories from the aspect of the morphology of each component during preparation: solution synthesis method and solid synthesis method. The so-called solution synthesis method is that the API and the CCF are in a fluid state during synthesis, including evaporation crystallization, cooling crystallization, and suspension method. The so-called solid synthesis method is that the API and the CCF are in a solid state during synthesis, including sublimation method, melting method, and grinding method. The solution synthesis method is simpler to operate than the solid synthesis method, and is more suitable for commercial production. Therefore, the NMN co-crystal prepared by the method has high safety, good flowability, and stability, and is simple to operate, which is more suitable for commercial production.

[0009] Preferably, the organic solvent is selected from methanol, isopropanol, and n-butanol.

[0010] By adopting the technical scheme, in the method for preparing the NMN co-crystal provided by the application, the mixed crystallization process is carried out in a mixed system of an organic solvent and water. The type of the organic solvent and the solid mass of NMN and proline, and whether the volume ratio of the mixed system used is appropriate, all play a key role in whether the co-crystal can be successfully precipitated. In the method for preparing the NMN co-crystal provided by the application, the organic solvent is preferably one of methanol, isopropanol, and n-butanol.

[0011] Preferably, the nicotinamide and the proline are in a molar ratio of 1:1-1.2.

[0012] By adopting the technical scheme, when the solution synthesis method is used to prepare the co-crystal, the measurement ratio of the API and the CCF will affect the amount of the co-crystal precipitated. If the measurement ratio is not appropriate, one of the substances may be precipitated alone, thereby affecting the precipitation rate of the co-crystal. In the method for preparing the nicotinamide mononucleotide co-crystal provided by the application, NMN and proline are preferably mixed in a theoretical molar ratio of 1:1. The amount of proline can be appropriately increased to a molar ratio of 1:1.2 of NMN to proline, so as to maximize the formation of the nicotinamide mononucleotide proline co-crystal.

[0013] Preferably, the mass ratio of the nicotinamide to the proline, and the volume ratio of the mixed solvent of the organic solvent and water is 1:30-60.

[0014] Preferably, in the mixed system of the organic solvent and water, the volume ratio of water to the organic solvent is 1:1-3.

[0015] By adopting the technical solution, it is ensured that no solid is precipitated during the mixing process.

[0016] Preferably, the mixed system of the organic solvent and water is obtained by slowly adding the organic solvent into water, and the NMN and proline are dissolved in water before the organic solvent is added into water.

[0017] By adopting the technical solution, in the method for preparing the NMN co-crystal provided by the application, the mixed system of the organic solvent and water can be obtained by directly mixing the organic solvent and water, or by slowly adding the organic solvent into water. For the former, the NMN and proline can be added after the organic solvent and water are mixed; for the latter, the NMN and proline should be dissolved in water before the organic solvent is added into water. In the method for preparing the NMN co-crystal provided by the application, the mixed system of the organic solvent and water is obtained by the latter, i.e., the NMN and proline are dissolved in water, and then the organic solvent is slowly added into water. This method can further improve the bulk density of the crystal, so that the crystal has better flowability.

[0018] Preferably, the crystallization process is carried out under stirring after the temperature of the mixed system of the organic solvent and water is reduced to 51-10℃.

[0019] By adopting the technical solution, the solid can be completely precipitated at room temperature, and the appearance and physicochemical indexes of the precipitated crystal are not significantly different from those at a lower temperature.

[0020] The inventor finally developed the method for preparing the NMN co-crystal provided by the application through a large number of experimental explorations and creative labor. It is proved through repeated tests that the method can successfully prepare a new crystal form of NMN in the form of a co-crystal, and is beneficial to scale-up production.

[0021] The NMN co-crystal obtained by the application is subjected to powder X-ray diffraction measurement, Cu-Kα radiation is adopted, and an X-ray powder diffractometer is used for detection. There are diffraction peaks at 5.5±0.2° and 16.8±0.2°, and a typical powder X-ray diffraction pattern is shown in Figure 1 The differential scanning calorimetry (DSC) analysis diagram has a regional endothermic peak at 129-150℃, and a DSC analysis diagram is shown inFigure 2 .

[0022] Further, the NMN-proline co-crystal prepared by the above method provided by the present application has diffraction peaks at 5.5±0.2°, 16.8±0.2°, 20.0±0.2°, 22.5±0.2°, 25.0±0.2°, 28.2±0.2°, 34.1±0.2°, and 40.0±0.2° in X powder diffraction 2θ angle.

[0023] It is found by experiments that the NMN-proline co-crystal prepared by the above method for preparing NMN co-crystal provided by the present application has higher bulk density and better content uniformity than single-component NMN, thereby significantly improving the flowability of NMN and the quality uniformity of the preparation process and the preparation product.

[0024] In summary, the present application has the following beneficial effects:

[0025] 1. The present application provides a preparation method of a new crystal of NMN in the form of NMN-proline co-crystal, which uses natural amino acid proline as a co-crystal former, and solves the safety problem of using isoniazid, a carcinogen of class 3, as a co-crystal former in the prior art.

[0026] 2. The new crystal form of NMN provided by the present application has higher bulk density than single-component NMN, thereby significantly improving the flowability of NMN, solving the mechanical handling property of the material in the production process of oral solid preparation, and solving the quality uniformity problem of the end product. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is an X diffraction pattern of NMN co-crystal;

[0028] Figure 2 is a DSC pattern of NMN co-crystal. DETAILED DESCRIPTION

[0029] The present application will be further described in detail below with reference to the accompanying drawings.

[0030] The present embodiment is only an explanation of the present application, and is not a limitation of the present application. Those skilled in the art can make modifications to the present embodiment without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

[0031] The materials, reagents, etc. used in the examples are commercially available. The NMN is consistent with the anhydrous crystal form 1 disclosed in patent CN108137639B.

[0032] Example 1

[0033] 90 g nicotinamide mononucleotide and proline 35 g were dissolved in 2.5 L water, the temperature was kept at 40-50 °C, 2.5 L of methanol was slowly added, stirring for 5-10 min, cooling 5-10 °C, stirring to crystallize, filtering, drying, to obtain the nicotinamide mononucleotide-proline co-crystal provided by the application.

[0034] Example 2

[0035] 90 g nicotinamide mononucleotide and proline 35 g were dissolved in 2.5 L water and 2.5 L of methanol to form a mixed solvent, the temperature was kept at 40-50 °C, stirring for 5-10 min, cooling 5-10 °C, stirring to crystallize, filtering, drying, to obtain the nicotinamide mononucleotide-proline co-crystal provided by the application.

[0036] Example 3

[0037] 90 g nicotinamide mononucleotide and proline 35 g were dissolved in 2.5 L water, the temperature was kept at 40-50 °C, 4 L of isopropyl alcohol was slowly added, stirring for 5-10 min, cooling 5-10 °C, stirring to crystallize, filtering, drying, to obtain the nicotinamide mononucleotide-proline co-crystal provided by the application.

[0038] Example 4

[0039] 90 g nicotinamide mononucleotide and proline 35 g were dissolved in 2.5 L water, the temperature was kept at 40-50 °C, 2.5 L of n-butanol was slowly added, stirring for 5-10 min, cooling 5-10 °C, stirring to crystallize, filtering, drying, to obtain the nicotinamide mononucleotide-proline co-crystal provided by the application.

[0040] Example 5

[0041] 90 g nicotinamide mononucleotide and proline 36 g were dissolved in 2.5 L water, the temperature was kept at 40-50 °C, 5 L of methanol was slowly added, stirring for 5-10 min, cooling 5-10 °C, stirring to crystallize, filtering, drying, to obtain the nicotinamide mononucleotide-proline co-crystal provided by the application.

[0042] The nicotinamide mononucleotide-proline prepared in the above examples was subjected to powder X-ray diffraction, using SHIMADZU XRD-7000, Cu-Ka radiation, wavelength 1.54 Å, divergence slit 1 °, X-ray tube voltage 45 KV, X-ray tube current 40 mA, scanning range 3-50 ° (2θ). The powder sample was pressed flat on a micro sample disc and then detected. The results showed that the key characteristic diffraction angle 2θ corresponding to the peak position was all in the range of ±0.2 °, and the intensity was in the range of ±5 %, proving that the crystal form was consistent.

[0043] The nicotinamide mononucleotide-proline co-crystal provided by the present application has an X-ray powder diffraction pattern, and a typical pattern is shown in Figure 1 The X-ray powder diffraction data of Examples 1-5 are shown in Table 1 below.

[0044] Table 1 Summary of main X-ray powder diffraction data of nicotinamide mononucleotide-proline

[0045]

[0046] The nicotinamide mononucleotide-proline co-crystal prepared in Example 1 above was measured for differential scanning calorimetry curve (DSC):

[0047] The DSC measurement was carried out in a NETZSCH (DSC214) instrument with a sealed disc device, and the sample was about 3 mg, weighed in an aluminum disc to the nearest one hundredth of a milligram, the instrument was purged with nitrogen at 50 ml / min, the protection gas flow rate was 70 ml / min, the temperature was raised at a heating rate of 10 ℃ / min between room temperature and 220 ℃, and the absorption peak was plotted downward, the abscissa was temperature ℃, and the ordinate was heat flow per unit mass Mw / mg, and the DSC graph is shown in Figure 2 .

[0048] Example 6

[0049] Bulk density determination

[0050] NMN single component (anhydrous crystal) and NMN-proline co-crystal obtained in Example 1 above were sieved, accurately weighed, and the bulk density was investigated according to the first method solid mass method of bulk density and tap density determination method in <0993> of Chinese Pharmacopoeia 2020 edition, and the results are shown in Table 2 below. It can be seen that the bulk density of the co-crystal is significantly improved compared with the single component NMN, that is, the flowability is improved. Considering the measurement error, the bulk density of the isoniazid co-crystal disclosed in the patent CN113292619B example is consistent (0.68 g / ml).

[0051] Example 7

[0052] Dose difference determination

[0053] NMN single component (anhydrous crystal) and NMN-proline co-crystal obtained in Example 1 above were sieved through a 200 mesh sieve, and were filled into capsules according to the marked amount of 0.2 g using 2# capsule shells. The obtained capsules were determined by reference to the dose difference determination method in <0103> of Chinese Pharmacopoeia 2020 edition, and the dose difference results are shown in Table 2 below. It can be seen that the dose difference of the co-crystal obtained by the present application is small, and when used for preparing tablets or capsules, there is no problem of uneven content and large quality difference of single NMN.

[0054] Table 2. Results of bulk density, fill weight variation investigation

[0055]

Claims

1. A method of preparing a nicotinamide mononucleotide co-crystal, characterized by, The nicotinamide mononucleotide is mixed with proline in an organic solvent and a water mixed system by solution synthesis method, and crystallization is carried out; The nicotinamide mononucleotide-proline co-crystal prepared is subjected to X-ray powder diffraction using Cu-Ka radiation, and diffraction peaks are present at 5.5±0.2°, 16.8±0.2°, 20.0±0.2°, 22.5±0.2°, 25.0±0.2°, 28.2±0.2°, 34.1±0.2° and 40.0±0.2° in terms of 2θ angle.

2. A process for preparing a nicotinamide mononucleotide co-crystal as claimed in claim 1, wherein, The organic solvent is selected from methanol, isopropanol and n-butanol.

3. A method of preparing a nicotinamide mononucleotide co-crystal according to claim 1, wherein, The nicotinamide and the proline are in a molar ratio of 1:1-1.

2.

4. A method of preparing a nicotinamide mononucleotide co-crystal according to claim 1, wherein, The solid mass of the nicotinamide and the proline is in a mass-to-volume ratio of 1:30-60 with the mixed solvent of the organic solvent and water.

5. A method of preparing a nicotinamide mononucleotide co-crystal according to claim 1 or 2, wherein, The volume ratio of water to the organic solvent in the mixed system of the organic solvent and water is 1:1-3.

6. A method of preparing a nicotinamide mononucleotide co-crystal according to claim 1, wherein, The mixed system of the organic solvent and water is obtained by slowly adding the organic solvent to water, and the nicotinamide mononucleotide and the proline are dissolved in water before the organic solvent is added to water.

7. A method of preparing a nicotinamide mononucleotide co-crystal according to claim 1, wherein, The crystallization process is carried out under stirring after the temperature of the mixed system of the organic solvent and water is reduced to 51°C.

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