Freeze-dried injectable formulation comprising beta-nicotinamide mononucleotide, injectable composition comprising same, and preparation method therefor

Abstract

The present invention relates to a freeze-dried injectable formulation comprising beta-nicotinamide mononucleotide, an injectable composition comprising same, and a preparation method therefor. In addition, the present invention relates to a pain relief injectable composition and a preparation method therefor, the composition comprising beta-nicotinamide mononucleotide and hyaluronidase, and having a pH of greater than 4 and 8 or less.

Description

Injectable lyophilized formulation containing beta-nicotinamide mononucleotide, injectable composition containing the same, and method for preparing the same

[0001] The present invention relates to an injectable lyophilized formulation comprising beta-nicotinamide mononucleotide, an injectable composition comprising the same, and a method for preparing the same.

[0002] Globally, the desire for anti-aging or reversal of aging is intensifying due to the increase in average lifespan and improvements in living standards. However, aging is the next stage of maturation and is an inevitable process of all life that occurs irreversibly over time and leads to death. Aging is influenced by genetic, lifestyle, and environmental factors. Although there have been improvements in lifestyle and the environment over the past few hundred years, the fact that maximum average lifespan has not increased significantly suggests that genetic factors are the most significant. Considering this, improving genetic factors can slow down the rate of aging and allow one to achieve the maximum lifespan bestowed by nature. The genetic factor of aging is explained by the accumulation of DNA damage, and maintaining DNA stability is a crucial method for inhibiting aging and extending lifespan.

[0003] Nicotinamide mononucleotide (NMN) began to be known as a future anti-aging drug in 2016 after a team led by Professor Shinichiro Imai in Washington, USA, confirmed that the lifespan of mice administered oral NMN increased by 16%. NMN exists in the body and plays a role in activating Sirtuin, a gene related to longevity, but levels of NMN decrease with aging. As a nucleotide, NMN is naturally formed through a reaction between a nucleoside containing ribose and nicotinamide (NAM) and a phosphate group; it has two isomers, NMN alpha and beta, with the beta isomer being the active form.

[0004] These beta-NMNs have been sold as oral dietary supplements in the United States. However, oral administration presents the problem of requiring large doses because only a portion is absorbed as the drug must pass through various digestive organs. While sublingual administration exhibits a bioavailability of up to 30%, there are significant individual variations due to the characteristics of oral administration and the retention time of the sublingual dose. Additionally, while the current dosage for beta-NMN supplements in the U.S. is up to 1250 mg per day, there are limitations to consuming large amounts daily due to the high cost of the drug.

[0005] Compared to oral administration, administration via injectable formulation is the optimal method for rapidly delivering a precise dose and effectively controlling systemic effects. Additionally, it offers the advantage of producing the same effect with a lower dose than that of oral administration. However, the stability and safety of injectable formulations must be given critical consideration.

[0006] Accordingly, research is needed to improve stability and safety when developing injectable formulations containing beta-nicotinamide mononucleotide. In particular, optimization studies are required to minimize pain induction in subcutaneous or intramuscular administration methods.

[0007] The present invention aims to provide an injectable lyophilized formulation containing beta-nicotinamide mononucleotide.

[0008] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0009] The present invention provides an injectable lyophilized formulation comprising beta-nicotinamide mononucleotide.

[0010] The above-mentioned lyophilized formulation for injection may further comprise one or more stabilizers selected from the group consisting of monosaccharides; disaccharides; and sugar alcohols.

[0011] The above-mentioned lyophilized formulation for injection may have a pH of 3 to 8.

[0012] The moisture content of the above-mentioned lyophilized formulation may be 5% or less.

[0013] In one embodiment of the present invention, an injectable composition is provided in which the freeze-dried formulation is redissolved in an aqueous solution.

[0014] The above-mentioned injectable composition may be for administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, and arterial.

[0015] In the above-mentioned injectable composition, the dose of the beta-nicotinamide mononucleotide may be administered at 1 / 20 to 1 / 2 times the dose for which a therapeutic effect was confirmed for oral administration.

[0016] In the above injectable composition, the beta-nicotinamide mononucleotide may be administered at a dose of 5 mg / kg to 40 mg / kg per day.

[0017] In another embodiment of the present invention, a method for preparing an injectable lyophilized formulation is provided, comprising the steps of: (a) preparing a solution containing beta-nicotinamide mononucleotide; and (b) freeze-drying the solution.

[0018] In another embodiment of the present invention, a method for preparing an injectable composition is provided, comprising the steps of: (a) preparing a solution containing beta-nicotinamide mononucleotide; (b) freeze-drying the solution to prepare an injectable lyophilized formulation; and (c) adding an aqueous solution to the injectable lyophilized formulation to redissolve it.

[0019] In another embodiment of the present invention, a pain-relieving injectable composition is provided, comprising beta-nicotinamide mononucleotide and hyaluronidase, characterized by having a pH greater than 4 and a pH of 8.

[0020] The above pain-relieving injection composition may have a pH of 5 to 7.

[0021] The osmotic pressure of the above pain-relieving injection composition may be 800 mOsmol / kg to 1,300 mOsmol / kg.

[0022] The above pain-relieving injectable composition may be for subcutaneous or intramuscular administration.

[0023] The above pain-relieving injectable composition may further include one or more stabilizers selected from the group consisting of monosaccharides; disaccharides; and sugar alcohols.

[0024] The above pain-relieving injectable composition may further include one or more alkalizing agents selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH)2), potassium hydroxide (KOH), tromethamine, and sodium bicarbonate, or HCl.

[0025] After administering the above pain-relieving injection composition, blood was collected at 5, 30, 60, 120, and 360 minutes and analyzed using an NAD+ / NADH analysis kit, and the results showed that i) the average blood NAD+ concentration at each time point increased by 200% to 400% compared to placebo administration, and ii) the average blood NADH concentration at each time point increased by 150% to 300% compared to placebo administration.

[0026] In another embodiment of the present invention, a method for preparing a pain-relieving injectable composition is provided, comprising: (a) preparing a solution containing beta-nicotinamide mononucleotide and hyaluronidase; (b) freeze-drying the solution to prepare a freeze-dried formulation; and (c) adding an aqueous solution to the freeze-dried formulation, wherein the pH is greater than 4 and has a pH of 8.

[0027] The injectable lyophilized formulation according to the present invention (particularly, a lyophilized formulation maintaining optimal pH conditions) comprises beta-nicotinamide mononucleotide, which is characterized by excellent stability. Accordingly, when an injectable composition is prepared using the injectable lyophilized formulation according to the present invention (particularly, a lyophilized formulation maintaining optimal pH conditions), there is an advantage in that its stability and safety can be improved. Furthermore, there is an advantage in that excellent pharmacological efficacy can be demonstrated with a lower dose compared to oral administration.

[0028] In addition, the pain-relieving injectable composition according to the present invention comprises beta-nicotinamide mononucleotide and hyaluronidase, and is characterized by having a pH greater than 4 and a pH of 8, thereby improving stability and safety, and in particular, having the advantage of minimizing pain induction.

[0029] As such, the injectable composition or pain-relieving injectable composition according to the present invention can optimize the NAD+ / NADH ratio in the body, thereby having an excellent effect in preventing aging.

[0030] Figure 1(a) shows the results of analyzing the blood NAD+ concentration when the beta-NMN injectable composition prepared in Example I-2 was administered intravenously (IV formulation (2)), and Figure 1(b) shows the results of analyzing the blood NADH concentration when the beta-NMN injectable composition prepared in Example I-2 was administered intravenously (IV formulation (2)).

[0031] Figure 2 shows the results of visual observation of the appearance after 8 weeks of storing an injectable composition (SC formulation) using the lyophilized formulations of beta-NMN and hyaluronidase prepared in Comparative Example II-1 and Examples II-1 to II-3 at 25°C for 8 weeks.

[0032] Figure 3(a) shows the results of analyzing blood NAD+ concentration upon subcutaneous administration (SC formulation) of the beta-NMN and hyaluronidase pain-relieving injection composition prepared in Example II-1, and Figure 3(b) shows the results of analyzing blood NADH concentration upon subcutaneous administration (SC formulation) of the beta-NMN and hyaluronidase pain-relieving injection composition prepared in Example II-1.

[0033] The inventors confirmed that in developing an injectable containing beta-nicotinamide mononucleotide, applying a lyophilized formulation (particularly a lyophilized formulation that maintains optimal pH conditions) can not only improve stability and safety but also yield excellent animal pharmacokinetic (PK) evaluation results even at low doses.

[0034] The inventors have confirmed that in developing an injectable agent comprising beta-nicotinamide mononucleotide and hyaluronidase, using an optimal lyophilized formulation (in particular, a lyophilized formulation that maintains optimal pH and osmotic pressure conditions) can improve stability and safety while, in particular, minimizing pain induction.

[0035]

[0036] The present invention will be described in detail below.

[0037]

[0038] Injectable lyophilized formulation / injectable composition

[0039]

[0040] The present invention provides an injectable lyophilized formulation comprising beta-nicotinamide mononucleotide.

[0041] In addition, the present invention provides an injectable composition in which the above-mentioned freeze-dried formulation is redissolved in an intravenous solution.

[0042]

[0043] The injectable lyophilized formulation according to the present invention is characterized by comprising beta-nicotinamide mononucleotide as a pre-formulation for preparing an injectable composition.

[0044] The above beta-nicotinamide mononucleotide is also called beta-NMN, and its specific structure is as follows:

[0045] [Chemical Formula 1]

[0046]

[0047] The above-mentioned beta-nicotinamide mononucleotide is a precursor of NAD+ and can be converted into NAD+ in the body; it possesses various benefits, including anti-aging, energy enhancement, improvement of cardiovascular health, protection of cognitive function, improvement of metabolism, and reduction of inflammation and oxidative stress.

[0048] In particular, according to the present invention, the beta-nicotinamide mononucleotide is prepared in a freeze-dried formulation, and then an injectable composition is prepared. When the injectable composition prepared in this manner is administered into the body, it lowers NADH activity while simultaneously increasing NAD+ activity to maintain an optimal ratio, thereby having an excellent anti-aging effect. That is, the injectable composition is characterized by improving the NAD+ / NADH ratio in the body.

[0049] Meanwhile, if the above-mentioned beta-nicotinamide mononucleotide is prepared in a liquid form rather than a freeze-dried form, there is a problem due to reduced stability as the change in the content of the beta-nicotinamide mononucleotide becomes relatively large when the liquid form is stored at 4°C for 8 weeks. In addition, when an injectable composition is prepared from the above-mentioned liquid form, there is a problem of generating a significant amount of insoluble particulates, particularly when the pH is 6 or higher. Consequently, there is a limitation in that it is not effective in improving the NAD+ / NADH ratio in the body.

[0050]

[0051] The above-described lyophilized formulation for injection may additionally include a stabilizer to prevent hydrolysis of the beta-nicotinamide mononucleotide. The stabilizer may be a stabilizer well known in the art, and is preferably one or more selected from the group consisting of monosaccharides (glucose, etc.); disaccharides (trehalose, sucrose, lactose, maltose, isomaltose, etc.); and sugar alcohols (mannitol, xylitol, sorbitol, maltitol, erythritol, lactitol, etc.), and is more preferably a sugar alcohol (mannitol, xylitol, sorbitol, maltitol, erythritol, lactitol, etc.), but is not limited thereto.

[0052] Specifically, the content of the stabilizer may be 50% to 200% by weight relative to the content of the beta-nicotinamide mononucleotide, and preferably 80% to 100% by weight, but is not limited thereto.

[0053]

[0054] The above-mentioned lyophilized formulation for injection can maintain acidic conditions and may have a pH of 3 to 8, preferably a pH of 3 or higher and less than 6, and preferably a pH of 3 to 5, but is not limited thereto. Thus, the above-mentioned lyophilized formulation for injection can have the advantage of excellent stability as it can cause almost no change in the content of the beta-nicotinamide mononucleotide when stored at 4°C for 8 weeks. On the other hand, in the above-mentioned lyophilized formulation for injection, if the pH is 6 or higher, there is a problem in that the change in the content of the beta-nicotinamide mononucleotide increases significantly when stored at 4°C for 8 weeks.

[0055] In addition, the above-mentioned lyophilized formulation for injection has a minimized moisture content, and the moisture content may be 5% or less, and preferably 3% or less, but is not limited thereto. The above-mentioned lyophilized formulation for injection may be formed into a cake with a minimized moisture content.

[0056]

[0057] In addition, the injectable lyophilized formulation according to the present invention needs to be redissolved or diluted in an intravenous solution to control the concentration of the beta-nicotinamide mononucleotide.

[0058] The above-mentioned fluid may be water for injection, or may additionally contain an isotonic agent such as sodium chloride or glucose in water for injection (e.g., 0.1–2.0% physiological saline solution, 1–10% glucose solution, or lactose-added Ringer's solution).

[0059] Thus, the beta-nicotinamide mononucleotide in the injectable composition according to the present invention can maintain a concentration of 1 mg / mL to 1,000 mg / mL, and preferably maintains a concentration of 50 mg / mL to 100 mg / mL, but is not limited thereto. Accordingly, it is particularly suitable for intravenous administration.

[0060]

[0061] Meanwhile, the above-mentioned injectable composition may be intended for administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, and arterial, and is preferably intended for intravenous administration but is not limited thereto. Such intravenous administration has the advantage of rapidly administering an accurate amount to affect the entire body while being well controlled, but stability and safety must be considered as primary factors for direct injection into blood vessels.

[0062] In the above-described injectable composition, the dose of the beta-nicotinamide mononucleotide may be administered at 1 / 20 to 1 / 2 times the dose for which a therapeutic effect has been confirmed for oral administration, and preferably at 1 / 20 to 1 / 4 times. Even with such a low dose administered, it can have an equivalent or greater effect and can have an immediate drug release effect in the body.

[0063] In the above injectable composition, the beta-nicotinamide mononucleotide may be administered at a dose of 0.5 mg / kg to 500 mg / kg per day.

[0064] After administering the above-mentioned injectable composition (after administering beta-NMN at a dose of 500 mg / kg / day for 3 days), blood was collected at 5, 30, 60, 120, and 360 minutes and analyzed using an NAD+ / NADH analysis kit, and the results showed that i) the average blood NAD+ concentration at each time point increased by 200% to 400% (especially 250% to 350%) compared to placebo administration, and ii) the average blood NADH concentration at each time point increased by 150% to 300% (especially 150% to 250%) compared to placebo administration.

[0065] In particular, after administering the above-mentioned injectable composition (after administering beta-NMN at a dose of 500 mg / kg / day for 3 days), blood was collected at 5, 30, and 60 minutes and analyzed using a NAD+ / NADH analysis kit, and the results showed that i) the average blood NAD+ concentration at each initial time point increased by 350% to 450% compared to placebo administration, and ii) the average blood NADH concentration at each initial time point increased by 200% to 300% compared to placebo administration.

[0066] In addition, when blood is collected 30 minutes after administering the above-mentioned injectable composition and analyzed using an NAD+ / NADH analysis kit, the blood NAD+ concentration may increase by 550% to 600% compared to placebo administration. Meanwhile, when blood is collected 5 minutes after administering the above-mentioned injectable composition and analyzed using an NAD+ / NADH analysis kit, the blood NADH concentration may increase by 350% to 400% compared to placebo administration.

[0067] In other words, when the above-mentioned injectable composition is administered, the increase in the average / maximum blood NAD+ concentration compared to placebo administration can be seen as relatively higher than the increase in the average / maximum blood NADH concentration compared to placebo administration. In particular, when the above-mentioned injectable composition is administered intravenously, the NAD+ / NADH ratio in the body can be optimized, which can have an excellent effect in preventing aging.

[0068]

[0069] Method for preparing an injectable lyophilized formulation / injectable composition

[0070]

[0071] The present invention provides a method for preparing an injectable lyophilized formulation, comprising the steps of: (a) preparing a solution containing beta-nicotinamide mononucleotide; and (b) freeze-drying the solution.

[0072] In addition, the present invention provides a method for preparing an injectable composition comprising the steps of: (a) preparing a solution containing beta-nicotinamide mononucleotide; (b) freeze-drying the solution to prepare an injectable freeze-dried formulation; and (c) adding an aqueous solution to the injectable freeze-dried formulation to redissolve it.

[0073]

[0074] First, the present invention comprises the step of preparing a solution containing beta-nicotinamide mononucleotide [step (a)].

[0075] The above solution contains beta-nicotinamide mononucleotide. Since the beta-nicotinamide mononucleotide has been described above, a redundant explanation will be omitted.

[0076] A stabilizer may be additionally included to prevent hydrolysis of the above-mentioned beta-nicotinamide mononucleotide, and since the stabilizer has also been described above, a redundant explanation will be omitted.

[0077] In the above solution, nitrogen-substituted water for injection may be used as a solvent. Such nitrogen-substituted water for injection can be prepared through a process known in the art and has an advantage in cake formation after freeze-drying as oxygen is replaced with nitrogen through the dissolution and filling process.

[0078]

[0079] Next, the present invention comprises the step of freeze-drying the solution to produce an injectable freeze-dried formulation [step (b)].

[0080] Prior to the freeze-drying step, a stabilization step may be performed, which can be carried out at 1°C to 10°C for 10 to 200 minutes under atmospheric pressure.

[0081] The freeze-drying step described above is divided into a freezing step and a drying step, wherein the freezing step may be performed for 100 minutes to 1000 minutes at -100°C to -1°C (preferably -80°C to -5°C) under atmospheric pressure. To minimize bubble generation, the freezing step may be performed in two stages, wherein the first stage may be performed at -10°C to -1°C, and the second stage may be performed at -100°C to -50°C. Meanwhile, the drying step may be performed for 500 minutes to 3000 minutes at 5°C to 50°C (preferably 10°C to 25°C) under 0.1 Pa to 1 Pa. To form a cake with minimized moisture content, the drying step may be performed in two stages, wherein the first stage may be performed at 5°C to 15°C, and the second stage may be performed at 20°C to 50°C.

[0082] As the above-mentioned lyophilized formulation for injectable use has been described previously, a redundant explanation will be omitted.

[0083]

[0084] Next, the present invention includes the step of adding an aqueous solution to the above-mentioned lyophilized formulation to redissolve it [step (c)]. By doing so, the injectable composition according to the present invention can be finally prepared.

[0085] The above-mentioned intravenous solution is intended to regulate the concentration of the above-mentioned beta-nicotinamide mononucleotide; since specific details have been previously described, a redundant explanation will be omitted.

[0086] Thus, the beta-nicotinamide mononucleotide in the injectable composition according to the present invention can maintain a concentration of 50 mg / mL to 500 mg / mL. In addition, the injectable composition according to the present invention can significantly reduce the number of insoluble particulates even after 24 hours of preparation, thereby ensuring stability as an injectable.

[0087]

[0088] Pain relief injection composition

[0089]

[0090] The present invention provides a pain-relieving injectable composition comprising beta-nicotinamide mononucleotide and hyaluronidase, characterized by having a pH greater than 4 and a pH of 8.

[0091]

[0092] In the "pain-relieving injectable composition" of this specification, pain relief means reducing unpleasant sensations associated with actual and potential tissue damage, the cause of such pain is due to the injection needle, and the pain site may be a local area centered on the tissue penetrated by the injection needle.

[0093]

[0094] First, the pain-relieving injectable composition according to the present invention comprises beta-nicotinamide mononucleotide.

[0095] Specifically, since the above-mentioned beta-nicotinamide mononucleate has been described above, a redundant explanation will be omitted.

[0096] Thus, the beta-nicotinamide mononucleotide in the pain-relieving injectable composition according to the present invention can maintain a concentration of 1 mg / mL to 1,000 mg / mL, and preferably maintains a concentration of 100 mg / mL to 500 mg / mL, but is not limited thereto. Accordingly, it is particularly suitable for subcutaneous or intramuscular administration.

[0097]

[0098] In addition, the pain-relieving injectable composition according to the present invention may further include hyaluronidase. This hyaluronidase is a substance added to enable the beta-nicotinamide mononucleotide to act more effectively in the body, and can produce a synergistic effect in improving the NAD+ / NADH ratio of the beta-nicotinamide mononucleotide in the body.

[0099] Specifically, the hyaluronidase is a large family of enzymes that degrade hyaluronic acid. Hyaluronic acid is a key component of the extracellular matrix and a major component of the interstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, the hyaluronidase lowers its viscosity, thereby increasing tissue permeability and diffusion. Therefore, the hyaluronidase is used as an adsorption agent or dispersant in combination with other reagents, drugs, proteins, etc., to enhance dispersibility and delivery.

[0100] That is, the hyaluronidase has the effect of facilitating the effective dispersion, delivery, and effect of the beta-nicotinamide mononucleotide within the body by promoting (or catalyzing) the hydrolysis of the hyaluronic acid.

[0101] In addition, the hyaluronidase may include any hyaluronidase of non-human origin, hyaluronidase of human origin, etc.

[0102] At this time, hyaluronidase of non-human origin may include hyaluronidase of origin from rodents, dogs, cats, rabbits, birds, cattle, sheep, pigs, horses, fish, frogs, fungi, leeches, other parasites, crustaceans, etc. For example, hyaluronidase of non-human origin may include hyaluronidase of origin from cattle, wasps, honeybees, white-faced bumblebees, giant hornets, mice, pigs, rats (white mice), rabbits, sheep, orangutans, Philippine monkeys, guinea pigs, Staphylococcus aureus, Streptococcus, Clostridium perfringens, etc.

[0103] In addition, hyaluronidase of human origin may include HYAL1, HYAL2, HYAL3, HYAL4, PH20, or variants thereof.

[0104] The content of the hyaluronidase in the pain-relieving injection composition according to the present invention may be 5 IU / mL to 1,500 IU / mL, and it is preferable to maintain a concentration of 50 IU / mL to 1,000 IU / mL, but is not limited thereto.

[0105]

[0106] The pain-relieving injectable composition according to the present invention must maintain optimal pH conditions. Accordingly, it is preferable that the pH be greater than 4 to 8, more preferable that the pH be 5 to 7, and most preferable that the pH be 6 (±0.5), but is not limited thereto. By doing so, the pain-relieving injectable composition has the advantage of significantly reducing pain when injected.

[0107]

[0108] In addition, the osmotic pressure of the pain-relieving injectable composition according to the present invention may be 800 mOsmol / kg to 1,300 mOsmol / kg, preferably 800 mOsmol / kg to 1,200 mOsmol / kg, and more preferably 1,000 mOsmol / kg to 1,200 mOsmol / kg, but is not limited thereto. This osmotic pressure can be controlled by adding an osmotic pressure regulator as needed, but the content of the osmotic pressure regulator needs to be limited so as not to excessively lower the osmotic pressure.

[0109]

[0110] In particular, since the pain-relieving injectable composition according to the present invention is prepared from a freeze-dried formulation, it has the advantage of excellent stability and safety as it can cause almost no change in the content of the beta-nicotinamide mononucleotide when stored at 25°C for 8 weeks. When preparing the pain-relieving injectable composition, if freeze-drying is omitted and a liquid formulation is used, there is a problem in that the change in the content of the beta-nicotinamide mononucleotide increases significantly when stored at 25°C for 8 weeks, thereby greatly reducing stability and safety.

[0111]

[0112] Meanwhile, the pain-relieving injectable composition according to the present invention may additionally include a stabilizer to prevent hydrolysis of the beta-nicotinamide mononucleotide. The stabilizer may be a stabilizer well known in the art, and is preferably one or more selected from the group consisting of monosaccharides (glucose, etc.); disaccharides (trehalose, sucrose, lactose, maltose, isomaltose, etc.); and sugar alcohols (mannitol, xylitol, sorbitol, maltitol, erythritol, lactitol, etc.), and is more preferably a sugar alcohol (mannitol, xylitol, sorbitol, maltitol, erythritol, lactitol, etc.), but is not limited thereto.

[0113] Specifically, the content of the stabilizer may be 20% to 50% by weight relative to the content of the beta-nicotinamide mononucleotide, and preferably 30% to 40% by weight, but is not limited thereto.

[0114]

[0115] In addition, the pain-relieving injectable composition according to the present invention may further include a pH adjuster to maintain optimal pH conditions, and may further include one or more alkalizing agents selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH)2), potassium hydroxide (KOH), tromethamine, and sodium bicarbonate, or HCl. In this case, it is preferable that the alkalizing agent be one or more selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH)2), potassium hydroxide (KOH), tromethamine, and sodium bicarbonate, and it is more preferable that the alkalizing agent be one or more selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH)2), and potassium hydroxide (KOH) in terms of suppressing the generation of insoluble fine particles and ensuring stability, but is not limited thereto.

[0116] For example, to satisfy the optimal pH conditions of the pain-relieving injection composition according to the present invention, the alkalizing agent may be used in combination with HCl, or the alkalizing agent may be omitted and HCl alone may be used.

[0117]

[0118] Optionally, the pain-relieving injectable composition according to the present invention may additionally include additives such as osmotic pressure regulators or surfactants to the extent that physical properties are not altered. Hydroxy-gamma-cyclodextrine (HP-Gamma-Cyclodextrine), Captisol, etc. may be used as the osmotic pressure regulator, and in order not to excessively lower the osmotic pressure, the content thereof may be 10% to 20% by weight relative to the content of beta-nicotinamide mononucleotide, and is preferably 10% to 15% by weight, but is not limited thereto. In addition, polysorbate-based surfactants, etc. may be used as the surfactant, and the content thereof may be 1% to 5% by weight relative to the content of beta-nicotinamide mononucleotide, and is preferably 1% to 2% by weight, but is not limited thereto.

[0119]

[0120] In addition, the lyophilized formulation for injection according to the present invention needs to be diluted in an intravenous solution to control the concentration of the beta-nicotinamide mononucleotide. Specifically, the intravenous solution may be water for injection, or may additionally contain an isotonic agent such as sodium chloride or glucose in the water for injection (e.g., 0.1–2.0% physiological saline solution, 1–10% glucose solution, or lactose-added Ringer's solution). Thus, the beta-nicotinamide mononucleotide in the pain-relieving injection composition according to the present invention can maintain a concentration of 1 mg / mL to 1,000 mg / mL, and it is preferable to maintain a concentration of 100 mg / mL to 500 mg / mL, but is not limited thereto. Accordingly, it is particularly suitable for subcutaneous or intramuscular administration.

[0121]

[0122] Meanwhile, the pain-relieving injectable composition according to the present invention may be intended for subcutaneous or intramuscular administration, and while subcutaneous administration is preferred, it is not limited thereto. Unlike oral formulations, this injectable formulation has the advantage of rapidly administering an accurate amount to affect the entire body while maintaining good control.

[0123] However, stability and safety must be given primary consideration when injectable formulations are to be directly injected into blood vessels. In particular, unlike intravenous administration, subcutaneous or intramuscular administration methods allow for rapid administration in emergencies; specifically, subcutaneous administration offers the advantage of enabling patients to self-administer. However, since patient aversion due to severe pain is a concern with these administration methods, consideration for pain relief is crucial. The pain-relieving injectable composition according to the present invention has the advantage of minimizing pain induction by utilizing a lyophilized formulation that maintains optimal pH and osmotic pressure conditions.

[0124] In the pain-relieving injectable composition according to the present invention, the dose of the beta-nicotinamide mononucleotide may be administered at 1 / 20 to 1 / 2 times the dose for which a therapeutic effect has been confirmed for oral administration, and preferably at 1 / 20 to 1 / 4 times. Even when administered at such a low dose, it may have an effect equivalent to or greater than that and may have an immediate drug release effect in the body.

[0125] That is, in the pain-relieving injectable composition according to the present invention, the beta-nicotinamide mononucleotide can be administered at a dose of 0.5 mg / kg to 500 mg / kg per day.

[0126] After administering the pain-relieving injection composition according to the present invention (after administering a dose of 500 mg / kg / day of beta-NMN for 3 days), blood is collected at 5, 30, 60, 120, and 360 minutes and analyzed using an NAD+ / NADH analysis kit, i) the average blood NAD+ concentration at each time point may increase by 200% to 400% (particularly 250% to 350%) compared to placebo administration, and ii) the average blood NADH concentration at each time point may increase by 150% to 300% (particularly 150% to 250%) compared to placebo administration.

[0127] In particular, after administering the pain-relieving injection composition according to the present invention (after administering a dose of beta-NMN 500 mg / kg / day for 3 days), blood is collected at 5, 30, and 60 minutes and analyzed using a NAD+ / NADH analysis kit, i) the average value of blood NAD+ concentration at each initial time point may increase by 350% to 450% compared to placebo administration, and ii) the average value of blood NADH concentration at each initial time point may increase by 250% to 350% compared to placebo administration.

[0128] In addition, after administering the pain-relieving injection composition according to the present invention, blood is collected at 5 minutes and analyzed using an NAD+ / NADH analysis kit, and the results show that i) the blood NAD+ concentration value increases by 500% to 550% compared to placebo administration, and ii) the blood NADH concentration value increases by 450% to 500% compared to placebo administration.

[0129] In other words, when the pain-relieving injectable composition according to the present invention is administered, the increase in the average / maximum blood NAD+ concentration compared to placebo administration can be seen to be relatively higher than the increase in the average / maximum blood NADH concentration compared to placebo administration. In particular, when the pain-relieving injectable composition according to the present invention is administered subcutaneously, the NAD+ / NADH ratio in the body can be optimized, thereby providing an excellent anti-aging effect.

[0130]

[0131] Method for preparing a pain-relieving injection composition

[0132]

[0133] The present invention provides a method for preparing a pain-relieving injectable composition, comprising the steps of: (a) preparing a solution containing beta-nicotinamide mononucleotide and hyaluronidase; (b) freeze-drying the solution to prepare a freeze-dried formulation; and (c) adding an aqueous solution to the freeze-dried formulation, wherein the pH is greater than 4 and has a pH of 8.

[0134]

[0135] First, the present invention comprises the step of preparing a solution containing beta-nicotinamide mononucleotide and hyaluronidase [step (a)].

[0136] First, the above solution contains beta-nicotinamide mononucleotide; since the beta-nicotinamide mononucleotide has been previously described, a redundant explanation will be omitted. Additionally, the above solution contains hyaluronidase so that the beta-nicotinamide mononucleotide can produce a synergistic effect in improving the NAD+ / NADH ratio in the body; since the hyaluronidase has also been previously described, a redundant explanation will be omitted.

[0137] Optionally, a stabilizer may be additionally included to prevent hydrolysis of the beta-nicotinamide mononucleotide; as specific types have been described above, a redundant explanation will be omitted.

[0138] If necessary, additives such as pH adjusters (alkaliizing agents and / or HCl), osmotic pressure regulators, and surfactants may be additionally included; as specific types have been described above, a redundant explanation will be omitted.

[0139] In the above solution, nitrogen-substituted water for injection may be used as a solvent. Such nitrogen-substituted water for injection can be prepared through a process known in the art and has an advantage in cake formation after freeze-drying as oxygen is replaced with nitrogen through the dissolution and filling process.

[0140]

[0141] Next, the present invention comprises the step of freeze-drying the solution to produce a freeze-dried formulation [step (b)].

[0142] Prior to the freeze-drying step, a stabilization step may be performed, which can be carried out at 1°C to 10°C for 10 to 200 minutes under atmospheric pressure.

[0143] The freeze-drying step described above is divided into a freezing step and a drying step, wherein the freezing step may be performed for 100 minutes to 1000 minutes at -100°C to -1°C (preferably -80°C to -5°C) under atmospheric pressure. To minimize bubble generation, the freezing step may be performed in two stages, wherein the first stage may be performed at -10°C to -1°C, and the second stage may be performed at -100°C to -50°C. Meanwhile, the drying step may be performed for 500 minutes to 3000 minutes at 5°C to 50°C (preferably 10°C to 25°C) under 0.1 Pa to 1 Pa. To form a cake with minimized moisture content, the drying step may be performed in two stages, wherein the first stage may be performed at 5°C to 15°C, and the second stage may be performed at 20°C to 50°C.

[0144] The above freeze-dried formulation has a minimized moisture content, and the moisture content may be 5% or less, preferably 3% or less, but is not limited thereto. The above freeze-dried formulation may be formed into a cake with a minimized moisture content.

[0145]

[0146] Next, the present invention includes the step of adding an aqueous solution to the freeze-dried formulation [step (c)]. By doing so, the pain-relieving injectable composition according to the present invention can be finally prepared with a pH greater than 4 and a pH of 8.

[0147] The above-mentioned intravenous solution is intended to control the concentration of the beta-nicotinamide mononucleotide; since specific details have been previously described, a redundant explanation will be omitted. Accordingly, the beta-nicotinamide mononucleotide in the pain-relieving injectable composition according to the present invention can maintain a concentration of 1 mg / mL to 1,000 mg / mL, and while it is preferable to maintain a concentration of 100 mg / mL to 500 mg / mL, it is not limited thereto. Accordingly, it is particularly suitable for subcutaneous or intramuscular administration.

[0148]

[0149] As reviewed above, the injectable lyophilized formulation according to the present invention (particularly, a lyophilized formulation maintaining optimal pH conditions) comprises beta-nicotinamide mononucleotide, which is characterized by excellent stability. Accordingly, when an injectable composition is prepared using the injectable lyophilized formulation according to the present invention (particularly, a lyophilized formulation maintaining optimal pH conditions), there is an advantage in that its stability and safety can be improved. Furthermore, there is an advantage in that excellent pharmacological efficacy can be demonstrated with a lower dose compared to oral administration.

[0150] In addition, the pain-relieving injectable composition according to the present invention comprises beta-nicotinamide mononucleotide and hyaluronidase, and is characterized by having a pH greater than 4 and a pH of 8, thereby improving stability and safety, and in particular, having the advantage of minimizing pain induction.

[0151] As such, the injectable composition or pain-relieving injectable composition according to the present invention can optimize the NAD+ / NADH ratio in the body, thereby having an excellent effect in preventing aging.

[0152]

[0153] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0154]

[0155] [Example]

[0156] <Example I> Preparation of Beta-NMN Injectable Composition [IV Formulations (1) and (2)]

[0157] Examples I-1 to I-3: Preparation of Beta-NMN Injectable Lyophilized Formulation

[0158] 67 g of beta-nicotinamide mononucleotide (beta-NMN) and 60 g of mannitol were added to 80 mL of nitrogen-substituted water for injection, stirred for 60 minutes to dissolve, and then the pH was adjusted to collect up to 100 mL. The collected solution was filtered using a 0.2 µm cellulose acetate (CA) filter. 5 mL of the filtered liquid was placed in a 20 mL vial and freeze-dried using a freeze-dryer (JABA Lyoph-Pride LP20) to prepare the beta-NMN injectable freeze-dried formulation (moisture content ≤ 5%) as shown in Table 1. The freeze-drying process consisted of: 1) a loading step at 5°C under atmospheric pressure; 2) a stabilization step at 5°C under atmospheric pressure for 120 minutes; and 3) a freezing step at -25°C under atmospheric pressure for 720 minutes. 4) a first drying process at 10°C for 1440 minutes under 0.2 Pa; and 5) a second drying process at 25°C for 500 minutes under 0.2 Pa.

[0159]

[0160] Comparative Examples I-1 to I-3: Preparation of Beta-NMN Injectable Liquid Formulations

[0161] A beta-NMN injectable liquid formulation as shown in Table 1 was prepared by performing the same method as in Examples I-1 to I-3, but omitting freeze-drying.

[0162] Classification Freeze-dried Formulation Liquid Formulation Example I-1 Example I-2 Example I-3 Comparative Example I-1 Comparative Example I-2 Comparative Example I-3 Beta-NMN 67 g 67 g 67 g 67 g 67 g 67 g Mannitol 60 g 60 g 60 g 60 g 60 g Hydrochloric acid appropriate amount appropriate amount appropriate amount appropriate amount appropriate amount Sodium hydroxide appropriate amount appropriate amount appropriate amount appropriate amount pH pH 3~4 pH 4~5 pH 6~7 pH 3~4 pH 4~5 pH 6~7

[0163]

[0164] Experimental Example I-1: Evaluation of Stability of Beta-NMN Injectable Formulations

[0165] To evaluate the stability of the freeze-dried formulations prepared in Examples I-1 to I-3 and the liquid formulations prepared in Comparative Examples I-1 to I-3, the change in beta-NMN content was measured at 4°C for 8 weeks as shown below, and the results are shown in Table 2.

[0166] - Equipment: LC-PDA (Waters HPLC system)

[0167] - Conditions: Column (GL Sciences InerSustain C18, 250×4.6 mm, 5 µm), Mobile phase (10 mM phosphate buffer (pH 3.0) / methanol = 90 / 10), Flow rate (1.0 mL / min), Injection volume (20 µL)

[0168] Classification Freeze-dried Formulation Liquid Formulation Example I-1 Example I-2 Example I-3 Comparative Example I-1 Comparative Example I-2 Comparative Example I-3 Change in Beta-NMN Content Initial 99.8% 99.1% 94.2% 100.1% 99.3% 95.2% Week 4 99.7% 99.5% 90.3% 95.3% 93.3% 80.2% Week 8 99.9% 99.2% 85.3% 90.2% 83.3% 40.8%

[0169] As shown in Table 2, the freeze-dried formulations prepared in Examples I-1 to I-3 exhibited excellent stability compared to the liquid formulations prepared in Comparative Examples I-1 to I-3, as the change in beta-NMN content over 8 weeks at 4°C was negligible. In particular, among the freeze-dried formulations prepared in Examples I-1 to I-3, when maintaining a pH of 3 to 5, the stability was judged to be particularly excellent as the change in beta-NMN content was within 1% over 8 weeks at 4°C.

[0170]

[0171] Experimental Example I-2: Evaluation of Stability of Beta-NMN Injectable Composition

[0172] Water for injection was added to each of the freeze-dried formulations prepared in Examples I-1 to I-3 and the liquid formulations prepared in Comparative Examples I-1 to I-3 to prepare a beta-NMN injectable composition having a beta-NMN concentration of 67 mg / mL. After 24 hours, the stability of the injectable was evaluated by measuring the appearance and insoluble particulates (foreign substances of 10 μm, 25 μm, and 50 μm or larger) at 25°C. At this time, the appearance was observed visually, and the number of insoluble particulates was measured by microscopic counting, and the results are shown in Table 3 and Figure 1.

[0173] Classification Freeze-dried Formulation Liquid Formulation Example I-1 Example I-2 Example I-3 Comparative Example I-1 Comparative Example I-2 Comparative Example I-3 Appearance Clear solution Clear solution Light brown Clear solution Clear solution Light brown Insoluble fine particles 10 ㎛ 00 43 00 76 25 ㎛ 00 200 450 ㎛ 00 00 00

[0174] As shown in Table 3, the injectable composition obtained by redissolving the lyophilized formulations prepared in Examples I-1 to I-3 can significantly reduce the number of insoluble particulates after 24 hours compared to the injectable composition obtained by redissolving the liquid formulations prepared in Comparative Examples I-1 to I-3, thus ensuring stability and safety as an injectable. In particular, among the lyophilized formulations prepared in Examples I-1 to I-3, when a pH of 3 to 5 is maintained, the injectable composition obtained by redissolving it is confirmed to be in a clear solution state and does not generate any insoluble particulates. Therefore, it can be considered that the stability and safety as an injectable are particularly excellent.

[0175]

[0176] Experimental Example I-3: Animal pharmacokinetic (PK) evaluation of a beta-NMN injectable composition (IV formulation (1)).

[0177] A beta-NMN injectable composition with a beta-NMN concentration of 67 mg / mL was prepared by adding water for injection to the lyophilized formulation (pH 4-5) prepared in Example I-2. The prepared beta-NMN injectable compositions at 40 mg / kg, 20 mg / kg, and 10 mg / kg were intravenously administered to rats, and blood NAD levels were evaluated for 6 hours. Meanwhile, the lyophilized formulation (pH 4-5) prepared in Example I-2 was converted into an oral formulation at 80 mg / kg and 40 mg / kg and orally administered to rats, and blood NAD levels were evaluated for 6 hours. Blood samples of 0.5 mL were collected before administration, 5 minutes after administration, 10 minutes after administration, 15 minutes after administration, 30 minutes after administration, 45 minutes after administration, 60 minutes after administration, 90 minutes after administration, 2 hours after administration, 4 hours after administration, and 6 hours after administration, and blood NAD levels were evaluated as follows, and the results are shown in Table 4.

[0178] - Equipment: LC-MS / MS (Nexera X2 Ultra HPLC system), Triple Quadrupole Mass Spectrometer (LCMS-8060, Shimadzu)

[0179] - Conditions: Column (NMN-2 150 mm 2.0 mm, 2.2 µm, Shimadzu) 4℃, Mobile phase (Acetonitrile (A), 0.1% Formic acid (B)), Mobile phase conditions (0-2 min 1% B, 2-10 min 1-38.6 B, 10-12 min 95% B, 12-15 min 1% B), Flow rate (0.2 mL / min), Injection volume (2 µL)

[0180] - Analyzer conditions: Single reaction monitoring, 0.4 sec (1 ms pause), residence time (10-15 ms), interfacial voltage (3.5 KV), spray gas flow rate (3 L / min), heating gas (10 L / min), drying gas flow rate (10 L / min), interfacial temperature (300℃), desolvation line temperature (200℃), heating block temperature (350℃), collision-induced separation gas pressure (270 Kpa)

[0181] Classification Freeze-dried Formulation Oral Formulation Dosage (mg / kg) 40 20 10 16 0 80 AUC (ng / mL*hr) 45 89 325 41 49 5325 41 326 C max (min) 55 59 0 90 T 1 / 2 (min) 24 24 24 30 30

[0182] As shown in Table 4, the injectable composition obtained by redissolving the lyophilized formulation prepared in Example I-2 is confirmed to have an effect equivalent to or greater than that of the oral formulation even when administered at a dose of 1 / 2 or less (especially 1 / 8 or less). In particular, since it has an immediate drug release effect in the body in the case of intravenous administration, it can have a rapid effect compared to oral administration.

[0183]

[0184] Experimental Example I-4: Analysis of blood NAD+ / NADH concentrations upon intravenous administration of a beta-NMN injectable composition (IV formulation (2)).

[0185] A beta-NMN injectable composition having a beta-NMN concentration of 67 mg / mL was prepared by adding water for injection to the freeze-dried formulation (pH 4-5) prepared in Example 2.

[0186] After administering this to rats intravenously at a dose of 500 mg / kg / day of beta-NMN for 3 days (IV formulation (2)), blood samples were collected at 5, 30, 60, 120, and 360 minutes, and blood NAD+ and NADH concentrations were measured at each time using an ABCAM NAD+ / NADH assay kit (Cat No. ab65348).

[0187] At this time, the beta-NMN-only composition was compared with the case where it was orally administered to rats for 3 days at the same dose of 500 mg / kg / day (PO formulation), and the case where an injectable composition without beta-NMN was intravenously administered to rats for 3 days as a placebo (CONT) was used as a control, and the results are shown in Tables 5 and 6 and Figures 1(a) and (b).

[0188] Classification Blood NAD+ Concentration 0 min 5 min 30 min 60 min 120 min 360 min Average Value Initial Average Value IV 1.00 4.5 8 5.6 6 1.6 9 1.00 0.9 12.7 7 4.00 PO 1.00 2.2 7 3.1 2 1.6 5 0.5 6 1.9 5 1.9 12.3 5 CONT 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

[0189] As shown in Table 5 and Figure 1(a), for the IV formulation (2), the average blood NAD+ concentration at each time point was found to have increased significantly by 200% to 400% (particularly 250% to 350%) compared to placebo administration, which is significantly higher than that of the PO formulation. In particular, at the initial time points (5 min, 30 min, and 60 min), the average blood NAD+ concentration was found to have increased significantly by 350% to 450% compared to placebo administration. Additionally, the IV formulation (2) had a maximum blood NAD+ concentration at the 30-minute time point, and the value was found to have increased by 566% compared to placebo administration.

[0190] Category Blood NADH Concentration 0 min 5 min 30 min 60 min 120 min 360 min Average Value Initial Average Value IV 1.00 3.8 8 2.4 3 1.00 1.00 1.00 1.8 6 2.4 3 PO 1.00 1.00 2.1 20.7 8 0.5 6 1.00 1.09 1.3 3 CONT 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

[0191] As shown in Table 6 and Figure 1(b), for the IV formulation (2), the average blood NADH concentration at each time point was found to have increased relatively to 150% to 300% (specifically, 150% to 250%) compared to placebo administration, which is significantly higher than that of the PO formulation. In particular, at the initial time points (5 minutes, 30 minutes, and 60 minutes), the average blood NADH concentration was found to have increased to 200% to 300% compared to placebo administration. Additionally, the IV formulation (2) also had a maximum blood NADH concentration at the 5-minute time point, and the value was found to have increased by 388% compared to placebo administration.

[0192]

[0193] Synthesizing the above results, it can be seen that in the case of the IV formulation, the increase in the average / maximum blood NAD+ concentration compared to placebo administration is relatively higher than the increase in the average / maximum blood NADH concentration compared to placebo administration. In other words, by implementing an injectable composition using a beta-NMN lyophilized formulation into an IV formulation, the NAD+ / NADH ratio in the body can be optimized, thereby providing excellent anti-aging effects.

[0194]

[0195] <Example II> Preparation of Beta-NMN and Hyaluronidase Pain-Relief Injectable Composition [SC Formulation]

[0196] Preliminary Examples II-1 to II-8: Preparation of Beta-NMN and Hyaluronidase Lyophilized Formulations and Redissolved Products thereof

[0197] 900 mg of beta-nicotinamide mononucleotide (beta-NMN) and 300 IU of hyaluronidase (Source: Ningbo zlinzyme Biosciences, China) were added to 2.7 mL of nitrogen-substituted water for injection and stirred for 60 minutes to dissolve. Subsequently, a stabilizer, an osmotic pressure regulator, and a surfactant were optionally added and dissolved for 30 minutes. Then, an appropriate amount of an alkalizing agent or HCl was added as a pH regulator to adjust the pH, and the solution was collected up to 3 mL. The collected solution was filtered using a 0.2 μm cellulose acetate (CA) filter. The filtered liquid was placed in a 20 mL vial and freeze-dried using a freeze-dryer (JABA Lyoph-Pride LP20) to prepare the beta-NMN injectable lyophilized formulation (moisture content ≤ 5%) as shown in Table 7. At this time, freeze-drying was performed through 1) a loading process at 5°C under atmospheric pressure; 2) a stabilization process at 5°C under atmospheric pressure for 120 minutes; 3) a freezing process at -25°C under atmospheric pressure for 720 minutes; 4) a primary drying process at 10°C under 0.2 Pa for 1440 minutes; and 5) a secondary drying process at 25°C under 0.2 Pa for 500 minutes.

[0198] Subsequently, 3 mL of water for injection was added to prepare a beta-NMN and hyaluronidase re-lysate.

[0199] Preliminary Example II-1 Preliminary Example II-2 Preliminary Example II-3 Preliminary Example II-4 Preliminary Example II-5 Preliminary Example II-6 Preliminary Example II-7 Preliminary Example II-8 Beta-NMN 900 mg 900 mg 900 mg 900 mg 900 mg 900 mg 900 mg Hyaluronidase 300 IU 300 IU 300 IU 300 IU 300 IU 300 IU 300 IU Stabilizer Mannitol 300 mg 300 mg 300 mg 300 mg 300 mg 300 mg -- Sorbitol ------ 300 mg - Lactose ------- 300 mg Osmotic pressure regulator Hydroxy-gamma-cyclodextrine (HP-Gamma-Cyclodextrine) -- 100 mg-----Captisol---100 mg----PH regulator (1): Alkalinizing agent NaOH appropriate amount appropriate amount appropriate amount--appropriate amount Tromethamine----appropriate amount---Sodium bicarbonate----appropriate amount--PH regulator (2): HCl appropriate amount appropriate amount appropriate amount appropriate amount appropriate amount appropriate amount Surfactant Polysorbate 80 10 mg

[0200] To evaluate the short-term stability of the beta-NMN and hyaluronidase relysates prepared in Preliminary Examples II-1 to II-8, the number of insoluble particles was measured by microscopic counting at 25°C after 48 hours. The results are shown in Table 8.

[0201] Preliminary Example II-1 Preliminary Example II-2 Preliminary Example II-3 Preliminary Example II-4 Preliminary Example II-5 Preliminary Example II-6 Preliminary Example II-7 Preliminary Example II-8 Insoluble fine particles (48 hours) 10um 2 104 1025 0025um 000056 0050um 00000000

[0202] As shown in Table 8, the beta-NMN and hyaluronidase re-lysates prepared in Preliminary Examples II-1 to II-8 are judged to have short-term stability as they generate a small amount or almost no insoluble microparticles after 48 hours. However, as in Preliminary Examples II-5 and II-6, when tromethamine or sodium bicarbonate is used instead of NaOH as an alkalizing agent, the number of insoluble microparticles increases, and short-term stability is judged to be relatively lower.

[0203]

[0204] Comparative Examples II-1 to II-2, Examples II-1 to II-4: Preparation of injectable compositions (SC formulations) using lyophilized formulations of beta-NMN and hyaluronidase

[0205] 900 mg of beta-nicotinamide mononucleotide (beta-NMN) and 300 IU of hyaluronidase (Source: Ningbo zlinzyme Biosciences, China) were added to 2.7 mL of nitrogen-substituted water for injection and stirred for 60 minutes to dissolve. Subsequently, mannitol and captisol were optionally added and dissolved for 30 minutes; then, an appropriate amount of NaOH and / or HCl was added to adjust the pH, and the solution was collected up to 3 mL. The collected solution was filtered using a 0.2 µm cellulose acetate (CA) filter. The filtered liquid was placed in a 20 mL vial and freeze-dried using a freeze-dryer (JABA Lyoph-Pride LP20) to prepare a freeze-dried beta-NMN injectable formulation (moisture content ≤ 5%). At this time, the freeze-drying process included 1) a loading process at 5°C under atmospheric pressure; 2) a process of stabilizing at 5°C for 120 minutes under atmospheric pressure; 3) a process of freezing at -25°C for 720 minutes under atmospheric pressure; 4) a process of primary drying at 10°C for 1440 minutes under 0.2 Pa; and 5) a process of secondary drying at 25°C for 500 minutes under 0.2 Pa.

[0206] Subsequently, 3 mL of water for injection was added to prepare a beta-NMN and hyaluronidase injection composition (SC formulation) with a concentration of 300 mg / mL of beta-NMN and a concentration of 100 IU / mL of hyaluronidase as shown in Table 9, with controlled pH and osmotic pressure.

[0207] Comparative Example II-1 Comparative Example II-2 Example II-1 Example II-2 Example II-3 Example II-4 Beta-NMN 900 mg 900 mg 900 mg 900 mg 900 mg Hyaluronidase 300 IU 300 IU 300 IU 300 IU 300 IU 300 IU Mannitol 300 mg - 300 mg 300 mg 300 mg 300 mg Captisol 100 mg NaOH appropriate amount - appropriate amount appropriate amount - HCl appropriate amount - appropriate amount appropriate amount appropriate amount pH 3.346786 Osmotic pressure (mOsmol / kg) 1,1257201,1301,1151,133835

[0208] To evaluate the long-term stability of the injectable composition (SC formulation) using the lyophilized formulations of beta-NMN and hyaluronidase prepared in Comparative Examples II-1 to II-2 and Examples II-1 to II-4, the composition was stored at 25°C for 8 weeks as described below, and the change in beta-NMN content was measured after 8 weeks. The results are shown in Table 10.

[0209] - Equipment: LC-PDA (Waters HPLC system)

[0210] - Conditions: Column (GL Sciences InerSustain C18, 250×4.6 mm, 5 µm), Mobile phase (10 mM phosphate buffer (pH 3.0) / methanol = 90 / 10), Flow rate (1.0 mL / min), Injection volume (20 µL)

[0211] In addition, the appearance was observed visually after 8 weeks, and the results are also shown in Table 10 and Figure 3.

[0212] Test Item Comparison Example II-1 Comparison Example II-2 Example II-1 Example II-2 Example II-3 Example II-4 Content Initial Value 99.8 98.9 97.7 98.8 101.1 97.3 8 Weeks Later 99.7 96.7 97.9 96.2 96.7 97.7 Appearance After 4 Weeks Transparent Transparent Transparent Transparent Light Brown Transparent After 8 Weeks Transparent Transparent Transparent Light Brown Light Brown Transparent

[0213] As shown in Table 10 and Figure 3, the injectable compositions (SC formulations) using the lyophilized beta-NMN and hyaluronidase formulations prepared in Comparative Examples II-1 to II-2 and Examples II-1 to II-4 show excellent stability, as the change in beta-NMN content over 8 weeks at 25°C is negligible and they maintain a transparent or light brown appearance. On the other hand, in the case of Examples II-2 and II-3, as the pH increases, the change in beta-NMN content over 8 weeks at 25°C increases compared to Examples II-1 and II-4, and a light brown appearance is observed, so the stability can be considered to be somewhat reduced.

[0214]

[0215] Comparative Examples II-3 to II-8: Preparation of injectable compositions (SC formulations) using beta-NMN and hyaluronidase liquid formulations

[0216] A beta-NMN and hyaluronidase injection composition (SC formulation) was prepared using a liquid formulation, which was carried out in the same manner as Comparative Examples II-1 to II-2 and Examples II-1 to II-4 but without freeze-drying.

[0217] To evaluate the long-term stability of the injectable composition (SC formulation) using the liquid formulations of beta-NMN and hyaluronidase prepared in Comparative Examples II-3 to II-8, the composition was stored at 25°C for 8 weeks as described below, and the change in beta-NMN content was measured after 8 weeks, and the results are shown in Table 11.

[0218] - Equipment: LC-PDA (Waters HPLC system)

[0219] - Conditions: Column (GL Sciences InerSustain C18, 250×4.6 mm, 5 µm), Mobile phase (10 mM phosphate buffer (pH 3.0) / methanol = 90 / 10), Flow rate (1.0 mL / min), Injection volume (20 µL)

[0220] In addition, the appearance was observed visually after 8 weeks, and the results are also shown in Table 11.

[0221] Test Item Comparative Example II-3 Comparative Example II-4 Comparative Example II-5 Comparative Example II-6 Comparative Example II-7 Comparative Example II-8 Content Initial Value 98.9 100.6 99.9 99.3 99.6 101.1 After 8 Weeks 78.2 78.2 77.9 60.1 58.3 72.9 Appearance After 4 Weeks Transparent Transparent Transparent Dark Brown Dark Brown Brown After 8 Weeks Transparent Transparent Transparent Dark Brown Dark Brown Brown

[0222] As shown in Table 11, the injectable compositions (SC formulations) using the liquid formulations of beta-NMN and hyaluronidase prepared in Comparative Examples II-3 to II-8 were found to have significantly reduced stability, as the change in beta-NMN content over 8 weeks at 25°C was confirmed to be substantial.

[0223]

[0224] Experimental Example II-1: Pain Evaluation Experiment Upon Subcutaneous Administration - VAS (Visual Analogue Scale) Evaluation Experiment

[0225] When 3 mL of an injectable composition using the lyophilized formulations of beta-NMN and hyaluronidase prepared in Comparative Examples II-1 to II-2 and Examples II-1 to II-4 was administered into subcutaneous fat (SC formulation), the degree of pain was evaluated using the Visual Analogue Scale (VAS). The experiment was conducted using a double-blind method on 25 voluntary volunteers after obtaining sufficient explanation and informed consent prior to the procedure. All procedures were performed by a single operator, and neither the operator nor the voluntary volunteers were aware of the components of the injectable composition. The VAS score was graded from 0 to 10, with 0 representing no pain at all and 10 representing unbearable pain; the results are shown in Table 12 below.

[0226] Comparative Example II-1 Comparative Example II-2 Example II-1 Example II-2 Example II-3 Example II-4 VAS scores 6.2 6.8 2.8 2.4 2.6 2.1

[0227] As shown in Table 12, in the case of Comparative Examples II-1 and II-2, it was confirmed that pain was significant upon subcutaneous administration, which can be attributed to the low pH value. In particular, in the case of Comparative Example II-2, in which mannitol was omitted as a stabilizer, the pain was found to be even more severe. Meanwhile, in the case of Examples II-1 to II-4, it was found that pain upon subcutaneous administration could be significantly reduced by maintaining a pH greater than 4 to pH 8 (particularly pH 6 to pH 8).

[0228]

[0229] Experimental Example II-2: Analysis of blood NAD+ / NADH concentrations upon subcutaneous administration of a beta-NMN and hyaluronidase pain-relieving injection composition

[0230] A pain-relieving injectable composition using the lyophilized formulation of beta-NMN and hyaluronidase prepared in Example II-1 was administered to the subcutaneous fat of rats at a dose of 500 mg / kg / day of beta-NMN (SC formulation) for 3 days, and blood samples were collected at 5, 30, 60, 120, and 360 minutes, and blood NAD+ concentration and blood NADH concentration were measured at each time point using an ABCAM NAD+ / NADH analysis kit (Cat No. ab65348).

[0231] At this time, the beta-NMN alone composition was orally administered to rats for 3 days at the same dose of 500 mg / kg / day (PO formulation) and the injectable composition with beta-NMN and hyaluronidase omitted was intravenously administered to rats for 3 days as a placebo (CONT), and the results are shown in Tables 13 and 14 and Figures 3(a) and (b).

[0232] Classification Blood NAD+ Concentration 0 min 5 min 30 min 60 min 12 min 36 min Average Value Initial Average Value SC 1.00 5.16 5.10 1.76 1.00 1.47 2.90 4.01 PO 1.00 2.27 3.12 1.65 0.56 1.95 1.91 2.35 CONT 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

[0233] As shown in Table 13 and Figure 3(a), for the SC formulation, the average blood NAD+ concentration at each time point was found to have increased significantly by 200% to 400% (specifically, 250% to 350%) compared to placebo administration, which is significantly higher than that of the PO formulation. In particular, at the initial time points (5 minutes, 30 minutes, and 60 minutes), the average blood NAD+ concentration was found to have increased significantly by 350% to 450% compared to placebo administration. Additionally, the SC formulation showed a maximum blood NAD+ concentration at the 5-minute time point, with the value increasing by 516% compared to placebo administration.

[0234] Classification Blood NADH Concentration 0 min 5 min 30 min 60 min 120 min 360 min Average Value Initial Average Value SC 1.00 4.8 32.2 91.1 21.00 1.00 2.0 52.7 5 PO 1.00 1.00 2.1 20.7 80.5 61.00 1.09 1.3 0 CONT 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

[0235] As shown in Table 14 and Figure 3(b), for the SC formulation, the average blood NADH concentration at each time point was found to have increased relatively by 150% to 300% (specifically, 150% to 250%) compared to placebo administration, which is significantly higher than that of the PO formulation. In particular, at the initial time points (5 minutes, 30 minutes, and 60 minutes), the average blood NADH concentration was found to have increased by 250% to 350% compared to placebo administration. Additionally, the SC formulation also showed a maximum blood NADH concentration at the 5-minute time point, with the value increasing by 483% compared to placebo administration.

[0236]

[0237] Synthesizing the above results, it can be seen that in the case of the SC formulation, the increase in the average / maximum blood NAD+ concentration compared to placebo administration is relatively higher than the increase in the average / maximum blood NADH concentration compared to placebo administration. In other words, by implementing the pain-relieving injectable composition using the lyophilized beta-NMN and hyaluronidase formulation prepared in Example II-1 as an SC formulation, the NAD+ / NADH ratio in the body can be optimized, thereby providing excellent anti-aging effects.

[0238]

[0239] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. An injectable lyophilized formulation containing beta-nicotinamide mononucleotide.

2. In Paragraph 1, The above-mentioned lyophilized formulation for injection is characterized by further comprising one or more stabilizers selected from the group consisting of monosaccharides; disaccharides; and sugar alcohols.

3. In Paragraph 1, The above-mentioned injectable lyophilized formulation is characterized by having a pH of 3 to 8.

4. In Paragraph 1, An injectable lyophilized formulation characterized by having a moisture content of 5% or less.

5. An injectable composition obtained by redissolving the lyophilized formulation according to claim 1 in an intravenous solution.

6. In Paragraph 5, The above-described injectable composition is characterized by being for administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, and arterial.

7. In Paragraph 5, An injectable composition characterized in that, in the above injectable composition, the dose of the beta-nicotinamide mononucleotide is administered at 1 / 20 to 1 / 2 times the dose for which a therapeutic effect was confirmed with oral administration.

8. In Paragraph 5, An injectable composition characterized in that, in the above injectable composition, the beta-nicotinamide mononucleotide is administered at a dose of 0.5 mg / kg to 500 mg / kg per day.

9. In Paragraph 5, After administering the above-mentioned injectable composition, blood was collected at 5, 30, 60, 120, and 360 minutes and analyzed using an NAD+ / NADH analysis kit, and the results were as follows: i) At each time point, the average blood NAD+ concentration increases by 200% to 400% compared to placebo administration, but, ii) An injectable composition characterized by an average blood NADH concentration of 150% to 300% compared to placebo administration at each time point.

10. (a) a step of preparing a solution containing beta-nicotinamide mononucleotide; and (b) A method for preparing an injectable freeze-dried formulation comprising the step of freeze-drying the above solution. 11.(a) A step of preparing a solution containing beta-nicotinamide mononucleotide; (b) a step of freeze-drying the above solution to prepare an injectable freeze-dried formulation; and (c) A method for preparing an injectable composition comprising the step of adding a solution to the above-mentioned lyophilized formulation for injection.

12. Beta-nicotinamide mononucleotide; and Contains hyaluronidase, A pain-relieving injectable composition characterized by having a pH greater than 4 to a pH of 8.

13. In Paragraph 12, The above pain-relieving injectable composition is characterized by having a pH of 5 to 7.

14. In Paragraph 12, A pain-relieving injectable composition characterized by having an osmotic pressure of 800 mOsmol / kg to 1,300 mOsmol / kg.

15. In Paragraph 12, The above pain-relieving injectable composition is characterized by being for subcutaneous or intramuscular administration.

16. In Paragraph 12, The above pain-relieving injectable composition is characterized by further comprising one or more stabilizers selected from the group consisting of monosaccharides; disaccharides; and sugar alcohols.

17. In Paragraph 12, The above pain-relieving injectable composition is characterized by further comprising one or more alkalizing agents selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH)2), potassium hydroxide (KOH), tromethamine, and sodium bicarbonate, or HCl.

18. In Paragraph 12, After administering the above pain-relieving injection composition, blood samples were collected at 5, 30, 60, 120, and 360 minutes and analyzed using an NAD+ / NADH analysis kit, and the results were as follows: i) At each time point, the average blood NAD+ concentration increases by 200% to 400% compared to placebo administration, but, ii) A pain-relieving injectable composition characterized by an average blood NADH concentration of 150% to 300% compared to placebo administration at each time point. 19.(a) A step of preparing a solution containing beta-nicotinamide mononucleotide and hyaluronidase; (b) a step of preparing a freeze-dried formulation by freeze-drying the above solution; and (c) a step of adding a solution to the above freeze-dried formulation, and A method for preparing a pain-relieving injectable composition characterized by having a pH greater than 4 and a pH of 8.

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