Method for producing freeze-dried NMN preparation

Abstract

Provided is a method for producing a freeze-dried NMN preparation, which enables the production of a flocculent freeze-dried NMN preparation that can be produced smoothly during use. The method comprises: a solution preparation step for preparing a 5-200 mg / mL NMN (nicotinamide mononucleotide) solution; a subpackaging step in which an NMN solution in an amount corresponding to the NMN of 50 mg to 200 mg or 600 mg to 800 mg is subpackaged into penicillin bottles at a depth of 10 to 45 mm; a freeze-drying step for producing flocculent NMN by performing, under a pressure of 1-100 Pa, a first treatment in which the vial is held at-37 to-43 DEG C for 8 hours, a second treatment in which the vial is held at-27 to-33 DEG C for 3 hours, a third treatment in which the vial is held at-12 to-18 DEG C for 99 hours, a fourth treatment in which the vial is held at-3 to 3 DEG C for 6 hours, and a fifth treatment in which the temperature is raised to 27 to 33 DEG C; a gas phase formation step in which the upper space of the penicillin bottle is filled with a predetermined gas; and a sterilization step in which the flocculent NMN is sterilized with radiation.

Description

Technical Field

[0001] This invention relates to a method for manufacturing an NMN freeze-dried formulation. Background Technology

[0002] NMN (nicotinamide mononucleotide) can be converted into nicotinamide adenine dinucleotide (NAD) in the body. + It is a substance that activates the sirtuin gene, which is known as the longevity gene.

[0003] In recent years, people have been expecting NMN to delay aging and have adopted intravenous infusion to administer it (see Patent Document 1).

[0004] Existing technical documents Patent documents Patent Document 1: Japanese Patent No. 7195498 Summary of the Invention

[0005] The problem that the invention aims to solve However, in order to administer NMN to the target individual via intravenous infusion, NMN needs to be liquefied.

[0006] One method for liquefying NMN is, for example, manufacturing the NMN formulation as a liquid during the production of the NMN preparation. However, in this case, there are several points to note regarding the handling of the NMN preparation.

[0007] First, because NMN is unstable in solution, NMN preparations need to be kept frozen until just before use. Therefore, this is necessary not only in hospitals but also during distribution and storage, requiring careful consideration to maintain the stability of NMN.

[0008] In addition, thawing takes time and is a slow process. When administering the drug to the target, it is necessary to estimate the time and start thawing in advance so that the thawing is completed just before the drug is administered.

[0009] Moreover, once thawed, it cannot be frozen again to maintain its quality; if it is not used immediately after thawing, the preparation must be discarded.

[0010] Therefore, when manufacturing NMN formulations as liquids, precautions may arise due to the instability of NMN in the solution.

[0011] In contrast, if the NMN solution is not manufactured as a liquid, but rather dispensed into vials and then freeze-dried to precipitate NMN, it can be stored at room temperature for about 18 months without the instability issues of NMN mentioned above, making it more advantageous in terms of handling.

[0012] Moreover, when administering the drug, only purified water or saline solution needs to be injected into the vial to dissolve NMN. Once the target person is confirmed to be at the hospital, the drug can be prepared for use, thus minimizing the waste of NMN preparations.

[0013] In addition, not limited to NMN, a variety of freeze-dried formulations for intravenous infusion preparation have been provided to date.

[0014] Therefore, it can also be considered that as long as NMN is frozen and dried in accordance with the traditional freeze-drying technology, it is possible to manufacture freeze-dried formulations for intravenous infusion.

[0015] However, simply freeze-drying will only yield porous, blocky NMN formed by removing moisture through sublimation. Although freeze-dried products in this state have basic solubility, there is still room for improvement in terms of rapid dissolution.

[0016] That is, when administering drugs via intravenous infusion, drug residues are not allowed to dissolve and a homogeneous solution with no concentration deviation must be formed. However, NMN that has undergone conventional freeze-drying is difficult to dissolve to the desired level of rapid dissolution in a solvent for intravenous infusion within a short time.

[0017] Therefore, since it takes time for traditional freeze-dried NMN formulations to dissolve evenly, it is desirable to prepare formulations that can be used smoothly in a shorter time.

[0018] Other formulations besides NMN may have a flocculent appearance due to freeze-drying. Such formulations are expected to dissolve quickly compared to porous, blocky freeze-dried products. However, for NMN, no such preparation method is known to date.

[0019] The present invention was made in view of the above circumstances, and provides a method for manufacturing an NMN freeze-dried formulation, which can produce a flocculent NMN freeze-dried formulation that can be smoothly prepared for use.

[0020] Technical solutions for solving the problem To address the aforementioned existing problems, the present invention relates to a method for manufacturing a freeze-dried NMN preparation (1) comprising: a solution preparation step, wherein a 5-200 mg / mL NMN (nicotinamide mononucleotide) solution is prepared; a dispensing step, wherein an NMN solution of an amount equivalent to 50 mg-200 mg or 600 mg-800 mg of NMN is dispensed into vials at a depth of 10-45 mm; a freeze-drying step, wherein the vials are subjected to the following treatments at a pressure of 1-100 Pa to generate flocculent NMN: a first treatment of maintaining at -37 to -43°C for 8 hours, a second treatment of maintaining at -27 to -33°C for 3 hours, a third treatment of maintaining at -12 to -18°C for 99 hours, a fourth treatment of maintaining at -3 to 3°C for 6 hours, and a fifth treatment of raising the temperature to 27 to 33°C; a gas phase formation step, wherein a specified gas is filled into the upper space of the vials; and a sterilization step, wherein the flocculent NMN is sterilized by radiation.

[0021] Furthermore, the method for manufacturing the NMN freeze-dried formulation involved in this invention also has the following characteristics.

[0022] (2) The specified gas is a gas in which 1 to 50 v / v% argon (Ar) is mixed in nitrogen (N2).

[0023] (3) The vial is a brown glass vial.

[0024] Invention Effects According to the method for manufacturing the NMN freeze-dried formulation of the present invention, the method includes a solution preparation step for preparing an NMN (nicotinamide mononucleotide) solution of 5-200 mg / mL; a dispensing step for dispensing an NMN solution of an amount equivalent to 50 mg-200 mg or 600 mg-800 mg of NMN into vials at a depth of 10-45 mm; and a freeze-drying step for treating the vials under a pressure of 1-100 Pa to generate flocculent NMN by maintaining the solution at -37 to -43°C for 8 hours. The process includes a first treatment, a second treatment of maintaining at -27 to -33°C for 3 hours, a third treatment of maintaining at -12 to -18°C for 99 hours, a fourth treatment of maintaining at -3 to 3°C for 6 hours, and a fifth treatment of raising the temperature to 27 to 33°C; a gas phase formation step of filling the upper space of the vial with a specified gas; and a sterilization step of sterilizing the flocculent NMN with radiation. Therefore, a method for manufacturing an NMN freeze-dried preparation that can produce a flocculent NMN freeze-dried preparation that can be smoothly prepared in use can be provided.

[0025] Furthermore, if the specified gas is a gas in which 1 to 50 v / v% argon (Ar) is mixed in nitrogen (N2), preferably a gas in which 1 to 30 v / v%, more preferably 1 to 10 v / v% argon (Ar) is mixed, then free radicals generated by radiation irradiation can be suppressed, thereby preventing NMN from decomposing into substances other than NMN.

[0026] Furthermore, if the vial is made of brown glass, it can prevent NMN from turning into substances other than NMN due to light exposure. Detailed Implementation

[0027] This invention provides a method for manufacturing a freeze-dried NMN formulation, which can produce a flocculent freeze-dried NMN formulation that can be readily prepared for use.

[0028] The method for manufacturing NMN freeze-dried formulations described in this embodiment consists of a solution preparation process, a dispensing process, a freeze-drying process, a vapor phase formation process, and a sterilization process.

[0029] The solution preparation process is the process of preparing an NMN solution with a concentration of 5–200 mg / mL.

[0030] NMN in this specification includes both α- and β-stereoisomers. For example, β-NMN is a compound having the following structure, which, as previously described, is converted in vivo into nicotinamide adenine dinucleotide (NAD). + ), is a substance that activates deacetylase genes, also known as longevity genes.

[0031] [Chemical Formula 1] The solvent used to dissolve NMN (hereinafter also referred to as manufacturing water) is not particularly limited here; for example, pure water can be used.

[0032] The concentration of the solution is preferably 5–200 mg / mL. When the concentration is below 5 mg / mL, the amount of freeze-dried material produced per unit volume becomes too small, and therefore is not preferred. Furthermore, when the concentration exceeds 200 mg / mL, the freeze-dried NMN is difficult to form into flocculent particles, and therefore is also not preferred.

[0033] The dispensing process involves dispensing an amount of NMN solution, equivalent to 50mg to 200mg or 600mg to 800mg of NMN, into vials at a depth of 10 to 45mm.

[0034] The NMN solution is prepared by dispensing an amount of NMN equivalent to 50 mg to 200 mg (hereinafter referred to as the first range) or 600 mg to 800 mg (hereinafter referred to as the second range) into glass vials. There are no particular limitations on the dispensing method; for example, it can be done manually using a pipette or the like, or mechanically using a filling machine or the like.

[0035] When the equivalent amount of NMN in the dispensed NMN solution is taken as the first range, the following effect is achieved: compared to the second case, a looser, flocculent NMN can be prepared. Furthermore, when the equivalent amount of NMN is taken as the second range, the following effect is achieved: compared to the first case, a denser (tightly crystalline) flocculent NMN can be prepared.

[0036] Furthermore, considering the need to dry the NMN solution in the freeze-drying process described below into a flocculent state, it is preferable that the dispensing depth inside the vial is 10 to 45 mm. When the dispensing depth inside the vial is less than 10 mm, the density of the dried material will be locally uneven, making it impossible to form a uniform flocculent state, which is not preferred. When it exceeds 45 mm, even if the first to fifth processes described later are performed, drying will not be easy, which is also not preferred.

[0037] There are no particular restrictions as long as the vial is made of a material that can withstand freeze-drying; glass or resin products can be used.

[0038] Furthermore, as long as the vial is transparent, there is no particular limitation on the distinction between colored and colorless. As long as the dissolution of NMN can be visually confirmed when adding water for injection into the vial, colored transparent containers such as brown bottles can also be used.

[0039] When using brown transparent vials, the visibility of the contents is poor compared to when using colorless transparent vials, so it is foreseeable that it will be difficult to confirm the dissolution state of freeze-dried NMN.

[0040] Therefore, if it is traditional porous block-shaped NMN, there are concerns about whether to use brown vials due to the possibility of dissolution residue during preparation.

[0041] However, the NMN freeze-dried formulation manufactured by the method for manufacturing NMN freeze-dried formulations according to this embodiment has a flocculent morphology and the possibility of producing dissolved residues is extremely low.

[0042] Therefore, even though visibility is slightly sacrificed, it can be used with peace of mind because there is almost no concern about dissolution residue, and since the brown vial can be used without any worries, you can enjoy the UV blocking effect of the brown vial.

[0043] The freeze-drying process involves freezing and drying the NMN solution inside a vial. Freezing and drying can be performed, for example, using a commercially available freeze dryer.

[0044] The freeze-drying process is carried out by performing the following first to fifth treatments under a pressure of 1 to 100 Pa.

[0045] The first treatment involved adjusting the pressure of the NMN solution to 1–100 Pa at -37 to -43°C and maintaining this temperature for 8 hours. The second treatment involved maintaining the solution at -27 to -33°C for 3 hours, and the third treatment involved maintaining the solution at -12 to -18°C for 99 hours.

[0046] If the first to third treatments are performed in this manner, almost all the ice will be removed, resulting in a state where the first drying is complete. Crucially, this process immediately transitions to a second drying stage. This is to remove the water that did not freeze during pre-freezing but was incorporated into the solute as unfrozen water (bound water). Therefore, the second drying stage needs to be carried out shortly after the first drying stage.

[0047] The subsequent secondary drying is achieved through the fourth and fifth processes.

[0048] The fourth treatment involves stabilizing the vials at -3 to 3°C for 6 hours, and the fifth treatment involves heating to 27 to 33°C. This secondary drying process completely removes moisture from the vials and inhibits hydrolysis, allowing for transportation and storage at room temperature while maintaining quality.

[0049] The gas phase formation process is the process of filling the upper space of the vial with a specified gas. A gas mixed with 1 to 50 v / v% argon (Ar) in nitrogen (N2) (hereinafter referred to as the mixed gas) is introduced into the upper space of the vial for filling and sealing.

[0050] In general, the gas phase formation of freeze-dried formulations is carried out using only nitrogen gas. However, in the method for manufacturing NMN freeze-dried formulations according to this embodiment, the replacement gas is not only nitrogen gas (N2), but also a mixed gas combined with argon gas (Ar).

[0051] This configuration can prevent NMN from being degraded or decomposed by the free radicals of nitrogen (N2) generated during subsequent sterilization processes when exposed to radiation.

[0052] The sterilization process involves sterilizing the inside of the vial. This sterilization is performed using radiation, such as gamma rays or electron beams.

[0053] In the case of sterilization by gamma rays, it is desirable to irradiate with an energy level that does not affect the quality of NMN, such as 25 kGy.

[0054] As described above, the method for manufacturing NMN freeze-dried formulations according to this embodiment, by including these solution preparation steps, dispensing steps, freeze-drying steps, gas phase formation steps, and sterilization steps, can produce flocculent NMN freeze-dried formulations, thereby shortening the dissolution time and increasing the concentration uniformity during dissolution, and can provide a product most suitable for use as an intravenous infusion formulation.

[0055] The following description, based on experimental results, further explains the manufacturing method of the NMN freeze-dried formulation involved in this embodiment.

[0056] [First Embodiment] (1-1. Preparation of NMN freeze-dried formulation) 2.4 g of NMN was weighed into a 200 mL beaker and dissolved in 100 mL of purified water to prepare a 24 mg / mL NMN aqueous solution. Furthermore, no dissolved residue was observed at this concentration.

[0057] Next, in a clean bench, 5 mL of the NMN aqueous solution is dispensed into 10 mL amber glass vials using a pipette. Furthermore, in the following description of the freeze-drying and sterilization processes, the dispensed NMN solution, regardless of whether it is in liquid, solid, or dry state, is referred to as the "drying object".

[0058] Next, the NMN solution was freeze-dried using a freeze dryer. Specifically, the vials were placed on a tray, and the tray was placed together in the chamber of the freeze dryer and the door was closed.

[0059] Next, the temperature inside the chamber is lowered, and the dried object in the vial is frozen while being measured to be -37 to -43°C.

[0060] The pressure was reduced to 1–100 Pa, and the following first to fifth treatments were performed while maintaining this pressure. That is, the first treatment was performed by maintaining the temperature at -37 to -43°C for 8 hours to allow the solvent to sublimate from the frozen dried object.

[0061] Next, the object to be dried in the vial is heated to -27 to -33°C and maintained for 3 hours (second treatment), and then maintained at -12 to -18°C for 99 hours to complete the first drying process (third treatment).

[0062] Next, the sample in the vial was kept at -3 to 3°C for 6 hours (fourth treatment), and then the temperature was raised to 33°C to end the second drying process (fifth treatment). When the second drying process was completed, the sample in the vial was observed to be flocculent.

[0063] Next, a gas phase formation process is carried out by supplying a mixture of nitrogen (N2) and argon (Ar) in a volume ratio of 90:10 (nitrogen containing 10v / v% argon) into the chamber, allowing it to flow into and fill the upper space of the vial, and then sealing it with a sealing frame.

[0064] Next, the sealed vials were removed from the freeze dryer and sterilized by irradiating the object with gamma rays at a dose of 25 kGy using a radiation sterilization device, thereby obtaining the NMN freeze-dried formulation. It was also confirmed that the NMN freeze-dried formulation retained its flocculent morphology even at the end of the sterilization process.

[0065] Next, an experiment was conducted to test whether the NMN freeze-dried formulation prepared by the above (1-1. Preparation of NMN freeze-dried formulation) could dissolve rapidly to a degree that would allow for smooth preparation during use.

[0066] Specifically, 5 mL of physiological saline was added to a 10 mL volumetric vial of the NMN freeze-dried preparation prepared above and the vial was turned over and stirred until no dissolved residue was visible after a certain number of turns.

[0067] The results showed that the preparation in the vial dissolved almost instantly after the addition of physiological saline, and no dissolved residue was visible after the first inversion.

[0068] This confirms that the method for manufacturing the NMN freeze-dried formulation involved in this embodiment is a method for manufacturing an NMN freeze-dried formulation that can be smoothly prepared for use.

[0069] [Second Embodiment] Next, it was confirmed whether flocculent NMN could be generated under various deviated conditions.

[0070] (a. Solubility test of NMN freeze-dried formulation) In the solution preparation process, it was confirmed whether flocculent NMN could be generated even when the solution concentration deviated. Specifically, the study was conducted under the conditions shown in Table 1.

[0071] [Table 1] The results show that, as shown in Examples 2-1 and 2-2 in Table 1, when the value of solution concentration a in the solution preparation process deviates, the dried object does not appear flocculent.

[0072] (b. Verification experiment on the dispensing depth of NMN solution) In the dispensing process, it was confirmed whether flocculent NMN could be generated by changing the dispensing depth of the NMN solution. Specifically, the study was conducted under the conditions shown in Table 2.

[0073] [Table 2] The results show that, as shown in Examples 2-3 and 2-4 in Table 2, even when the NMN solution is dispensed to a depth of 1.0 to 4.5 cm in the dispensing process, the dried object can still be made into a flocculent state.

[0074] (c. Temperature test of freeze-drying process) In the freeze-drying process, it was confirmed whether flocculent NMN could be generated under deviated temperature conditions. Specifically, the study was conducted under the conditions shown in Table 3.

[0075] [Table 3] The results show that, as shown in Examples 2-5 and 2-6 in Table 3, even if the temperature of the first to fifth processing temperatures in the freeze-drying process is increased by 3°C or decreased by 3°C, the dried object can be made into a flocculent state.

[0076] [Third Embodiment] In the gas-phase formation process, the difference in residual NMN after γ-ray sterilization was confirmed between the use of a mixed gas of nitrogen and argon and the use of nitrogen alone.

[0077] Specifically, the residual NMN in the freeze-dried formulation prepared using the method of the first embodiment was quantified and evaluated by HPLC in both the case of using pure nitrogen and the case of using a mixed gas composed of nitrogen and argon. The results are shown in Table 4.

[0078] [Table 4] As shown in Example 3-1, when nitrogen gas was used for replacement alone, the residual amount of NMN decreased by 4.2% to 95.8% due to the influence of gamma ray irradiation. Although the mechanism of this phenomenon is not yet clear, the inventors believe that NMN deteriorates or decomposes due to gamma ray irradiation.

[0079] In contrast, when a mixed gas containing less than half the total amount of argon is used for replacement, the effect on the residual amount of NMN is suppressed and the residual amount increases even after gamma ray irradiation. It is particularly noteworthy that, compared with Example 3-1 which uses pure nitrogen, adding only 1% argon can significantly improve the residual amount (Example 3-2).

[0080] Furthermore, when the gas was replaced with a mixture containing 90v / v% nitrogen and 10v / v% argon (Example 3-3), a mixture containing 70v / v% nitrogen and 30v / v% argon (Example 3-4), and a mixture containing 50v / v% nitrogen and 50v / v% argon (Example 3-5), the residual amount of NMN increased compared to Example 3-1, which used pure nitrogen.

[0081] However, when replaced with a mixed gas containing 30 v / v% nitrogen and 70 v / v% argon (Examples 3-6), the residual amount showed a decreasing trend compared to Example 3-1, which used pure nitrogen.

[0082] In summary, based on the results of this third embodiment, it can be considered that the gas filled into the upper space of the vial in the gas phase formation process is preferably a mixture of nitrogen and argon, and the proportion of argon in the 100v / v% of the replacement gas is 1 to 50v / v, preferably 1 to 30v / v, and more preferably 1 to 10v / v.

[0083] Based on the first to third embodiments described above, it can be seen that the method for manufacturing the NMN freeze-dried preparation involved in this embodiment is a method for manufacturing a flocculent NMN freeze-dried preparation that can be smoothly prepared during use.

[0084] Finally, the above description of the embodiments is one example of the present invention, and the present invention is not limited to the above embodiments. Therefore, even in cases other than the above embodiments, various modifications can be made according to capacity, etc., as long as they do not depart from the technical concept involved in the present invention.

Claims

1. A method for manufacturing an NMN freeze-dried formulation, characterized in that, have: The solution preparation process involves preparing an NMN solution with a concentration of 5–200 mg / mL, wherein the NMN is nicotinamide mononucleotide; The dispensing process involves dispensing an NMN solution equivalent to 50mg to 200mg or 600mg to 800mg of NMN into vials at a depth of 10 to 45mm. The freeze-drying process involves treating the vial under a pressure of 1 to 100 Pa as follows to generate flocculent NMN: a first treatment at -37 to -43°C for 8 hours, a second treatment at -27 to -33°C for 3 hours, a third treatment at -12 to -18°C for 99 hours, a fourth treatment at -3 to 3°C for 6 hours, and a fifth treatment at 27 to 33°C. A gas-phase formation process, which fills the upper space of the vial with a specified gas; and The sterilization process involves sterilizing the flocculent NMN using radiation.

2. The method for manufacturing the NMN freeze-dried formulation according to claim 1, characterized in that, The specified gas is a gas in which 1 to 50 v / v% argon (Ar) is mixed in nitrogen (N2).

3. The method for manufacturing the NMN freeze-dried formulation according to claim 1 or 2, characterized in that, The vial is made of brown glass.

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