Novel benfotiamine choline salt

Abstract

The present invention relates to a novel benfotiamine choline salt, its preparation method, and its use. The novel benfotiamine choline salt according to the present invention exhibits improved stability compared to benfotiamine, has excellent aqueous solubility, and exhibits improved dissolution rate and bioavailability. Furthermore, the benfotiamine choline salt according to the present invention has the effect of improving cognitive function or preventing or treating degenerative brain diseases, and can therefore be useful as a pharmaceutical ingredient or a raw material for functional health foods.

Description

[Technical Field]

[0001] The present invention relates to a novel benfotiamine choline salt or a crystalline form of benfotiamine choline salt, its preparation method, and use. The benfotiamine choline salt or a crystalline form of benfotiamine choline salt according to the present invention has superior stability and significantly higher aqueous solubility than existing benfotiamine, and can exhibit improved dissolution and bioavailability, and has the effect of improving cognitive function or preventing, ameliorating, or treating degenerative brain diseases. [Background technology]

[0002] Thiamine is a compound called vitamin B1, which has the following chemical structure: Because thiamin is a water-soluble vitamin, even if administered in high doses, only a very small amount is stored in the body and is entirely excreted.

[0003] [ka]

[0004] Salts and derivatives have been developed to slow the excretion rate of thiamine, typically thiamine hydrochloride, thiamine nitrate, fursultiamine, benfotiamine, and bisbentiamine.

[0005] Benfotiamine is fat-soluble and reaches its peak concentration in the blood about five times faster than thiamine hydrochloride or thiamine nitrate, and has the advantage of being about 3.6 times more bioavailable.

[0006] Although benfotiamine is lipid-soluble and has the advantage of being slowly excreted from the body, it has the drawback of having extremely low solubility in water.

[0007] Meanwhile, the reason why crystalline polymorphism is important in the pharmaceutical field is that various crystalline polymorphs can exist depending on the drug, and a specific crystalline polymorph can often affect the ease of preparation of medicinal materials, solubility, storage stability, ease of preparation of finished products, and in vivo pharmacological activity. That is, there can be significant differences in bioavailability between different salts or crystalline forms of the same drug, and some crystalline forms of a drug can have higher biological activity than other crystalline forms.

[0008] As mentioned above, research into drug polymorphism is constantly ongoing in order to ensure higher biological activity and stable raw materials. Summary of the Invention [Problem to be solved by the invention]

[0009] One object of the present invention is to provide a benfotiamine choline salt of formula 1:

[0010] [ka]

[0011] Another object of the present invention is to provide a crystalline form of benfotiamine choline salt, which comprises 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 (PXRD).

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising the benfotiamine choline salt or a crystalline form of the benfotiamine choline salt.

[0013] Another object of the present invention is to provide a functional health food composition containing the benfotiamine choline salt or a crystalline form of the benfotiamine choline salt.

[0014] Another object of the present invention is to provide a method for preparing benfotiamine choline salt, which comprises the step of reacting benfotiamine with choline hydroxide.

[0015] Another object of the present invention is to provide a method for producing benfotiamine choline salt, which includes a first step of adding a lower alcohol having 1 to 4 carbon atoms to an aqueous solution of choline hydroxide and stirring the mixture; a second step of adding benfotiamine to the solution obtained in the first step and stirring the mixture; a third step of concentrating the solution obtained in the second step under reduced pressure and then adding a solvent to crystallize the solution; and a fourth step of filtering, washing, and drying the solution obtained in the third step.

[0016] Another object of the present invention is to provide a method for improving cognitive function or preventing or treating degenerative brain diseases, comprising the step of administering a therapeutically effective amount of benfotiamine choline salt to a subject in need thereof.

[0017] Another object of the present invention is to provide a use of benfotiamine choline salt for the manufacture of a medicament for improving cognitive function or preventing or treating degenerative brain diseases. [Means for solving the problem]

[0018] The present invention provides a benfotiamine choline salt of formula 1:

[0019] [ka]

[0020] In the present invention, benfotiamine, which is represented by the formula (1), is a fat-soluble S-acyl derivative of thiamine. Benfotiamine is dephosphorylated to S-benzoylthiamine by ecto-alkaliphosphate present in the intestinal mucosa and then hydrolyzed to thiamine by thioesterase in the liver. Benfotiamine has higher bioavailability than thiamine salts and provides higher levels of thiamine in the muscles, brain, liver, and kidneys. Benfotiamine is known to be useful for improving cardiac, renal, neurological, and brain health and for preventing aging and diabetic complications. In particular, benfotiamine has been shown to improve cognitive function in a mouse model of Alzheimer's disease by dose-dependently improving spatial memory and reducing the number of amyloid plaques and phosphorylated tau levels (Brain, Volume 133, Issue 5, May 2010, Pages 1342-1351). Benfotiamine is also 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;78(3):989~1010).

[0021] Meanwhile, in the present invention, choline in the structure of Chemical Formula 1 is a water-soluble vitamin and a type of amino acid that synthesizes lecithin, which constitutes cell membranes, and acetylcholine, which is related to memory, in the body. Choline activates metabolism in the body and plays a role in maintaining energy and brain function. Choline is a precursor to acetylcholine, a brain neurotransmitter that plays a central role in memory and learning. Replenishing acetylcholine in patients with brain dysfunction normalizes neurotransmission functions that have been impaired due to brain nerve damage. Patients with cognitive dysfunction or degenerative brain diseases such as Alzheimer's disease have reduced levels of not only choline but also acetylcholine compared to healthy individuals. Therefore, by taking this product, it is possible to increase the production of acetylcholine and improve the patient's symptoms.

[0022] In the present invention, the term "benfotiamine choline salt" refers not only to the compound represented by Chemical Formula 1, but also to its hydrate, amorphous, partially crystalline, or crystalline form.

[0023] Preferably, the benfotiamine choline salt of Formula 1 of the present invention may be an amorphous, partially crystalline or crystalline compound.

[0024] More preferably, the benfotiamine choline salt of Formula 1 of the present invention is in a crystalline form.

[0025] Preferably, the benfotiamine choline salt of Formula 1 of the present invention is characterized in that benfotiamine and choline are combined in a molar ratio of 1:1.

[0026] Preferably, the benfotiamine choline salt of the present invention represented by Chemical Formula 1 is 1 H nuclear magnetic resonance spectrum (NMR) peaks may be:

[0027] 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), 3.7 9(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.

[0028] Also, preferably, the benfotiamine choline salt of Chemical Formula 1 of the present invention may have a powder X-ray diffraction (PXRD) pattern including 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°.

[0029] Preferably, the benfotiamine choline salt of Chemical Formula 1 of the present invention has the following refractive indices: 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°, 21.64±0.2°, 22.36±0.2°, 23. 0.36±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°.

[0030] Preferably, the benfotiamine choline salt of the present invention may have peak positions in a powder X-ray diffraction (PXRD) pattern that are substantially the same as the peak positions in FIG.

[0031] The present invention also provides a crystalline form of benfotiamine choline salt, which comprises, in powder X-ray diffraction (PXRD), 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°.

[0032] Preferably, the crystalline forms of the benfotiamine choline salt have the following angles: 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°, 24.12±0.2°, The diffraction peaks may further include at 2θ values ​​of 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°.

[0033] The benfotiamine choline salt or crystalline form of benfotiamine choline salt according to the present invention is not a salt or crystalline compound that simply provides benfotiamine alone, but a complex compound that combines two active ingredients, benfotiamine and choline, in a single compound, and can be usefully used as a pharmaceutical ingredient or a raw material for functional health foods for improving cognitive function and preventing, ameliorating, or treating neurological diseases such as degenerative brain diseases.

[0034] The benfotiamine choline salt or crystalline form of benfotiamine choline salt according to the present invention exhibits excellent stability, a very high aqueous solubility compared to benfotiamine, and improved bioavailability. Furthermore, the benfotiamine choline salt or crystalline form of benfotiamine choline salt according to the present invention is a single complex compound, and therefore, it is only necessary to characterize, handle, and process one compound instead of two separate compounds, thereby simplifying the manufacturing and formulation processes. Furthermore, since benfotiamine and choline are bound in a 1:1 molar ratio, there is no problem of inhomogeneity within the formulation, and they are advantageously dissolved simultaneously according to an accurate stoichiometric ratio upon administration.

[0035] The present invention also provides a pharmaceutical composition comprising the benfotiamine choline salt of Formula 1 or a crystalline form of the benfotiamine choline salt as an active ingredient.

[0036] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives that are commonly contained in the pharmaceutical composition, such as at least one selected from the group consisting of an excipient, a disintegrant, a binder, a lubricant, and a coating agent, but is not limited thereto as long as the intended effect of the present invention is maintained.

[0037] The pharmaceutical composition of the present invention may be in the form of, but is not limited to, tablets, capsules, powders, granules, dropping pills, pulvis, boluses, tinctures, or poultices, and may be prepared by any method known in the art.

[0038] A pharmaceutical composition comprising the benfotiamine choline salt of Formula 1 or a crystalline form of benfotiamine choline salt of the present invention as an active ingredient can be useful as a pharmaceutical composition for improving cognitive function or preventing or treating degenerative brain diseases. The cognitive function improving effects and the preventive or therapeutic effects of benfotiamine and choline on degenerative brain diseases, as well as their pharmacological mechanisms, are well known in the art. In a specific example, treatment of hippocampal cells with the benfotiamine choline salt of the present invention effectively increased the levels of intracellular p-Akt, p-ERK, and NR2B, demonstrating that benfotiamine choline salt can support long-term memory consolidation (LPT) and improve cognitive function and memory.

[0039] The degenerative brain disease of the present invention may be one or more diseases selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, learning disability, cognitive impairment, neuroinflammation, neuronal damage, and memory impairment, but is not limited thereto.

[0040] The present invention also provides a functional health food composition containing the benfotiamine choline salt of Chemical Formula 1 or a crystalline form of the benfotiamine choline salt as an active ingredient.

[0041] In the present invention, the functional health food composition may be formulated in the dosage form of a conventional functional health food known in the art, such as, but not limited to, granules, tablets, capsules, pills, suspensions, emulsions, syrups, chewing gums, drinks, etc.

[0042] The functional health food composition of the present invention, which contains the benfotiamine choline salt of Formula 1 or a crystalline form of benfotiamine choline salt as an active ingredient, can be useful as a functional health food composition for improving cognitive function or preventing or ameliorating degenerative brain diseases. The effects of benfotiamine and choline on improving cognitive function and preventing or ameliorating degenerative brain diseases are as described above.

[0043] The pharmaceutical composition or health functional food composition of the present invention may further contain one or more active ingredients exhibiting the same or similar medicinal effects in addition to the benfotiamine choline salt of Chemical Formula 1 or a crystalline form of benfotiamine choline salt.

[0044] The present invention also provides a method for improving cognitive function or preventing or treating a degenerative brain disease, comprising administering to a subject in need thereof a therapeutically effective amount of benfotiamine choline salt or a crystalline form of benfotiamine choline salt of Chemical Formula 1. The subject may be a mammal, including a human.

[0045] The term "therapeutically effective amount" used herein refers to an amount of benfotiamine choline salt or benfotiamine choline salt crystalline compound effective in improving cognitive function or treating or preventing degenerative brain diseases. Specifically, "therapeutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors well known in the medical field, including the type and severity of the individual, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors. The pharmaceutical compositions of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. They may also be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy with the minimum amount without side effects, and this can be easily determined by those skilled in the art. The dosage of the pharmaceutical compositions of the present invention can be determined by a specialist depending on various factors, such as the patient's condition, age, sex, and comorbidities. The active ingredient of the pharmaceutical composition of the present invention is highly safe and may be used at a dose higher than the prescribed dose.

[0046] The present invention also provides use of the benfotiamine choline salt or crystalline benfotiamine choline salt compound of Formula 1 for use in the manufacture of a medicament for improving cognitive function or preventing or treating degenerative brain diseases. The benfotiamine choline salt or crystalline benfotiamine choline salt compound for the manufacture of the medicament may be mixed with a pharmaceutically acceptable adjuvant, diluent, carrier, etc., and may be prepared as a combined preparation with other active ingredients to achieve synergistic effects of the active ingredients.

[0047] The present invention also provides a method for producing benfotiamine choline salt, which comprises the step of reacting benfotiamine with choline hydroxide.

[0048] The present invention also provides a method for producing benfotiamine choline salt, which includes a first step of adding a lower alcohol having 1 to 4 carbon atoms to an aqueous choline hydroxide solution and stirring the mixture; a second step of adding benfotiamine to the solution obtained in the first step and stirring the mixture; a third step of concentrating the solution obtained in the second step under reduced pressure and then adding a solvent to crystallize the solution; and a fourth step of filtering, washing, and drying the solution obtained in the third step.

[0049] The details of the method for producing the benfotiamine choline salt may be the same as those described above for the benfotiamine choline salt of the present invention.

[0050] In the method for producing benfotiamine choline salt, benfotiamine and choline hydroxide may be reacted in a molar ratio of 1:1.

[0051] Preferably, the lower alcohol having 1 to 4 carbon atoms may be any one or more selected from the group consisting of methanol, ethanol, and isopropyl alcohol.

[0052] Preferably, the solvent is at least one selected from the group consisting of acetone, ethyl acetate, and cyclohexane.

[0053] More preferably, the solvent may be acetone or ethyl acetate.

[0054] The matters mentioned in the uses, compositions, and treatment methods of the present invention are equally applicable unless they contradict each other. [Effects of the Invention]

[0055] The novel benfotiamine choline salt or crystalline form of benfotiamine choline salt of the present invention exhibits improved stability compared to conventional benfotiamine and has significantly higher aqueous solubility than benfotiamine, which is known to be poorly soluble, and is expected to significantly increase dissolution rate and bioavailability. Furthermore, the novel benfotiamine choline salt or crystalline form of benfotiamine choline salt of the present invention is not a salt or crystalline compound simply providing benfotiamine alone, but a complex compound combining two active agents, benfotiamine and choline, in a single compound, thereby enabling benfotiamine and choline to be formulated and ingested simultaneously. Furthermore, the benfotiamine choline salt or crystalline form of benfotiamine choline salt of the present invention increases AMPA aqueous solution and enhances the signals of sub-signals p-Akt, p-ERK, and NR2B, thereby supporting long-term memory enhancement, and can therefore be useful as a pharmaceutical ingredient for improving cognitive function and memory, or for preventing or treating neurological disorders such as degenerative brain diseases. [Brief explanation of the drawings]

[0056] [Figure 1] 1 shows the results of nuclear magnetic resonance analysis ( 1 H NMR) of the benfotiamine choline salt prepared in Example 1. [Figure 2] 1 shows the results of nuclear magnetic resonance analysis ( 1 H NMR) of benfotiamine as a comparative example. [Figure 3] 1 shows the results of powder X-ray diffraction analysis of the benfotiamine choline salt prepared in Example 1. [Figure 4] 1 is a graph showing the results of a solubility test of the benfotiamine choline salt of Example 1 and benfotiamine as a comparative example. [Figure 5] 1 shows the results of immunoblotting in which the benfotiamine choline salt of Example 1 was treated in hippocampal cells to confirm the degree of activation of neuroplasticity factors. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention will be described in detail below with reference to examples and experimental examples, but these examples and experimental examples are provided merely for the purpose of illustration to aid in understanding the present invention, and the scope of the present invention is not limited by the following examples.

[0058] Materials and equipment

[0059] In the examples, 1 H NMR was performed using a Bruker UltraShield 400 (400 MHz), and HPLC was performed using an Agilent 1200 series. Benfotiamine used in the examples and comparative examples may be directly produced by known methods or may be commercially purchased, and various reagents and solvents used in the reactions were purchased from Aldrich, TCI, DeJon, etc.

[0060] Comparative Example: Preparation of Benfotiamine

[0061] Benfotiamine for comparison was prepared by purchasing commercially available benfotiamine.

[0062] Example 1: Preparation of benfotiamine choline salt

[0063] Under nitrogen, 10 g (36.3 mmol) of 44% aqueous choline hydroxide solution was dissolved in 45 mL of methanol, and then 17 g (36.3 mmol) of benfotiamine was added to completely dissolve the solution. After concentrating under reduced pressure, 90 mL of acetone was added to crystallize the solution. After the salt was formed, it was filtered under nitrogen, washed with 45 mL of acetone, and vacuum dried to obtain 18.0 g of benfotiamine choline salt.

[0064] 1H 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), 3.7 9(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.

[0065] Example 2: Preparation of benfotiamine choline salt

[0066] Under nitrogen, 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 to completely dissolve the solution. After concentrating under reduced pressure, 90 mL of acetone was added to crystallize the solution. After the salt was formed, it was filtered under nitrogen, washed with 45 mL of acetone, and vacuum dried to obtain 17.8 g of benfotiamine choline salt.

[0067] Example 3: Preparation of benfotiamine choline salt

[0068] Under nitrogen, 10 g (36.3 mmol) of 44% aqueous choline hydroxide solution was dissolved in 45 mL of methanol, and then 17 g (36.3 mmol) of benfotiamine was added to completely dissolve the solution. After concentrating under reduced pressure, 200 mL of ethyl acetate was added to crystallize the solution. After the salt was formed, it was filtered under nitrogen, washed with 50 mL of ethyl acetate, and vacuum dried to obtain 18.6 g of benfotiamine choline salt.

[0069] Example 4: Preparation of benfotiamine choline salt

[0070] Under nitrogen, 10 g (36.3 mmol) of 44% aqueous choline hydroxide solution was dissolved in 150 mL of ethanol, and then 17 g (36.3 mmol) of benfotiamine was added to completely dissolve the solution. After concentrating under reduced pressure, 200 mL of ethyl acetate was added to crystallize the solution. After the salt was formed, it was filtered under nitrogen, washed with 50 mL of ethyl acetate, and vacuum dried to obtain 19.0 g of benfotiamine choline salt.

[0071] Example 5: Preparation of benfotiamine choline salt

[0072] Under nitrogen, 10 g (36.3 mmol) of 44% choline hydroxide aqueous solution was dissolved in 45 mL of methanol, and then 17 g (36.3 mmol) of benfotiamine was added to completely dissolve the solution. After concentrating under reduced pressure, 100 mL of cyclohexane was added to crystallize the salt. After the salt was formed, it was filtered under nitrogen, washed with 50 mL of cyclohexane, and vacuum dried to obtain 19.5 g of benfotiamine choline salt.

[0073] Example 6: Preparation of benfotiamine choline salt

[0074] Under nitrogen, 10 g (36.3 mmol) of 44% choline hydroxide solution and 17 g (36.3 mmol) of benfotiamine were added to 500 mL of isopropyl alcohol and heated to dissolve. The solution was gradually cooled to room temperature, filtered, washed with 50 mL of isopropyl alcohol, and then vacuum dried to obtain 16.7 g of benfotiamine choline salt.

[0075] Experimental Example 1: Stability test (accelerated)

[0076] Pharmaceutical stability testing involves confirming the stability of a certain quality over time under certain conditions in order to determine the storage method and usage period of a pharmaceutical product. In other words, appropriate specifications are set, and significant changes are evaluated based on established analytical methods to evaluate compliance with the specifications, thereby determining the shelf life of the product.

[0077] The stability of the benfotiamine choline salt prepared in Example 1 of the present invention was compared with that of the benfotiamine salt of the comparative example.

[0078] Specifically, a stability test was conducted under severe conditions (60°C ± 2°C) according to the ICH guidelines, and the results were analyzed using high performance liquid chromatography (HPLC). Table 1 shows the results.

[0079] [Table 1]

[0080] As shown in Table 1 above, benfotiamine, which is a comparative example, showed a 2.5% purity change as a result of a 21-day stability test under harsh conditions. On the other hand, benfotiamine choline salt according to the present invention showed a 1.1% purity change as a result of a 21-day stability experiment under harsh conditions, and it was confirmed that the stability was more than twice that of benfotiamine.

[0081] Experimental Example 2: Solubility test

[0082] In order to evaluate the solubility of benfotiamine choline salt according to the present invention, the water solubility depending on the temperature of Example 1 and the comparative example was measured.

[0083] For the solubility test method, 1 mL each of purified water, solutions of pH 1.2, pH 4.0, and pH 6.8 was taken, and an excessive amount of benfotiamine choline salt of Example 1 or benfotiamine of the comparative example was added to saturate it. After ultrasonic extraction for 1 hour and filtration through a PVDF filter, an appropriate amount was diluted for the experiment. The standard solution was diluted with 50% acetonitrile, and a calibration curve (range: 1 to 1,000 mg / mL) was created by HPLC analysis in the USP process. The concentration of the saturated solution was determined from the linear function of the created calibration curve. The results of the test by the solubility test method are shown in Table 2 and Figure 4 below.

[0084] <HPLC Conditions> Detector: Ultraviolet absorption photometer (measurement wavelength 230 nm) Column: Aegispak C18 (4.6 mm × 150 mm, 5 μm) or a column equivalent thereto Column temperature: Constant temperature around 25°C Injection volume: 20 μL Flow rate: 1.0 mL / min Mobile phase: Mixture of 0.5% ammonium carbonate solution: methanol (8:2)

[0085] <W

Table 2

[0086] As shown in Table 2 and Figure 4, the benfotiamine of the Comparative Example exhibited low solubility of 3 to 6.6 mg / mL in purified water and solutions at pH 1.2, pH 4.0, and pH 6.8, whereas the benfotiamine choline salt of Example 1 exhibited very high solubility in purified water and all solutions at pH 1.2, pH 4.0, and pH 6.8. Specifically, the benfotiamine choline salt of Example 1 exhibited solubility that was approximately 124 to 222 times higher than that of the Comparative Example (benfotiamine). This confirms that the benfotiamine choline salt of the present invention can significantly improve the physicochemical properties of benfotiamine, a poorly soluble drug, thereby dramatically increasing the absorption and dissolution of the drug.

[0087] Experimental Example 3: PXRD (Powder X-ray diffraction) analysis

[0088] In powder X-ray diffraction, the diffraction pattern changes depending on the crystalline structure and chemical form of a substance. This can be used to confirm the crystalline structure of a substance by comparing it with a standard substance. Benfotiamine choline salt according to the present invention and comparative benfotiamine as a control were analyzed using a Rigaku MiniFlex 600.

[0089] As a result, as shown in FIG. 3, the benfotiamine choline salt of Example 1 of the present invention exhibited the following temperature values: 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°, 22.3° It was confirmed that the powder X-ray diffraction peak values ​​were characteristically at 23.6°, 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°.

[0090] Experimental Example 4: Immunoblot test

[0091] 1. Preparation of experimental cells

[0092] All cells used in the experiments were primary cultured hippocampal neurons. Hippocampal neurons were isolated from fetal Sprague-Dawley rats (SAMTAKO KOREA) between the 18th and 19th days of gestation. Using a dissecting microscope, fetal rat hippocampal neural tissue was physically isolated and then placed in HBSS (Hank's Balanced Salt Solution) containing 0.25% trypsin for 10 minutes at 37°C. The hippocampal neural tissue was washed with HBSS and then carefully pipetted to isolate single cells. The isolated cells were cultured in 60 mm culture dishes pre-coated with poly-L-lysine (0.5 mg / mL) and filled with Neurobasal / B27 medium (0.5 mM L-glutamine, 25 μM glutamic acid, 25 μM 2-mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin) at 37°C in 5% CO2. The medium was changed on days 4 and 11 of cell culture, and the cells were used for experiments on day 14 of culture.

[0093] 2. Confirmation of activation of neuroplasticity factors in hippocampal cells

[0094] To examine intracellular p-ERK, p-AKT, and NR2B expression, the medium of primary cultured hippocampal neurons cultured for 14 days was replaced with B-27-free medium at least 1 hour before drug treatment. The drugs used were AMPA, benfotiamine, or the benfotiamine choline salt described in Example 1. AMPA was used as a positive control in this experiment as an agonist of the AMPA receptor (α-amino-3-hydroxy-5-methyl-4-isoxazoleproionic acid receptor).

[0095] After treatment with 1 or 3 μg / mL benfotiamine choline salt for 10 minutes, cells were washed with cold PBS and crushed on ice. The crushed cell solution was quantified according to the protein quantification method, and then the proteins were electrophoresed on 8% and 10% SDS-polyacrylamide gels for immunoblotting. The membranes were incubated with 5% skim milk at room temperature for 1 hour. Primary antibodies were incubated with Tris-buffered saline at a dilution of 1:1000 and incubated at 4°C for 24 hours. After washing three times with TBST (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5), secondary antibodies were incubated with anti-rabbit or anti-mouse antibodies (HRP-conjugated) in 3% skim milk at a dilution of 1:5000 for 1 hour. The bound antibody complex was confirmed using West Femto Maximum Sensitivity Substrate (Thermo), and the results are shown in Figure 5.

[0096] As a result, as shown in Figure 5, benfotiamine choline salt effectively increased the levels of phosphorylated Akt, phosphorylated ERK, and NR2B, thereby confirming that benfotiamine choline salt can support long-term potentiation (LPT) and improve cognitive function and memory.

Claims

1. Benfotiamine choline salt of the following chemical formula 1. 【Chemical 1】

2. The benfotiamine choline salt of claim 1, wherein the benfotiamine choline salt of Chemical Formula 1 is a combination of benfotiamine and choline in a molar ratio of 1:

1.

3. The benfotiamine choline salt of Formula 1 is １ 2. The benfotiamine choline salt of claim 1, having a H nuclear magnetic resonance (NMR) spectrum peak: 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)、および2.11(s、3H)ppm。

4. 2. The benfotiamine choline salt of claim 1, wherein the benfotiamine choline salt of formula 1 has a powder X-ray diffraction (PXRD) 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 of Formula 1 had the following peaks: 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°, 21.64±0.2°, 22.36±0.2°, 23.36±0.2°, 23.79±0.2°, 24.01±0.2°, 24.62±0.2°, 2 ...25.91±0.2°, 26.01±0.2°, 26.01±0.2°, 27.01±0.2°, 2 2. The benfotiamine choline salt of claim 1, having a powder X-ray diffraction (PXRD) pattern comprising peaks at 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 crystalline form of benfotiamine choline salt comprising 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 (PXRD).

7. 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°, 24.12±0.2°, 25.37±0.2°, 26.82±0.2°, 28 7. The crystalline form of benfotiamine choline salt of claim 6, further comprising diffraction peaks at 2θ values ​​of 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 of any one of claims 1 to 5 as an active ingredient.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is for improving cognitive function or for preventing or treating a degenerative brain disease.

10. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is in the form of a tablet, capsule, powder, granules, drop pill, pulvis, bolus, tincture or poultice.

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, which is for improving cognitive function or preventing or ameliorating degenerative brain diseases.

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

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is for improving cognitive function or for preventing or treating degenerative brain diseases.

15. 14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is in the form of a tablet, capsule, powder, granules, drop pill, pulvis, bolus, tincture or plaster.

16. A functional health food composition comprising the crystalline form of benfotiamine choline salt according to claim 6 or 7 as an active ingredient.

17. The functional health food composition according to claim 16, which is for improving cognitive function or preventing or ameliorating degenerative brain diseases.

18. 1. A method for producing benfotiamine choline salt, comprising the step of reacting benfotiamine with choline hydroxide.

19. a first step of adding a lower alcohol having 1 to 4 carbon atoms to an aqueous choline hydroxide solution and stirring the mixture; a second step of adding benfotiamine to the solution of the first step and stirring; a third step of concentrating the solution obtained in the second step under reduced pressure and then adding a solvent to crystallize the solution; a fourth step of filtering, washing and drying the solution of the third step.

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

1.

21. 20. The method for producing 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 isopropyl alcohol.

22. 20. 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 a benfotiamine choline salt described in any one of claims 1 to 5 to a subject in need thereof.

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

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