Novel benfotiamine choline salts

Abstract

The invention relates to a novel benfotiamine choline salt as well as a preparation method and application thereof. The benfotiamine choline salt according to the present invention has excellent stability, and has significantly higher water solubility than conventional benfotiamine, thereby exhibiting improved solubility and bioavailability. Furthermore, the benfotiamine choline salt according to the present invention can effectively improve cognitive function or prevent or treat cranial nerve diseases, and thus can be used as a pharmaceutical raw material or a health functional food raw material.

Description

Technical Field

[0001] The present invention relates to a novel benfotiamine choline salt or a benfotiamine choline salt crystal form, a preparation method and use thereof. Compared with traditional benfotiamine, the benfotiamine choline salt or the benfotiamine choline salt crystal form according to the present invention has excellent stability and significantly higher water solubility, thereby being able to exhibit better solubility and bioavailability, and has the effect of improving cognitive function, or preventing, alleviating or treating degenerative brain diseases. Background Art

[0002] Thiamine is a compound called vitamin B1, and its chemical structure is shown below. Since thiamine is a water-soluble vitamin, even if taken in large doses, the amount stored in the body is very small and is completely excreted.

[0003]

[0004] In order to reduce the excretion rate of thiamine, various salts or derivatives have been developed, among which the representative ones are thiamine hydrochloride, thiamine nitrate, fursultiamine, benfotiamine and bisbenzamide.

[0005] Of these, fat-soluble benfotiamine reaches maximum blood concentrations about five times faster than thiamine hydrochloride or thiamine nitrate and has a bioavailability about 3.6 times higher.

[0006] However, although benfotiamine is fat-soluble and thus has the advantage of delaying excretion from the body, it has the problem of extremely low solubility in water.

[0007] At the same time, polymorphism is important in the pharmaceutical field because different drugs may exist in various forms of polymorphs, and specific polymorphs often affect the ease of preparation of drug raw materials, solubility, storage stability, ease of preparation of finished products, and in vivo pharmacological activity. Therefore, there may be significant differences in bioavailability between different salts and crystal forms of the same drug, and for a single drug, some crystal forms may have higher biological activity than other crystal forms.

[0008] As mentioned above, research on drug polymorphs has been ongoing to ensure higher biological activity and stable raw materials. Summary of the invention

[0009] Technical issues

[0010] One object of the present invention is to provide a benfotiamine choline salt as shown in the following formula 1:

[0011] [Formula 1]

[0012]

[0013] Furthermore, another object of the present invention is to provide a crystalline form of benfotiamine choline salt, which has diffraction peaks at 2θ values ​​of 5.39±0.2°, 8.86±0.2°, 10.59±0.2°, 13.12±0.2°, 15.46±0.2°, 18.11±0.2°, 18.51±0.2°, 22.36±0.2°, 26.16±0.2° and 30.98±0.2° in powder X-ray diffraction.

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising the benfotiamine choline salt or the benfotiamine choline salt crystal form.

[0015] In addition, another object of the present invention is to provide a health functional food composition, which contains the benfotiamine choline salt or the benfotiamine choline salt crystal form.

[0016] Furthermore, another object of the present invention is to provide a method for preparing benfotiamine choline salt, which comprises reacting benfotiamine with choline hydroxide.

[0017] In addition, another object of the present invention is to provide a method for preparing benfotiamine choline salt, which comprises: a first step, adding a lower alcohol having a carbon number of 1 to 4 to a choline hydroxide aqueous solution and stirring; a second step, adding benfotiamine to the solution obtained in the first step and stirring; a third step, concentrating the solution obtained in the second step under reduced pressure and crystallizing by adding a solvent; and a fourth step, filtering, washing and drying the solution obtained in the third step.

[0018] In addition, another object of the present invention is to provide a method for improving cognitive function or preventing and treating degenerative brain diseases, which comprises administering a therapeutically effective amount of benfotiamine choline salt to a subject in need thereof.

[0019] In addition, another object of the present invention is to provide a use of benfotiamine choline salt in the preparation of a medicament for improving cognitive function or preventing and treating degenerative brain diseases.

[0020] Technical Solution

[0021] The present invention provides a benfotiamine choline salt as shown in the following formula 1:

[0022] [Formula 1]

[0023]

[0024] In the present invention, the benfotiamine of the above formula 1 structure is a thiamine derivative, belonging to a fat-soluble S-acyl derivative. Benfotiamine is dephosphorylated by alkaline phosphatase in the intestinal mucosa to generate S-benzoylthiamine, and is hydrolyzed by thioesterase in the liver to generate thiamine. The bioavailability of benfotiamine is higher than that of thiamine salts, and it can provide higher levels of thiamine in muscles, brain, liver and kidneys. Benfotiamine is known to help enhance the health of the heart, kidneys, nerves and brain, and prevent aging and diabetic complications. In particular, it is known that in an Alzheimer's disease mouse model, benfotiamine improves spatial memory in a dose-dependent manner, reduces the number of amyloid plaques and the level of phosphorylated tau protein, thereby improving cognitive function (Brain, Vol. 133, No. 5, May 2010, pp. 1342-1351). Additionally, benfotiamine is known to be effective and safe in slowing the rate of functional decline in patients with mild cognitive impairment or Alzheimer's disease (J Alzheimers Dis 2020; Vol. 78, No. 3: pp. 989-1010).

[0025] At the same time, in the present invention, the choline in the above formula 1 structure is a water-soluble vitamin, which belongs to an amino acid and can synthesize lecithin that constitutes the cell membrane and acetylcholine related to memory in the body. Choline has the function of activating metabolism, maintaining energy and brain function in the body. Choline is a precursor of acetylcholine, which, as a brain neurotransmitter, plays a key role in memory and learning. Supplementing the acetylcholine that is lacking in patients with brain dysfunction can restore the neurotransmission function that is reduced due to brain nerve damage to normal. Compared with healthy people, patients with cognitive impairment or patients with degenerative brain diseases such as Alzheimer's disease not only lack choline, but also lack acetylcholine itself. Therefore, increasing the production of acetylcholine by intake is expected to improve the patient's symptoms.

[0026] In the present invention, the term "benfotiamine choline salt" may refer not only to the compound of the above-mentioned Formula 1, but also to its hydrate and its amorphous, partially crystalline or crystalline form.

[0027] Preferably, the benfotiamine choline salt of the above formula 1 of the present invention may be an amorphous, partially crystalline or crystalline compound.

[0028] More preferably, the benfotiamine choline salt of the above formula 1 of the present invention is in crystalline form.

[0029] Preferably, the benfotiamine choline salt of the above formula 1 of the present invention is characterized in that benfotiamine and choline are combined in a molar ratio of 1:1.

[0030] Preferably, the benfotiamine choline salt of the above formula 1 of the present invention may have the following characteristics: 1 H nuclear magnetic resonance (NMR) peaks:

[0031] 1 H NMR (400MHz, DMSO):7.91(s,1H),7.81(s,1H),7.69~7.48(m,5H),6.70(s,1H),4.34(s, 2H),3.79(s,2H),3.65(t,2H),3.40(s,2H),3.09(s,9H),2.58(t,2H),2.13(s,3H),and 2.11(s,3H)ppm.

[0032] In addition, preferably, the benfotiamine choline salt in the present invention has a powder X-ray diffraction (PXRD) pattern comprising peaks at 2θ values ​​of 5.39±0.2°, 8.86±0.2°, 10.59±0.2°, 13.12±0.2°, 15.46±0.2°, 18.11±0.2°, 18.51±0.2°, 22.36±0.2°, 26.16±0.2° and 30.98±0.2°.

[0033] In addition, preferably, the benfotiamine choline salt in the present invention has a powder X-ray diffraction (PXRD) pattern, including 2θ values ​​of 5.39±0.2°, 8.86±0.2°, 9.90±0.2°, 10.59±0.2°, 11.66±0.2°, 13.12±0.2°, 14.01±0.2°, 14.62±0.2°, 15.46±0.2°, 15.91±0.2°, 17.44±0.2°, 18.11±0.2°, 18.51±0.2°, 19.10±0.2°, 19.40±0.2°, 20.48±0.2°, 21. Peaks at 64±0.2°, 22.36±0.2°, 23.36±0.2°, 23.79±0.2°, 24.12±0.2°, 25.37±0.2°, 26.16±0.2°, 26.82±0.2°, 28.18±0.2°, 28.63±0.2°, 29.39±0.2°, 30.27±0.2°, 30.98±0.2°, 31.92±0.2°, 33.25±0.2°, 33.97±0.2°, 35.02±0.2°, 35.75±0.2°, 37.65±0.2° and 38.42±0.2°.

[0034] Preferably, the peak position of the powder X-ray diffraction (PXRD) pattern of the benfotiamine choline salt in the present invention is Figure 3 Basically the same.

[0035] In addition, the present invention provides a crystalline form of benfotiamine choline salt, which has diffraction peaks at 2θ values ​​of 5.39±0.2°, 8.86±0.2°, 10.59±0.2°, 13.12±0.2°, 15.46±0.2°, 18.11±0.2°, 18.51±0.2°, 22.36±0.2°, 26.16±0.2° and 30.98±0.2° in powder X-ray diffraction.

[0036] Preferably, the crystalline form of benfotiamine choline salt can also include 2θ values ​​of 9.90±0.2°, 11.66±0.2°, 14.01±0.2°, 14.62±0.2°, 15.91±0.2°, 17.44±0.2°, 19.10±0.2°, 19.40±0.2°, 20.48±0.2°, 21.64±0.2°, 23.36±0.2°, 23.79±0.2°, 2 Diffraction peaks at 4.12±0.2°, 25.37±0.2°, 26.82±0.2°, 28.18±0.2°, 28.63±0.2°, 29.39±0.2°, 30.27±0.2°, 31.92±0.2°, 33.25±0.2°, 33.97±0.2°, 35.02±0.2°, 35.75±0.2°, 37.65±0.2° and 38.42±0.2°.

[0037] The benfotiamine choline salt or benfotiamine choline salt crystal form of the present invention is not a compound that simply exists in the form of salt or crystal form and is only used to provide benfotiamine, but a composite compound in which two synergists, benfotiamine and choline, are linked into a single compound. It can be used as a useful raw material for preparing a drug or a healthy functional food for improving cognitive function, or preventing, alleviating or treating brain and nerve diseases such as degenerative brain diseases.

[0038] Compared with benfotiamine, the benfotiamine choline salt or the benfotiamine choline salt crystal form of the present invention has excellent stability and extremely high water solubility, so that the bioavailability is improved. In addition, the benfotiamine choline salt or the benfotiamine choline salt crystal form of the present invention is a single composite compound, and the preparation and formulation process is simplified because only a single compound needs to be characterized, treated and processed, rather than two separate compounds. Since benfotiamine and choline are combined in a 1:1 molar ratio, there is no uniformity problem in the formulation, and they can be dissolved simultaneously in a precise stoichiometric ratio when administered, which is a major advantage.

[0039] In addition, the present invention provides a pharmaceutical composition, which uses the benfotiamine choline salt or the benfotiamine choline salt crystal form of the above formula 1 as an active ingredient.

[0040] The pharmaceutical composition of the present invention may further include common pharmaceutically acceptable additives. For example, the additive may further include any one or more selected from excipients, disintegrants, binders, lubricants and coating agents, as long as the expected effects of the present invention can be achieved, and are not limited thereto.

[0041] The pharmaceutical composition of the present invention can be in the form of tablets, capsules, powders, granules, dripping pills, powders, pills, tinctures or ointments, but is not limited thereto. The formulation can be carried out according to any method known in the art to which the present invention belongs.

[0042] The pharmaceutical composition of the present invention using the benfotiamine choline salt or benfotiamine choline salt crystal form of formula 1 as an active ingredient can be effectively used as a pharmaceutical composition for improving cognitive function or preventing and treating degenerative brain diseases. The effects of benfotiamine and choline on improving cognitive function and preventing and treating degenerative brain diseases and their pharmacological mechanisms are well known in the art. In addition, in a specific embodiment, hippocampal cells were treated with the benfotiamine choline salt of the present invention, and the results confirmed that the levels of intracellular p-Akt (phosphorylated protein kinase B), p-ERK (phosphorylated extracellular signal-regulated kinase) and NR2B (N-methyl-D-aspartate receptor 2B subunit) were effectively improved. It is thus confirmed that benfotiamine choline salt has the effect of improving cognitive function or memory by promoting long-term potentiation (LPT).

[0043] The degenerative brain disease described in the present invention may be at least one disease selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, learning disabilities, cognitive impairment, neuroinflammation, nerve cell damage and memory impairment. It may be a variety of diseases, but is not limited thereto.

[0044] In addition, the present invention provides a functional health food composition, which uses the benfotiamine choline salt or the benfotiamine choline salt crystal form of the above formula 1 as an active ingredient.

[0045] In the present invention, the health functional food composition can be formulated into conventional health functional food dosage forms known in the art, such as granules, tablets, capsules, pills, suspensions, emulsions, syrups, chewing gums, drinks, etc., but are not limited thereto.

[0046] The health functional food composition of the present invention, which uses the benfotiamine choline salt or the benfotiamine choline salt crystal form of Formula 1 as an active ingredient, can be effectively used as a health functional food composition for improving cognitive function or preventing and alleviating degenerative brain diseases. The effects of benfotiamine and choline on improving cognitive function and preventing and alleviating degenerative brain diseases are as described above.

[0047] The pharmaceutical composition or health functional food composition of the present invention may further comprise at least one active ingredient having the same or similar medicinal effect in addition to the benfotiamine choline salt or benfotiamine choline salt crystal of Formula 1.

[0048] In addition, the present invention provides a method for improving cognitive function or preventing or treating degenerative brain diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of the benfotiamine choline salt or the benfotiamine choline salt crystalline form of the above formula 1. The subject may be a mammal including a human.

[0049] The term "therapeutically effective amount" used in the present invention refers to the amount of benfotiamine choline salt or benfotiamine choline salt crystalline compound that is effective for improving cognitive function or treating and preventing degenerative brain diseases. Specifically, "therapeutically effective amount" refers to a sufficient amount required to treat a disease at a reasonable benefit / risk ratio suitable for medical treatment, and the effective dosage level can be determined based on factors well known in the medical field, including subject type, disease severity, age, gender, disease type, drug activity, sensitivity to drugs, administration time, administration route, excretion rate, duration of treatment, and concurrently used drugs. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent, or it can be used in combination with other therapeutic agents, or it can be administered sequentially or simultaneously with commercially available therapeutic agents. In addition, it can be administered in a single dose or multiple doses. Taking into account all of the above factors, it is crucial to obtain the maximum effect with the minimum dose without side effects, and the dosage can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention can be determined by experts based on various factors such as the patient's condition, age, gender, and complications. Since the active ingredient of the pharmaceutical composition of the present invention has excellent safety, it is okay to use an amount higher than the determined dose.

[0050] In addition, the present invention provides the use of the benfotiamine choline salt or the benfotiamine choline salt crystal compound of the above formula 1 in the preparation of a drug for improving cognitive function or preventing and treating degenerative brain diseases. The benfotiamine choline salt or the benfotiamine choline salt crystal used for preparing the drug can be mixed with a pharmaceutically acceptable adjuvant, diluent, carrier, etc., and can be combined with other active agents to prepare a composite preparation to achieve a synergistic effect of the active ingredients.

[0051] Furthermore, the present invention provides a method for preparing a choline salt of benfotiamine, which comprises reacting benfotiamine with choline hydroxide.

[0052] In addition, the present invention provides a method for preparing benfotiamine choline salt, which comprises: a first step, adding a lower alcohol having a carbon number of 1 to 4 to a choline hydroxide aqueous solution and stirring; a second step, adding benfotiamine to the solution obtained in the first step and stirring; a third step, concentrating the solution obtained in the second step under reduced pressure and crystallizing by adding a solvent; and a fourth step, filtering, washing and drying the solution obtained in the third step.

[0053] The details of the preparation method of benfotiamine choline salt can be applied according to the description of benfotiamine choline salt of the present invention. Specifically, benfotiamine choline salt is as described above.

[0054] In the preparation method of benfotiamine choline salt, preferably, benfotiamine and choline hydroxide may be reacted in a molar ratio of 1:1.

[0055] In addition, preferably, the lower alcohol having 1 to 4 carbon atoms may be at least one selected from methanol, ethanol and isopropanol.

[0056] In addition, preferably, the solvent may be at least one selected from acetone, ethyl acetate and cyclohexane.

[0057] More preferably, the solvent may be acetone or ethyl acetate.

[0058] Unless contradictory, all details mentioned in the uses, compositions and methods of treatment of the present invention apply equally.

[0059] Advantageous Effects of the Invention

[0060] Compared with traditional benfotiamine, the novel benfotiamine choline salt or benfotiamine choline salt crystal of the present invention has improved stability, and compared with the known poorly soluble benfotiamine, its water solubility is significantly higher, so it is expected to greatly improve the dissolution rate and bioavailability. The benfotiamine choline salt or benfotiamine choline salt crystal of the present invention is not just a compound that exists in the form of salt or crystal and is only used to provide benfotiamine, but a composite compound in which two synergists, benfotiamine and choline, are connected into a single compound, which has the effect of formulating and ingesting benfotiamine and choline at the same time. In addition, the benfotiamine choline salt or benfotiamine choline salt crystal of the present invention can increase AMPA receptors (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors), strengthen the signal intensity of p-Akt, p-ERK and NR2B, which are sub-signals, thereby contributing to long-term potentiation. Therefore, it can be used as a useful pharmaceutical raw material for improving cognitive function or memory, or preventing and treating brain and nerve diseases such as degenerative brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The NMR analysis of the benfotiamine choline salt prepared in Example 1 is shown (1 H NMR) results.

[0062] Figure 2 The NMR analysis of benfotiamine as a comparative example is shown ( 1 H NMR) results.

[0063] Figure 3 The powder X-ray diffraction analysis results of the benfotiamine choline salt prepared in Example 1 are shown.

[0064] Figure 4 It is a graph showing the solubility test results of the benfotiamine choline salt of Example 1 and benfotiamine as a comparative example.

[0065] Figure 5 The results of immunoblotting for confirming the degree of activation of neuroplasticity factors after treating hippocampal cells with the benfotiamine choline salt of Example 1 are shown. DETAILED DESCRIPTION

[0066] Hereinafter, the present invention will be described in detail by way of examples and experimental examples. However, these examples and experimental examples are only for illustration to help understand the present invention, and the protection scope of the present invention is not limited by the following examples.

[0067] Materials and instruments

[0068] In each example, Bruker UltraShield TM 400 (400 MHz) hydrogen nuclear magnetic resonance ( 1 H NMR) analysis, and high performance liquid chromatography (HPLC) analysis was performed using Agilent 1200 series. Benfotiamine used in the examples and comparative examples can be prepared by known methods or purchased from the market, and various reagents and solvents used in the reaction were purchased from Aldrich, Tokyo Chemical Industry Co., Ltd. (TCI), Daejeong and other companies.

[0069] Comparative Example: Preparation of Benfotiamine

[0070] Benfotiamine in the comparative example was obtained by purchasing a commercially available product.

[0071] Example 1: Preparation of benfotiamine choline salt

[0072] Under nitrogen atmosphere, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution was dissolved in 45 ml of methanol, followed by the addition of 17 g (36.3 mmol) of benfotiamine and complete dissolution. After concentration under reduced pressure, 90 ml of acetone was added for crystallization. After salt formation, the resulting mixture was filtered under nitrogen atmosphere, washed with 45 ml of acetone, and then vacuum dried to obtain 18.0 g of benfotiamine choline salt.

[0073] 1 H NMR (400 MHz, dimethyl sulfoxide (DMSO)): 7.91 (s, 1H), 7.81 (s, 1H), 7.69-7.48 (m, 5H), 6.70 (s, 1H), 4.34 (s, 2H), 3.79 (s, 2H), 3.65 (t, 2H), 3.40 (s, 2H), 3.09 (s, 9H), 2.58 (t, 2H), 2.13 (s, 3H), and 2.11 (s, 3H) ppm.

[0074] Example 2: Preparation of benfotiamine choline salt

[0075] Under nitrogen protection, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution was dissolved in 150 ml of ethanol, and then 17 g (36.3 mmol) of benfotiamine was added and completely dissolved. After reduced pressure concentration, 90 ml of acetone was added for crystallization. After salt formation, the resulting mixture was filtered under nitrogen environment, washed with 45 ml of acetone, and then vacuum dried to obtain 17.8 g of benfotiamine choline salt.

[0076] Example 3: Preparation of benfotiamine choline salt

[0077] Under nitrogen protection, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution was dissolved in 45 ml of methanol, followed by the addition of 17 g (36.3 mmol) of benfotiamine and complete dissolution. After concentration under reduced pressure, 200 ml of ethyl acetate was added for crystallization. After salt formation, the resulting mixture was filtered under nitrogen environment, washed with 50 ml of ethyl acetate, and then vacuum dried to obtain 18.6 g of benfotiamine choline salt.

[0078] Example 4: Preparation of benfotiamine choline salt

[0079] Under nitrogen protection, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution was dissolved in 150 ml of ethanol, followed by the addition of 17 g (36.3 mmol) of benfotiamine and complete dissolution. After concentration under reduced pressure, 200 ml of ethyl acetate was added for crystallization. After salt formation, the resulting mixture was filtered under nitrogen, washed with 50 ml of ethyl acetate, and then vacuum dried to obtain 19.0 g of benfotiamine choline salt.

[0080] Example 5: Preparation of benfotiamine choline salt

[0081] Under nitrogen protection, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution was dissolved in 45 ml of methanol, and 17 g (36.3 mmol) of benfotiamine was added and completely dissolved. After concentration under reduced pressure, 100 ml of cyclohexane was added for crystallization. After salt formation, the resulting mixture was filtered under nitrogen environment, washed with 50 ml of cyclohexane, and then vacuum dried to obtain 19.5 g of benfotiamine choline salt.

[0082] Example 6: Preparation of benfotiamine choline salt

[0083] Under nitrogen protection, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution and 17 g (36.3 mmol) of benfotiamine were added to 500 ml of isopropanol and heated to dissolve. The resulting mixture was slowly cooled to room temperature, filtered, washed with 50 ml of isopropanol, and then vacuum dried to obtain 16.7 g of benfotiamine choline salt.

[0084] Experimental Example 1: Stability Test (Accelerated Test)

[0085] The stability testing of drugs aims to determine whether the drug maintains stable quality over time under specific conditions in order to set the storage method and shelf life of the drug. Therefore, by setting appropriate specifications and evaluating significant changes based on prescribed analytical methods to determine whether the specifications are met, the shelf life of the product is determined.

[0086] The stabilities of the benfotiamine choline salt prepared in Example 1 of the present invention and the benfotiamine in the comparative example were compared.

[0087] Specifically, according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, stability testing was performed under strict conditions (60°C ± 2°C) and tested by high performance liquid chromatography (HPLC) analysis. The results are shown in Table 1.

[0088] [Table 1]

[0089] initial 3 days 7 days 14 days 21 days Benfotiamine choline salt 99.3 99.2 99.1 99.0 98.2 Benfotiamine 99.5 99.1 98.6 97.5 97.0

[0090] As shown in Table 1, the purity change of benfotiamine in the comparative example was 2.5% after the stability test under harsh conditions for 21 days. On the other hand, the purity change of benfotiamine choline salt of the present invention was 1.1% after the stability test under the same harsh conditions for 21 days, which proved that its stability was more than twice that of benfotiamine.

[0091] Experimental Example 2: Solubility Test

[0092] In order to evaluate the solubility of the benfotiamine choline salt of the present invention, the water solubility of Example 1 and the comparative example at different temperatures was measured.

[0093] Regarding the solubility test method, 1 mL of purified water and solutions with pH values of 1.2, 4.0, and 6.8 were taken, and an excessive amount of thiamine phosphosulfide choline salt of Example 1 or thiamine phosphosulfide of the comparative example was added thereto until saturation was reached. After ultrasonic extraction for 1 hour, filtration was carried out using a polyvinylidene fluoride (PVDF) membrane filter, and appropriate dilution was performed. After diluting the control solution with 50% acetonitrile, a calibration curve (range: 1 - 1000 mg / mL) was prepared by analysis using a high performance liquid chromatography (HPLC) according to the United States Pharmacopeia (USP) standard, and the concentration of the saturated solution was obtained from the first-order function of the prepared calibration curve. The results obtained by this solubility test method are shown in Table 2 and Figure 4 as shown below.

[0094] <HPLC Conditions>

[0095] Detector: Ultraviolet spectrophotometer (measurement wavelength 230 nm)

[0096] Chromatographic column: Aegispak C18 (4.6 mm × 150 mm, 5 μm) or an equivalent chromatographic column

[0097] Column temperature: Constant temperature at about 25°C

[0098] Sample injection volume: 20 μL

[0099] Flow rate: 1.0 mL / min

[0100] Mobile phase: A mixture of 0.5% ammonium carbonate solution and methanol (volume ratio 8:2)

[0101] [Table 2]

[0102]

[0103] As shown in Table 2 and Figure 4 as shown below, the solubility of thiamine phosphosulfide in the comparative example was low in purified water and solutions with pH values of 1.2, 4.0, and 6.8, only 3 - 6.6 mg / mL, while the thiamine phosphosulfide choline salt of Example 1 showed extremely high solubility in purified water and all solutions with pH values of 1.2, 4.0, and 6.8. Specifically, compared with the comparative example (thiamine phosphosulfide), the solubility of the thiamine phosphosulfide choline salt of Example 1 was about 124 - 222 times higher. Thus, it was confirmed that the thiamine phosphosulfide choline salt of the present invention can significantly improve the absorption and dissolution of the drug by greatly improving the physical and chemical properties of the poorly soluble drug thiamine phosphosulfide.

[0104] Experimental Example 3: Powder X-ray Diffraction (PXRD) Analysis

[0105] In the powder X-ray diffraction method, when the crystal structure of the material and the compound form are different, the shape of the diffraction pattern will change. Using this property, the crystal structure of the material can be compared and confirmed with the reference material. The benfotiamine choline salt of the present invention and the benfotiamine in the comparative example as a control were analyzed using Rigaku MiniFlex 600.

[0106] The results show that Figure 3 As shown, it is confirmed that the choline salt of benfotiamine of Example 1 of the present invention has a 2θ value of 5.39°, 8.86°, 9.90°, 10.59°, 11.66°, 13.12°, 14.01°, 14.62°, 15.46°, 15.91°, 17.44°, 18.11°, 18.51°, 19.10°, 19.40°, 20.48°, 21.64°, 22. There are characteristic powder X-ray diffraction peaks at 36°, 23.36°, 23.79°, 24.12°, 25.37°, 26.16°, 26.82°, 28.18°, 28.63°, 29.39°, 30.27°, 30.98°, 31.92°, 33.25°, 33.97°, 35.02°, 35.75°, 37.65° and 38.42°.

[0107] Experimental Example 4: Immunoblotting

[0108] 1. Preparation of Experimental Cells

[0109] All cells used in the experiment were primary cultured hippocampal neurons. Hippocampal neurons were isolated from embryos of SD rats (Sprague-Dawley rats, Samtaco, Korea) on the 18th and 19th days of pregnancy. The hippocampal nerve tissue of the fetal rat was physically separated using a dissecting microscope, and then placed in HBSS (Hank's balanced salt solution) supplemented with 0.25% trypsin and treated at 37°C for 10 minutes. After washing the hippocampal nerve tissue with HBSS, single cells were carefully separated by pipetting. The isolated cells were seeded into a 60 mm cell culture dish pre-coated with poly-L-lysine (0.5 mg / ml), and Neurobasal / B27 medium (containing 0.5 mM L-glutamine, 25 μM glutamate, 25 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin) was added and cultured in 5% carbon dioxide at 37°C. The medium was replaced on the 4th and 11th days of cell culture, and cells cultured to the 14th day were used for experiments.

[0110] 2. Confirmation of activation of neuroplasticity factors in hippocampal cells

[0111] In order to confirm p-ERK, p-AKT and NR2B in cells, the culture medium of primary hippocampal neurons cultured for 14 days was replaced with a medium without B-27 at least 1 hour before drug treatment. The drugs used were AMPA, benfotiamine or benfotiamine choline salt of Example 1, wherein AMPA is an agonist of AMPA receptor (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor) and was used as a positive control in this experiment.

[0112] After treating the cells with 1 or 3 μg / mL of benfotiamine choline salt for 10 minutes, the cells were washed with cold phosphate-buffered saline (PBS) and lysed on ice. The lysed cell solution was quantified according to the protein quantification method, and then protein analysis (immunoblotting) was performed, and the proteins were electrophoresed on 8% and 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). 5% skim milk was added, and the membrane was reacted for 1 hour at room temperature. The primary antibody was treated with Tris-buffered saline at a ratio of 1:1000, reacted at 4°C for 24 hours, and then washed three times with TBST (25mM Tris-HCl, 140mM NaCl, 0.1% Tween 20, pH7.5). After that, horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse secondary antibodies were reacted in 3% skim milk at a ratio of 1:5000 for 1 hour. The coupled antibody complex was confirmed using West Femto Maximum Sensitivity Substrate (Thermo) and the results were as shown. Figure 5 shown.

[0113] like Figure 5 As shown, the results confirmed that benfotiamine choline salt can effectively increase the levels of phosphorylated Akt (protein kinase B), phosphorylated ERK (extracellular signal-regulated kinase) and NR2B, thereby indicating that benfotiamine choline salt has the effect of improving cognitive function or memory by promoting long-term potentiation (LPT).

Claims

1. The choline salt of benfotiamine as shown in the following formula 1: [Formula 1] 2. The benfotiamine choline salt according to claim 1, wherein benfotiamine and choline are combined in a molar ratio of 1:

1.

3. The benfotiamine choline salt according to claim 1, wherein the benfotiamine choline salt has the following 1H nuclear magnetic resonance spectrum peaks: 1 1H NMR (400 MHz, DMSO): δ 7.91 (s, 1H), 7.81 (s, 1H), 7.69 - 7.48 (m, 5H), 6.70 (s, 1H), 4.34 (s, 2H), 3.79 (s, 2H), 3.65 (t, 2H), 3.40 (s, 2H), 3.09 (s, 9H), 2.58 (t, 2H), 2.13 (s, 3H), and 2.11 (s, 3H) ppm.

4. The benfotiamine choline salt according to claim 1, wherein the benfotiamine choline salt represented by Formula 1 has a powder X-ray diffraction pattern comprising peaks at 5.39±0.2°, 8.86±0.2°, 10.59±0.2°, 13.12±0.2°, 15.46±0.2°, 18.11±0.2°, 18.51±0.2°, 22.36±0.2°, 26.16±0.2°, and 30.98±0.2°.

5. The benfotiamine choline salt according to claim 1, wherein the benfotiamine choline salt has a powder X-ray diffraction pattern comprising 5.39±0.2°, 8.86±0.2°, 9.90±0.2°, 10.59±0.2°, 11.66±0.2°, 13.12±0.2°, 14.01±0.2°, 14.62±0.2°, 15.46±0.2°, 15.91±0.2°, 17.44±0.2°, 18.11±0.2°, 18.51±0.2°, 19.10±0.2°, 19.40±0.2°, 20.48±0.2°, Peaks at 21.64±0.2°, 22.36±0.2°, 23.36±0.2°, 23.79±0.2°, 24.12±0.2°, 25.37±0.2°, 26.16±0.2°, 26.82±0.2°, 28.18±0.2°, 28.63±0.2°, 29.39±0.2°, 30.27±0.2°, 30.98±0.2°, 31.92±0.2°, 33.25±0.2°, 33.97±0.2°, 35.02±0.2°, 35.75±0.2°, 37.65±0.2° and 38.42±0.2°.

6. A benfotiamine choline salt crystal form, which has diffraction peaks at 2θ values ​​of 5.39±0.2°, 8.86±0.2°, 10.59±0.2°, 13.12±0.2°, 15.46±0.2°, 18.11±0.2°, 18.51±0.2°, 22.36±0.2°, 26.16±0.2° and 30.98±0.2° in powder X-ray diffraction.

7. The benfotiamine choline salt crystal form according to claim 6, wherein the 2θ values ​​are 9.90±0.2°, 11.66±0.2°, 14.01±0.2°, 14.62±0.2°, 15.91±0.2°, 17.44±0.2°, 19.10±0.2°, 19.40±0.2°, 20.48±0.2°, 21.64±0.2°, 23.36±0.2°, 23.79±0.2°, There are diffraction peaks at 24.12±0.2°, 25.37±0.2°, 26.82±0.2°, 28.18±0.2°, 28.63±0.2°, 29.39±0.2°, 30.27±0.2°, 31.92±0.2°, 33.25±0.2°, 33.97±0.2°, 35.02±0.2°, 35.75±0.2°, 37.65±0.2° and 38.42±0.2°.

8. A pharmaceutical composition comprising the benfotiamine choline salt according to any one of claims 1 to 5 as an active ingredient.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is used to improve cognitive function or prevent or treat degenerative brain diseases.

10. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is in the form of tablets, capsules, powders, granules, pills, powders, boluses, tinctures or plasters.

11. A functional health food composition comprising the benfotiamine choline salt according to any one of claims 1 to 5 as an active ingredient. 12 . The functional health food composition according to claim 11 , wherein the functional health food composition is used to improve cognitive function or prevent or improve degenerative brain diseases.

13. A pharmaceutical composition comprising the benfotiamine choline salt crystal form of claim 6 or 7 as an active ingredient.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is used to improve cognitive function or prevent or treat degenerative brain diseases.

15. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is in the form of tablets, capsules, powders, granules, pills, powders, boluses, tinctures or plasters.

16. A functional health food composition comprising the benfotiamine choline salt crystal form of claim 6 or 7 as an active ingredient. 17 . The functional health food composition according to claim 16 , wherein the functional health food composition is used to improve cognitive function or prevent or improve degenerative brain diseases.

18. A process for preparing the choline salt of benfotiamine, the process comprising reacting benfotiamine with choline hydroxide.

19. A method for preparing benfotiamine choline salt, the method comprising: The first step is to add a lower alcohol having 1 to 4 carbon atoms to a choline hydroxide aqueous solution and stir; Step 2: Add benfotiamine to the solution obtained in the first step and stir; Step 3: Concentrate the solution obtained in the second step under reduced pressure and crystallize by adding a solvent; Step 4: Filter, wash and dry the solution obtained in step 3.

20. The method for preparing benfotiamine choline salt according to claim 18 or 19, wherein benfotiamine and choline hydroxide are reacted in a molar ratio of 1:

1.

21. The method for preparing benfotiamine choline salt according to claim 19, wherein the lower alcohol having 1 to 4 carbon atoms is at least one selected from the group consisting of methanol, ethanol and isopropanol.

22. The method for preparing benfotiamine choline salt according to claim 19, wherein the solvent is at least one selected from the group consisting of acetone, ethyl acetate and cyclohexane.

23. A method for improving cognitive function or preventing or treating degenerative brain diseases, comprising administering a therapeutically effective amount of the benfotiamine choline salt of any one of claims 1 to 5 to a subject in need thereof.

24. Use of the benfotiamine choline salt according to any one of claims 1 to 5 in the preparation of a medicament for improving cognitive function or preventing or treating degenerative brain diseases.

Images