A coenzyme i crystal form and application thereof

Abstract

The application discloses a coenzyme I crystal form and application thereof, relates to the technical field of nicotinamide adenine dinucleotide preparation, and belongs to the technical field of nicotinamide adenine dinucleotide preparation. The coenzyme I crystal form is a triclinic crystal system and has a Pl or Pl space group. The crystal lattice constant is a = 8.5894 Å, b = 8.8623 Å, c = 11.2009 Å, alpha = 109.803 DEG, beta = 90.697 DEG and gamma = 103.709 DEG. The coenzyme I crystal form provided by the application is a hydrate crystal form with high crystallinity, has good solid-state properties, the content of an effective component is obviously higher than that of a freeze-dried raw material sample, the content of maximum single substance and total impurities is obviously lower than that of the freeze-dried raw material sample, and with the prolongation of storage time, the content of maximum single substance and total impurities does not obviously increase after storage for 12 months.

Description

Technical Field

[0001] This invention relates to the field of nicotinamide adenine dinucleotide preparation technology, specifically to a coenzyme I crystal form and its application. Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD) is a physiological substance found in all living cells, including human cells. It has no side effects on the body and is a cofactor for many enzymes that catalyze redox reactions, hence the name coenzyme I. Coenzyme I participates in the metabolism of substances and energy in organisms, produced in the citric acid cycle during glycolysis and cellular respiration. It also acts as a carrier of biological hydrogen and an electron donor, transferring energy to ATP synthesis via oxidative phosphorylation on the inner mitochondrial membrane. Today, coenzyme I is widely used in chemical catalysis, pharmaceutical raw material production, health product manufacturing, and cosmetics. With in-depth research into the treatment and prevention of diseases, the importance of coenzyme I as a pharmaceutical and health product is increasingly prominent, and market demand is increasing year by year.

[0003] Existing methods for preparing coenzyme I are mainly divided into chemical synthesis and biocatalysis. Biocatalysis includes bio-fermentation and enzyme catalysis, and due to its advantages of being green, environmentally friendly, and pollution-free compared to chemical synthesis, it has gradually become the mainstream approach. The crude product solution (enzyme reaction solution or bio-fermentation broth) obtained by preparing coenzyme I via biocatalysis contains a large number of impurities in addition to coenzyme I. After a series of purification and drying processes, coenzyme I is obtained.

[0004] However, existing coenzyme I is mainly in lyophilized powder form. Tablets prepared from lyophilized powder contain a certain amount of impurities, and as the storage time increases, the impurities in the tablets prepared from lyophilized powder slowly increase, affecting the quality of the product. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a coenzyme I crystal form and its application, solving at least one technical problem mentioned in the background art.

[0006] (II) Technical Solution In a first aspect, the present invention provides a coenzyme I crystal form, wherein the coenzyme I crystal form is triclinic and has a P1 or P1 spatial group; the lattice constants are: a = 8.5894 Å, b = 8.8623 Å, c = 11.2009 Å, α = 109.803°, β = 90.697° and γ = 103.709°.

[0007] Preferably, the weight gain of the coenzyme I crystal form at relative humidity from 0% to 80% is 2.43%.

[0008] Secondly, the present invention provides a method for preparing coenzyme I crystal form, wherein the specific steps of the preparation method are as follows: Adjust the pH of the NAD enzymatic reaction solution to 3.0-3.2, and then filter using a nanofiltration membrane; The filtered NAD enzymatic reaction solution was desalted. The desalted NAD enzymatic reaction solution was concentrated to a final concentration of 100-150 g / L. The concentrated NAD enzyme reaction solution was added to an organic solvent in batches and stirred at low speed to crystallize. After crystallization stops, solid-liquid separation is performed, followed by rinsing with the same organic solvent used for crystallization and drying to obtain the coenzyme I crystal form.

[0009] Preferably, the volume percentage of organic solvent in the concentrate is 10%-80% by adding it in batches.

[0010] Thirdly, the present invention provides a coenzyme I solid dosage form prepared using any of the coenzyme I crystal forms described above.

[0011] Preferably, the coenzyme I solid dosage form includes tablets, capsules, or powders.

[0012] Fourthly, the present invention provides a coenzyme I tablet, wherein the tablet comprises any of the coenzyme I crystal forms described above. Diluents, disintegrants, gliding agents, lubricants, and film coating premixes.

[0013] Preferably, the contents of each component of the tablet are as follows: The coenzyme I crystal form is 30-45%, the diluent is 40-60%, the wetting agent is 0.5-1%, the disintegrant is 2-6%, the flow aid is 0.1-3%, the lubricant is 0.1-3%, and the membrane coating premix is ​​1-3%.

[0014] Preferably, the film coating premix has the following composition: 1-5% hydroxypropyl methylcellulose, 1-5% talc, 1-4% polyethylene glycol, 1-4% titanium dioxide and 85-90% 60% ethanol.

[0015] Preferably, the diluent is selected from one, two or more of microcrystalline cellulose, mannitol, and lactose; And / or the wetting agent is selected from 20% to 50% aqueous ethanol solution; And / or the disintegrant is selected from one, two or more of sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, and crosporopyrrolidone; And / or the flow aid is selected from one or a combination of two of micronized silica gel and talc; And / or the lubricant is selected from one or a combination of two of magnesium stearate and calcium stearate.

[0016] Preferably, the diluent is selected from microcrystalline cellulose, and / or the disintegrant is selected from low-substituted hydroxypropyl methylcellulose, and / or the flow aid is selected from micronized silica gel, and / or the lubricant is selected from magnesium stearate.

[0017] (III) Beneficial Effects This invention provides a coenzyme I crystal form and its application, which has the following advantages compared with the prior art: The coenzyme I crystal form provided by this invention has a highly crystalline hydrate crystal form with good solid-state properties. The content of effective ingredients is significantly higher than that of the freeze-dried raw material (amorphous) sample, and the maximum content of elemental and total impurities is significantly lower than that of the freeze-dried raw material (amorphous) sample. Furthermore, as the storage time is extended, the maximum content of elemental and total impurities does not increase significantly even after 12 months of storage. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is a crystal structure diagram of coenzyme I. Figure 2 The XRPD pattern was obtained by simulating single-crystal diffraction data of coenzyme I crystal form. Specific implementation methods To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. To better understand the above technical solution, the following will provide a detailed description of the technical solution in conjunction with the accompanying drawings and specific implementation methods: This invention provides a method for preparing coenzyme I crystal form, specifically: Adjust the pH of the NAD enzymatic reaction solution to 3.0-3.2, and then filter using a nanofiltration membrane; The filtered NAD enzymatic reaction solution was desalted. The desalted NAD enzymatic reaction solution was concentrated to a final concentration of 100-150 g / L. The concentrated NAD enzyme reaction solution was added to an organic solvent in batches and stirred at low speed to crystallize. After crystallization stops, solid-liquid separation is performed, followed by rinsing with the same organic solvent used for crystallization and drying to obtain the coenzyme I crystal form.

[0019] Furthermore, the detailed steps of the above-mentioned method for preparing the coenzyme I crystal form are as follows: (1) pH adjustment: A certain amount of NAD enzymatic reaction solution was measured and the pH was adjusted to 3.0-3.2 (25℃) using hydrochloric acid, and then filtered through a 0.22µm pore size filter membrane. The NAD enzymatic reaction solution was obtained by synthesizing the substrate nicotinamide mononucleotide (NMN) (12-16.0 g / L) and adenosine triphosphate (ATP) (20-29.9 g / L) in a water bath at 33-37℃ under the action of nicotinamide mononucleotide adenosine transferase (NMNAT enzyme) for 2-3 hours.

[0020] (2) Desalination: The filtered NAD enzyme reaction solution is desalted using a membrane device with a pore size of 300-500Dal. Cooling circulating water is used to cool the solution throughout the desalination process so that the temperature of the solution does not exceed 25℃.

[0021] (3) Concentration: The desalted NAD enzyme reaction solution is concentrated using a rotary evaporator. The vacuum degree is set to 10-30 mbar, the temperature to 30-35℃, and the rotation speed to 50-100 rpm. The final concentration after concentration should reach 100-150 g / L.

[0022] (4) Crystallization: Using an organic solvent, the volume percentage of the organic solvent in the concentrate is increased to 10%-80% by adding it in batches. Then, the concentrate is placed at 2-8℃ and stirred at a low speed to crystallize. The specific organic solvent can be ethers, esters, ketones or glycol derivatives, such as methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.

[0023] (5) Collecting crystals: Stop crystallization after 20-48 hours. After solid-liquid separation, rinse with 10%-80% concentration of the same organic solvent as the crystallizer, with a volume of 1-4 times that of the crystallization liquid.

[0024] (6) Drying: The solid obtained in (5) above is placed in a vacuum drying oven at 30-35°C and dried for 2-6 hours. After being taken out and crushed, the coenzyme I crystal of the crystal form of the present invention is obtained.

[0025] The following detailed explanation is provided with reference to specific embodiments: Example 1 Preparation of Coenzyme I Crystals The NAD enzymatic reaction solution was obtained by synthesizing the substrate nicotinamide mononucleotide (NMN) (140 g / L) and adenosine triphosphate (ATP) (25 g / L) in a 35°C water bath with nicotinamide mononucleotide adenosine transferase (NMNAT enzyme) for 2.5 hours. pH adjustment: Measure a certain amount of NAD enzyme reaction solution and adjust the pH to 3.1 (25℃) using hydrochloric acid. Filter through a 0.22µm pore size membrane. Desalination: The filtered NAD enzymatic reaction solution is desalted using a 400Dal pore size membrane device. Cooling circulating water is used to cool the solution throughout the desalination process to ensure that the temperature of the solution does not exceed 25°C.

[0026] Concentration: The desalted NAD enzyme reaction solution was concentrated using a rotary evaporator, with a vacuum of 20 mbar, a temperature of 32°C, and a rotation speed of 80 rpm. The final concentration after concentration should reach 125 g / L.

[0027] Crystallization: An organic solvent is added in batches to achieve a volume percentage of 45% in the concentrate. The solution is then placed at 5°C and stirred at a low speed to allow crystallization. The specific organic solvent is ethanol.

[0028] Crystal collection: After crystallization for 40 hours, stop the process. After solid-liquid separation, rinse with a 45% concentration of the same organic solvent as that used for crystallization, with a volume twice that of the crystallization liquid.

[0029] Drying: The solid obtained above is placed in a vacuum drying oven at 33°C and dried for 4 hours. After being taken out and crushed, the coenzyme I crystals of the crystal form of this invention are obtained.

[0030] In this embodiment of the invention, the above-mentioned coenzyme I crystals are used as raw materials for single-crystal culture. The specific single-crystal culture steps are as follows: Approximately 50 mg of coenzyme I crystals were weighed and dissolved in 1 mL of water. The solution was then filtered, and the filtrate was placed in a sample vial. The vial was then left open in an environment containing acetonitrile for gas-liquid diffusion, resulting in a block crystal. This block crystal is the coenzyme I crystal form of this embodiment of the invention. The structure of this coenzyme I crystal form was analyzed, and the results are as follows. Single crystals obtained through gas-liquid diffusion in the water-acetonitrile system were subjected to single crystal detection and analysis: The present invention provides a coenzyme I crystal form, wherein the coenzyme I crystal form is triclinic and has a P1 or P1 spatial group; the lattice constants are: a = 8.5894 Å, b = 8.8623 Å, c = 11.2009 Å, α = 109.803°, β = 90.697° and γ = 103.709°.

[0031] Specifically, the relevant crystallographic data and structural refinement parameters are shown in the table below, and the X-ray diffraction patterns obtained through simulation using single-crystal diffraction data are shown in [the table below]. Figure 2 Crystal structure diagram is shown below. Figure 1 ; The values ​​in parentheses in the table above are the error ranges. For example, a=8.5894 (12) means a=8.5894±0.0012.

[0032] Hygroscopicity studies and structural analysis were conducted on the coenzyme I crystal form according to embodiments of the present invention. The hygroscopicity study results showed that the weight gain of the coenzyme I crystal form from 0% to 80% RH was 2.43%. Single-crystal structural analysis revealed that the coenzyme I crystal form contains four water molecules, classifying it as a tetrahydrate.

[0033] Using a lyophilized raw material (amorphous) as a reference, the lyophilized raw material was a water-based lyophilized material prepared using existing technology. The quality standards of the coenzyme I crystal form and the lyophilized raw material (amorphous) in this embodiment of the invention were tested. The testing methods were performed according to the testing items in the Chinese Pharmacopoeia, and the results were obtained uniformly under 25°C conditions. Based on the table above, it can be seen that although freeze-dried raw materials (amorphous) meet the quality standards, the maximum amount of elemental and total impurities increases continuously with the extension of storage time.

[0034] The preparation method of the freeze-dried raw material (amorphous) is as follows: NAD enzymatic reaction solution is obtained by synthesizing the substrate nicotinamide mononucleotide (NMN) (140 g / L) and adenosine triphosphate (ATP) (25 g / L) in a 35°C water bath under the action of nicotinamide mononucleotide adenosine transferase (NMNAT enzyme) for 2.5 hours; the obtained NAD enzymatic reaction solution is filtered through a nanofiltration membrane to obtain a coenzyme I concentrate, which is then freeze-dried. The specific freeze-drying process is as follows: Lower the plate temperature to -45℃±2 and the coenzyme I concentrate temperature to below -35℃, and keep it at this temperature for 3 hours. Open the condenser valve to lower the temperature of the rear chamber condenser to below -40℃. First sublimation drying: Turn on the vacuum pump and butterfly valves. When the vacuum degree of the freeze-drying chamber is <15 Pa, sublimate the product. Increase the plate temperature to -30℃ over 3 hours and keep it at this temperature for 1-2 hours. Continue increasing the temperature for 3 hours to -20℃ and keep it at this temperature for 1-2 hours. Continue increasing the temperature for 4 hours to -10℃ and keep it at this temperature for 1-3 hours. Continue increasing the temperature for 3-4 hours to 8℃. Second sublimation: Heat the product to dry it. With the vacuum degree of the freeze-drying chamber <15 Pa, continue increasing the temperature for 3 hours to 25℃ and keep it at this temperature for 1-3 hours to obtain freeze-dried raw material (amorphous).

[0035] The coenzyme I crystal form in this application embodiment is: It is understood that the effective component content of the embodiments of the present invention is significantly higher than that of the freeze-dried raw material (amorphous) sample, and the maximum elemental content and total impurity content are significantly lower than those of the freeze-dried raw material (amorphous) sample. Furthermore, as the storage time is extended, the maximum elemental content and total impurity content do not significantly increase even after 12 months of storage.

[0036] Furthermore, embodiments of the present invention provide applications of the above-mentioned coenzyme I crystal form, and embodiments of the present invention provide coenzyme I solid dosage forms prepared by the above-mentioned coenzyme I crystal form, wherein the coenzyme I solid dosage forms include tablets, capsules or powders.

[0037] This invention provides a coenzyme I tablet, the tablet's components including any of the coenzyme I crystal forms and dilutions described above. Disintegrants, disintegrants, glidants, lubricants, and film coating premixes.

[0038] Specifically, the contents of each component of the tablet are as follows: 30-45% coenzyme I crystal form, 40-60% diluent, 0.5-1% wetting agent, 2%-6% disintegrant, 0.1%-3% glidant, and so on. The lubricant is 0.1-3% and the film coating premix is ​​1-3%.

[0039] In one embodiment, the film coating premix comprises the following components: 1-5% hydroxypropyl methylcellulose, 1-5% talc, 1-4% polyethylene glycol, 1-4% titanium dioxide, and 85-90% 60% ethanol.

[0040] In one embodiment, the diluent is selected from one, two, or a combination of more than one of microcrystalline cellulose, mannitol, and lactose; And / or the wetting agent is selected from 20% to 50% aqueous ethanol solution; And / or the disintegrant is selected from one, two or more of sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, and crosporopyrrolidone; And / or the flow aid is selected from one or a combination of two of micronized silica gel and talc; And / or the lubricant is selected from one or a combination of two of magnesium stearate and calcium stearate.

[0041] In one embodiment, the diluent is selected from microcrystalline cellulose, and / or the disintegrant is selected from low-substituted hydroxypropyl methylcellulose, and / or the flow aid is selected from micronized silica gel, and / or the lubricant is selected from magnesium stearate.

[0042] Example 2 Preparation of Coenzyme I tablets Coenzyme I tablets were prepared using this coenzyme I crystal form, specifically: (1) Weigh out the prescribed amount of coenzyme I crystal raw material, namely 40% of coenzyme I crystal prepared in Example 1 above, 3% of disintegrant sodium carboxymethyl starch, 50% of diluent mannitol, and 1.5% of flow aid talc powder, put them into a mixer and mix them evenly. Add 0.7% of 40% ethanol aqueous solution as wetting agent, wet granulate, and granulate with an 18-mesh sieve. (2) After the material is dried, add 2% magnesium stearate lubricant and mix and pass through a 16-mesh sieve; (3) Press the film to the specified weight to obtain the raw film; (4) Coating: Add 2.8% film coating premix. Coating process parameters: compressed air pressure 0.3 MPa, hot air temperature 105℃, sheet bed temperature 30~40℃, coating pan rotation speed 5-6 r / min, spraying speed 0.5~1 g / min. Specifically, the film coating premix consists of the following components: 3% hydroxypropyl methylcellulose, 4% talc, 3% polyethylene glycol, 3% titanium dioxide, and 87% 60% ethanol.

[0043] Tablets were prepared using the aforementioned lyophilized raw material (amorphous) and the aforementioned coenzyme I crystal form, respectively. The tableting process for the lyophilized raw material (amorphous) was the same as that for the coenzyme I crystal form tablets, except that the coenzyme I crystals in the above components were replaced with the lyophilized raw material (amorphous). The quality inspection results at 25°C are as follows: It was found that tablets prepared from freeze-dried raw materials showed a significant increase in the maximum content of elemental compounds and total impurities after 12 months of storage, even increasing by tens of times. However, tablets prepared from the aforementioned coenzyme I crystal form raw materials did not show a significant increase in the maximum content of elemental compounds and total impurities even after prolonged storage.

[0044] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A coenzyme I crystal form, characterized in that, The coenzyme I crystal form is triclinic and has Pl or Pl spatial groups; the lattice constants are: a = 8.5894 Å, b = 8.8623 Å, c = 11.2009 Å, α = 109.803°, β = 90.697° and γ = 103.709°.

2. The coenzyme I crystal form according to claim 1, characterized in that, The weight gain of the coenzyme I crystal form at relative humidity from 0% to 80% was 2.43%.

3. A method for preparing a coenzyme I crystal form, characterized in that, The specific steps of the preparation method are as follows: Adjust the pH of the NAD enzymatic reaction solution to 3.0-3.2, and then filter using a nanofiltration membrane; The filtered NAD enzymatic reaction solution was desalted. The desalted NAD enzymatic reaction solution was concentrated to a final concentration of 100-150 g / L. The concentrated NAD enzyme reaction solution was added to an organic solvent in batches and stirred at low speed to crystallize. After crystallization stops, solid-liquid separation is performed, followed by rinsing with the same organic solvent used for crystallization and drying to obtain the coenzyme I crystal form.

4. The method for preparing the coenzyme I crystal form according to claim 3, characterized in that, The volume percentage of organic solvent in the concentrate is increased to 10%-80% by adding it in batches.

5. A solid dosage form of coenzyme I, characterized in that, Prepared using the coenzyme I crystal form according to any one of claims 1 or 2 Prepare.

6. The coenzyme I solid dosage form according to claim 5, characterized in that, The coenzyme I solid dosage form includes tablets and gels. Capsules or powders.

7. A coenzyme I tablet, characterized in that, The tablet's components include the excipients described in either claim 1 or 2. Enzyme I crystal form, diluent, disintegrant, flow aid, lubricant, and film coating premix.

8. The coenzyme I tablet according to claim 7, characterized in that, The contents of each component of the tablet are as follows: The coenzyme I crystal form is 30-45%, the diluent is 40-60%, the wetting agent is 0.5-1%, the disintegrant is 2-6%, the flow aid is 0.1-3%, the lubricant is 0.1-3%, and the membrane coating premix is ​​1-3%.

9. The coenzyme I tablet according to claim 8, characterized in that, The film coating premix consists of the following components: 1-5% hydroxypropyl methylcellulose, 1-5% talc, 1-4% polyethylene glycol, 1-4% titanium dioxide, and 85-90% 60% ethanol.

10. The coenzyme I tablet according to claim 8, characterized in that, The diluent is selected from one, two or more of microcrystalline cellulose, mannitol and lactose; And / or the wetting agent is selected from 20% to 50% aqueous ethanol solution; And / or the disintegrant is selected from one, two or more of sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, and crosporopyrrolidone; And / or the flow aid is selected from one or a combination of two of micronized silica gel and talc; And / or the lubricant is selected from one or a combination of two of magnesium stearate and calcium stearate; Preferably, the diluent is selected from microcrystalline cellulose, and / or the disintegrant is selected from low-substituted hydroxypropyl methylcellulose, and / or the flow aid is selected from micronized silica gel, and / or the lubricant is selected from magnesium stearate.

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