KR101801478B1 - Composition for preventing or treating neurodegenerative disease comprising biochanin A as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain disease, containing biochanin A or a pharmaceutically acceptable salt thereof as an active ingredient. More specifically, biochanin A inhibits activities of beta-secretase 1 (BACE1) and suppresses production of beta-amyloid plaque, thereby being useful for preventing or treating degenerative brain disease.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating neurodegenerative diseases,

The present invention relates to a composition for preventing or treating degenerative brain diseases comprising biocanin A as an active ingredient.

According to the National Statistical Office (NSO), 7.9 persons are able to support one elderly person in 2005, but 6.6 persons in 2010, 2.6 persons in 2030, 1.4 persons in 2050 and 1.2 persons in 2060 to be. This shows the reality of an aging society that emerges as a serious social problem in recent years, and shows the need for a response to an aging society (National Statistical Office, 2011).

Alzheimer's disease is characterized by atrophy of the brain and enlargement of the ventricles, accompanied by pathological features such as neurofibrillary tangles and senile plaques. Among these, β-amyloid plaque (hereinafter referred to as "Aβ"), a constituent of the elderly, is a peptide composed of 39-43 amino acids. Aβ is produced by sequential hydrolysis of β-secretase and γ-secretase to the β-site of amyloid precursor protein (APP) (Park SH et al., 2014). In normal individuals, the α-secretase cleaves the 15th and 16th amino acids at the N-terminus of the APP before the action of the γ-secretase, and the γ-enzyme acts to release the short peptide that is not toxic (Efrev I et al., 2012). In patients with dementia, the production of Aβ is high due to high β- and γ-enzyme activities. The resulting Aβ deposits in the pericellular blood vessels, secondary to the inflammatory reaction, resulting in a widespread impairment of brain function (Kim JH, 2000).

There are four treatments approved by the US FDA and acetylcholinesterase (AChE) inhibitors are the main treatments for Alzheimer's dementia. Until now, treatment has only been symptomatic or delayed, and the disease itself has not been treated (Lee HS, 2012). In order to solve the fundamental problem, Aβ has been suggested as a candidate substance for inhibition of aggregation, but there is no proven therapeutic agent (Lee MY, 2005).

1. U.S. Published Patent Application No. 14/069740

1. Biradar SM, Joshi H, Chheda TK. Biochanin-A ameliorates behavioral and neurochemical derangements in cognitive-deficit mice for the betterment of Alzheimer's disease. Hum Exp Toxicol. 2014 Apr; 33 (4): 369-82. 2. Ditkoff EC, Cassidenti DL, Paulson RJ, Sauer MV, Paul WL, Rivier J, et al. The gonadotropin-releasing hormone antagonist (Nal-Glu) acutely blocks the luteinizing hormone surge but allows resumption of folliculogenesis in normal women. Am J Obstet Gynecol. 1991 Dec; 165 (6 Pt 1): 1811-7. 3. Efremov IV, Vajdos FF, Borzilleri KA, Capetta S, Chen H, Dorff PH, et al. Discovery and Optimization of a Novel Spiropyrrolidine Inhibitor of I²-Secretase (BACE1) Through Fragment-Based Drug Design. J Med Chem. 11/08/2012; 55 (21): 9069-88. 4. Evin G, Hince C. BACE1 as a therapeutic target in Alzheimer's disease: rationale and current status. Drugs Aging. 2013 Oct; 30 (10): 755-64. 5. Kandalepas PC, Vassar R. The normal and pathologic roles of the Alzheimer's beta-secretase, BACE1. Curr Alzheimer Res. 2014; 11 (5): 441-9. 6. Paganini-Hille, Henderson VW. Estrogen replacement therapy and risk of Alzheimer disease. Arch Intern Med. 1996 Oct 28; 156 (19): 2213-7. 7. Park SH, Yang EJ, Kim SI, Song KS. beta-Secretase (BACE1) -inhibited C-methylrotenoids from Abronia nana suspension cultures. Bioorg Med Chem Lett. 2014 Jul 1; 24 (13): 2945-8. 8. Kim Jin-hong. Symposium / Alzheimer's disease and estrogen. Journal of Korean Society of Menopause. 2000; 6 (1): 3-11. 9. Lee Min Young. Articles: Alzheimer 's Disease and Aromatherapy. Journal of Clinical Oncology. 2005; 1 (2): 53-63. 10. Lee Han Sup. A study on the percutaneous permeability of Tacrine, a treatment for Alzheimer type dementia. Journal of the Korean Petrochemical Society. 2012; 29 (4): 552-60.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising biocanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a health functional food for preventing or ameliorating a degenerative brain disease comprising biocanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases comprising biocanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the biocanin A may have a structure represented by the following formula (1).

[Chemical Formula 1]

In one embodiment of the present invention, the biocanin A may inhibit the activity of BACE1 (beta-secretase 1) to inhibit the formation of beta-amyloid plaques.

In one embodiment of the present invention, the degenerative brain disease may be Alzheimer's disease, Huntington's disease or Parkinson's disease.

The present invention also provides a health functional food for preventing or ameliorating a degenerative brain disease comprising biocanin A or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the biocanin A may inhibit the activity of BACE1 (beta-secretase 1) to inhibit the formation of beta-amyloid plaques.

In one embodiment of the present invention, the degenerative brain disease may be Alzheimer's disease, Huntington's disease or Parkinson's disease.

Biocanin A of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, acid addition salts formed by pharmaceutically acceptable free acids are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving biocanin A in an excess amount of an aqueous acid solution and then using this salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile Followed by precipitation. By heating the same amount of biocanin A and an acid or alcohol in water, then evaporating the mixture and drying, or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, Biocanin A of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving Biocanin A in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile or the like, adding an excessive amount of organic acid Adding an aqueous acid solution of an inorganic acid, and precipitating or crystallizing it. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

When the composition of the present invention is used as a medicine, the pharmaceutical composition comprising biocanin A or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated into various oral or parenteral dosage forms at the time of clinical administration, But is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, Silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols), in addition to the active ingredient (s). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing the biocanin A of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be administered parenterally, and parenteral administration can be carried out by subcutaneous injection, intravenous injection, intramuscular injection, It depends on the injection method. In this case, in order to formulate a formulation for parenteral administration, a pharmaceutical composition comprising biocanin A or a pharmaceutically acceptable salt thereof as an active ingredient is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, Ampicillin or vial unit dosage forms. The compositions may comprise sterilized and / or contain adjuvants such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure and / or buffers, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the composition of the present invention to the human body may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient. When the patient is 60 kg body weight, And may be 0.01 to 500 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

In order for the biocanin A of the present invention or a pharmaceutically acceptable salt thereof to be used for the prevention and improvement of degenerative brain diseases as described above, it may be prepared by various methods known in the field of pharmacy or pharmacy, Can be prepared in any food form that can be ingested orally by mixing with pharmaceutically acceptable carriers, excipients, diluents, and the like. Preferably in the form of beverage, ring, granule, tablet or capsule.

The health functional food of the present invention may further comprise ingredients that are conventionally added at the time of food production and which are pharmaceutically acceptable. For example, in the case of beverage preparation, one or more components may be further included in citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, etc., in addition to biocanin A of the present invention or pharmaceutically acceptable salts thereof.

The amount of the active ingredient of the health functional food according to the present invention may be appropriately selected depending on the age, sex, weight, condition and symptom of a person who desires to prevent or improve the degenerative brain disease, Day to 0.01 g to 10.0 g. By taking the health functional food having such a content, prevention and improvement of degenerative brain diseases can be obtained.

It has been confirmed that biocanin A according to the present invention can effectively inhibit the formation of beta-amyloid plaques through inhibition or inhibition of BACE1 (beta-secretase 1), which is an enzyme producing beta-amyloid plaques, Degenerative brain diseases induced by beta-amyloid plaques, especially Alzheimer ' s dementia, can be effectively prevented or treated.

Fig. 1 shows the chemical structure of biocanin A. Fig.
Fig. 2 shows the inhibitory effect of BACE1 (beta-secretase) upon treatment with biocanin A. Fig.
Figure 3 shows IC ₅₀ values for biocanin A;
FIG. 4 shows the BACE1 inhibition pattern of biocanin A according to the substrate concentration in a Dixon plot.
Figure 5 shows the inhibition of BACEl of biocanin A by Lineweaver-Burk plot.

Hereinafter, the present invention will be described in detail with reference to embodiments and drawings. However, these examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Experimental material

Biocinin A (≥97%) was purchased from Sigma-aldrich and the kit required for the experiment to determine if biocanin A inhibited BACE1 (beta-secretase 1) was purchased from Pan Vera Inc. . Serine proteases and TACE (alpha secretase) required for BACE1 specificity and selectivity experiments were purchased from Sigma-aldrich.

Example  2. Of BACE1 (beta-secretase 1)  Measure inhibition effects and identify inhibition patterns

BACE1 (beta-secretase 1) inhibition experiments were conducted to verify the preventive and therapeutic effects of Biocanin A on Alzheimer's dementia. The substrate (Rh-EVNLDAEFK-Quencher) is diluted to 750 nM using BACE1 assay buffer and the inhibitor is diluted to 10, 25, 50 and 100 μM in buffer. BACE1 was diluted to 1.0 U / mL, and the enzyme, inhibitor and substrate were placed in a 384-black micro well plate and reacted at 25 ° C for 60 minutes. The fluorescence was measured at 530 nm for excitation and 590 nm for emission using a fluorescence microplate reader, and the inhibition rate was calculated by the following equation.

Inhibition (%) = [1 - ( S - S ₀ ) / ( C - C ₀ )] 100

C : Fluorescence of control after 60 min of incubation (enzyme, assay buffer and substrate)

C ₀ : Fluorescence of the control at time 0

S : Fluorescence of control after 60 min of incubation (enzyme, sample solution and substrate)

S ₀ : Fluorescence of the tested samples ( S ) at time 0

In order to examine BACE1 inhibition pattern of biocanin A, the inhibition patterns of 250, 500, and 750 nM were measured at different substrate concentrations. Then, sigma plot ver. 12.3 program using a Dixon plot and a Lineweaver-Burk plot.

Resoratrol, a known inhibitor of BACE1, was used as a positive control. Biotanin A was found to be 21.20 ± 0.44%, 33.50 ± 0.91% for BACE1 at concentrations of 10, 25, 50 and 100 μM, , 54.03 ± 130% and 62.85 ± 1.88%, respectively (FIG. 2). Biocanin A inhibited BACE1 in a dose dependent manner and showed a significant difference ( p <0.001). Especially, at the concentration of 50 μM, the effect was similar to that of resveratrol.

The enzyme inhibition pattern of biocanin A was studied using a Dixon plot. As a result, IC ₅₀ value was 6.2 × 10 ^-5 M (FIG. 3) and K _i was 4.9 × 10 ^-5 M 4 and 5). Therefore, considering that K _i is a χ intercept, biocanin A was found to be a substance that inhibits BACE1 in a non-competitive manner with BACE1 substrate.

Example  3. BACE1 (beta-secretase 1)  Selectivity / specificity study

Inhibition of serine proteases such as trypsin, chymotrypsin and elastase was investigated to see if biocanin A selectively inhibited BACE1 alone. Trypsin inhibitory activity Measurement of chymotrypsin and elastase dehydratase is 0.05 M Tris-HCl using _{(pH 8.2, 0.02 M CaCl 2} buffer) was the substrate is 1.25 mM z -Arg- p NA, 1.25 mM z -L-Tyr , respectively - p NA, 3.6 mM N- Suc- (Ala) ₃ - p NA. The optical density (OD) was measured at 410 nm using a 96-well plate. In addition, 50 μL of recombinant human TACE (0.1 ppm in 25 mM Tris buffer), 48 μL of sample, and Mca-PLAQAV-DpA-RSSSR-NH were measured to determine the degree of TACE (alpha secretase) inhibition and activity against non-amylodogenic pathway ₂ substrate was incubated at 25 ° C for 60 minutes and the fluorescence intensity was measured at 320 nm for excitation and 405 nm for emission using a fluorescence ELISA. The fluorescence intensity was measured using the same method as BACE1 in Example 2 To calculate the inhibition rate.

In order to observe the enzyme specificity for BACE1, we examined the inhibitory ability of biocanin A on TACE and serine proteases (trypsin, chymotrypsin and elastase) involved in the non-amyloidogenic pathway. As a result, biocanin A showed no inhibitory effect on TACE, trypsin, chymotrypsin and elastase (Table 1).

Therefore, it can be confirmed that biocanin A is a specific inhibitor of BACE1 against BACE1.

Concentration (μM) TACE Trypsin Chymotrypsin Elastase 50 18.33 + - 2.14 11.69 ± 3.25 8.33 ± 1.89 15.01 + - 3.02 100 22.71 + - 2.54 14.14 ± 1.39 7.99 ± 1.78 17.78 ± 1.89

Claims (7)

A method of inhibiting BACE1 (beta-secretase 1) activity by treating biotanin A represented by the following formula 1 with BACE1 (beta-secretase 1) in Invitro.
[Chemical Formula 1]

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