KR20200008724A - Composition for preventing or treating neurodegenerative disease comprising cardamonin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the purpose of preventing or treating neurodegenerative diseases, containing cardamonine as an active component. Cardamonine of the present invention has excellent activity of inhibiting Aβ-induced neurotoxicity, thereby being able to be usefully used for pharmaceuticals, functional foods, and the like for preventing or treating (alleviating) degenerative neurological diseases including Alzheimer′s disease. Especially, cardamonine of the present invention is a substance derived from a finger root, which is a natural product, thereby having stability without cytotoxicity. Therefore, a composition containing cardamonine as an active component has an advantage of being safe even for long-term use without side effects to the human body.

Description

Composition for preventing or treating neurodegenerative disease comprising cardamonin, including cardamonine as an active ingredient

The present invention relates to a pharmaceutical composition for the purpose of preventing or treating neurodegenerative diseases comprising cardamonine as an active ingredient.

Neurons are constantly apoptotic in the process of development and reconstruction of synapses, and apoptosis by stress and cytotoxic drugs is a major factor in degenerative brain diseases. Oxidative stress has been associated with many causes of degenerative brain diseases such as Parkinson's disease, stress, aging, stroke, and Huntington's disease. Recent studies have shown that chronic and oxidative stress are hypothalamic. Induces oxidative stress in the pituitary-adrenal cortex, hippocampus, striatum, medulla and anterior cerebral cortex to increase cell death and reduce neurons and growth factors resulting in Parkinson's disease, stress, aging, stroke and Huntington's It is known to cause.

In particular, free radicals liberated in oxygen are known as the main causes of tissue damage, and the types of oxidative radicals related to neurotoxicity include hydrogen peroxide, hydrogen peroxide anion and hydroxyl group, among which hydrogen peroxide is the most important precursor of highly reactive free radicals. It is known to be a substance and is likely to cause apoptosis of the central nervous system.

Oxidative stress on cerebral neurons triggers free radical species, which results in the release of cytochrome C and caspase-3 activation in mitochondria. At the same time, ROS leads to activation of glutamate, especially NMDA receptors, leading to an increase in Ca ²⁺ ions by metabotrophical cascade, and an increase in intracellular Ca ²⁺ associated with ROS is also caspase-2 Activation of DNA causes DNA damage.

In addition, the elderly population is increasing all over the world, and Korea has already entered an aging society in 2000. In 2010, the proportion of people aged 65 and over rose to 11%. With the expectation that 2018 will be an aging society and 2026 an aging society, the solution of senile diseases of the elderly population is emerging as an urgent social problem.

In particular, Alzheimer's disease, which accounts for about half of senile dementia diseases, has no effective treatment and prevention methods at present, and is one of the most urgent neurological diseases to be solved in an aging society. .

Korean Patent Registration No. 0838253

Accordingly, an object of the present invention is to provide a pharmaceutical composition useful for preventing or treating degenerative neurological diseases including Alzheimer's, having excellent activity of inhibiting Aβ-induced neurotoxicity to cardamonine.

It is another object of the present invention to provide a dietary supplement that is useful for preventing or improving degenerative neurological diseases including Alzheimer's, having excellent activity of inhibiting Aβ-induced neurotoxicity to cardamonine.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

 In one embodiment of the present invention, the compound may be included in a concentration of 0.01 to 70μM relative to the composition.

In one embodiment of the present invention, the compound may inhibit the generation of free radicals (ROS) or apoptosis due to Aβ induced toxicity.

In one embodiment of the present invention, the compound may inhibit or inhibit the activity of BACE1 by Aβ induced toxicity.

In one embodiment of the present invention, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, muscular dystrophy, sclerosis, multiple sclerosis, immune system dysfunction, neurodegenerative disorders, metabolic brain disease, It may be selected from the group consisting of dementia due to Neiman-Pick disease, cerebral ischemia and cerebral hemorrhage.

The present invention also provides a health functional food for the prevention or improvement of degenerative neurological disease comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

The cardamonine of the present invention has excellent activity of inhibiting Aβ-induced neurotoxicity and thus may be usefully used in pharmaceutical medicines, functional foods, and the like for the prevention or treatment (improvement) of degenerative neurological diseases including Alzheimer's disease. In particular, the cardamonine of the present invention is a substance derived from a finger root which is a natural product, and thus has a stability without cytotoxicity, so that the composition of the present invention containing it as an active ingredient has a safe side effect even for long-term use without side effects to the human body.

Figure 1 shows the chemical structural formula of cardamonine.
2 is a result confirming the BACE1 inhibitory effect of cardamonine.
Figure 3 is a Dixon plot (Dixon plot) is a result showing the BACE1 inhibition pattern of cardamonin according to the substrate concentration (substrate concentration).
Figure 4 is a lineweaver-Burk plot (Lineweaver-Burk plot) is a result showing the inhibition pattern of BACE1 of cardamonine.
5 shows the specificity of cardamonine for BACE1.

The present invention relates to a novel use of cardamonine, and is characterized by providing a composition for preventing or treating neurodegenerative diseases comprising cardamonine as an active ingredient.

The cardamonin of the present invention is a substance isolated from the finger roots, and is known to protect skin damage from ultraviolet rays and improve the deposition of pigments such as blemishes, blemishes, and blotch, which helps clean skin. There is no known literature that can be used for degenerative brain diseases such as Alzheimer's dementia.

Accordingly, the inventors of the present invention have clarified the fact that it is possible to effectively inhibit Aβ-induced neurotoxicity to cardamonin during various studies for the treatment of Alzheimer's disease.

This fact can be confirmed through one embodiment of the present invention. As a result of examining the inhibitory effect of BACE1 by cardamonine treatment, cardamonine showed a concentration-dependent inhibition pattern, and 35.68 ± 0.18% at 1, 10, and 25μ, respectively. , 62.33 ± 9.11%, 95.07 ± 5.19% showed very excellent inhibitory effect. The IC ₅₀ value of the cardamonine was 4.35 × 10 ⁻⁶ M, indicating that the inhibitory activity was more than three times stronger than the IC ₅₀ value (1.5 × 10 ⁻⁵ M) of the positive control resveratrol.

In addition, cardamonin showed no inhibitory effect on trypsin, chymotrypsin, elastase and TACE, and was found to be a specific inhibitor of BACE1.

Therefore, we have experimentally demonstrated that cardamonine can very effectively inhibit BACE1 of Aβ induction.

Therefore, the composition of the present invention containing cardamonine as an active ingredient can effectively prevent or treat neurodegenerative diseases.

In the present invention, the cardamonine may be a compound represented by the following Chemical Formula 1.

<Formula 1>

The compound represented by Formula 1 according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is preferable, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is, but is not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The cardamonine represented by Formula 1 according to the present invention may be commercially available, or may be one that is separated from nature or manufactured by a chemical synthesis method known in the art.

The composition of the present invention is a pharmaceutical composition comprising the cardamonine as an active ingredient may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to such an active ingredient, and the adjuvant may include an excipient and a disintegrant. , Sweeteners, binders, coatings, swelling agents, lubricants, lubricants, or flavoring agents may be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.

Formulation forms of the pharmaceutical compositions may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solutions, maltodextrin solutions, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

In one embodiment of the invention, the cardamonine of the present invention may be included in the composition at a concentration of 0.01 to 70μM.

Degenerative neurological diseases in which the pharmaceutical composition of the present invention may have a therapeutic effect include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Amyotrophic lateral sclerosis, Multiple sclerosis, Immune system dysfunction, progressive neurodegenerative disease , Metabolic brain disease, Neiman-Pick disease, cerebral ischemia and dementia due to cerebral hemorrhage, and the like, but are not limited thereto, and may be Alzheimer's disease in more detail.

The present invention also provides the use of a composition comprising cardamonine as an active ingredient for the manufacture of a medicament for preventing or treating neurodegenerative diseases. The composition of the present invention containing the cardamonine as an active ingredient can be used for the manufacture of a medicament for the prevention or treatment of degenerative neurological diseases.

The present invention also provides a method for preventing or treating neurodegenerative diseases comprising administering cardamonine to a mammal.

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.

As used herein, the term “therapeutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by one skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient It can be adjusted according to various factors including the sex, diet, time of administration, route of administration and rate of composition, duration of treatment, and drugs used simultaneously. In the treatment method of the present invention, in the case of an adult, the cardamonine of the present invention is preferably administered at a dose of 0.01 mg / kg to 250 mg / kg once or several times a day.

In the treatment method of the present invention, the composition comprising the cardamonine of the present invention as an active ingredient may be administered through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It may be administered in a conventional manner.

In addition, the present invention provides a health functional food for the prevention or improvement of degenerative neurological diseases containing cardamonine as an active ingredient.

The health functional food of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and improving degenerative neurological diseases.

In the present invention, "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and nutrients for the structure and function of the human body. It is meant to be consumed for the purpose of regulating or obtaining a useful effect for health use such as physiological action.

The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive is related to the item according to the General Regulations and General Test Act of the Food Additives approved by the Food and Drug Administration, unless otherwise specified. Judging by the standards and standards.

Examples of the items listed in the "Food Additive Reduction" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark blue, licorice extract, crystalline cellulose, high amount of pigment and guar gum; And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.

For example, the health functional food in the form of tablets is a mixture of excipients, binders, disintegrants and other additives with cardamonin, an active ingredient of the present invention, granulated in a conventional manner, and then compressed with a lubricant and the like. Or the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.

Hard capsules among the health functional foods in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of cardamonine, which is an active ingredient of the present invention, with additives such as excipients, and soft capsules with cardamonine as excipients. The mixture mixed with the additive of may be prepared by filling in a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The health functional food in the form of a cyclic form may be prepared by molding a mixture of a cardamonine, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method. It may be avoided, or the surface may be coated with materials such as starch, talc.

The health functional food in the form of granules may be prepared by granulating a mixture of cardamonine, an active ingredient of the present invention, an excipient, a binder, a disintegrating agent, and the like by a conventionally known method. And the like.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

<Example 1>

Experimental Materials and Methods

<1-1> Experimental material

BACE1 inhibition experiment was performed using cardamonine (≥98%) purchased from Sigma-aldrich. The kit for the BACE1 inhibition experiment was purchased from invitrogen. The serine proteases and TACE required for specificity and selectivity for BACE1 were purchased from Sigma-aldrich.

<1-2> Measurement of β-Secretase (BACE1) Inhibitory Effect and Identification of Inhibition Pattern

Β-Secretase (BACE1) inhibition experiments were performed to verify the prophylactic and therapeutic effects of cardamonin against Alzheimer's dementia. Substrates (Rh-EVNLDAEFK-Quencher) were dispensed at 750 nM using BACE1 assay buffer, and inhibitors were diluted by 10, 25, 50, and 100 μM concentrations in buffer. Thereafter, BACE1 was diluted to 1.0 U / mL and placed in a 384-black micro well plate in the order of enzymes, inhibitors, and substrates, and reacted at 25 ° C. for 60 minutes. Ex. using fluorescence microplate reader Fluorescence was measured at 530 nm and Em 590 nm, and the inhibition rate was calculated by the following equation.

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

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

C ₀ : Fluorescence of the control at time 0

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

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

In addition, in order to determine the BACE1 inhibition pattern of cardamonine, the inhibitory ability of each (250, 500, 750 nM) was measured at different substrate concentrations, and then constructed using a Dixon plot and a Lineweaver-Burk plot.

<1-3> Selectivity and Specificity Study Method for BACE1

In order to determine whether cardamonine selectively inhibits BACE1, an experiment for inhibition of serine proteases such as trypsin, chymotrypsin, and elastase was conducted.

Trypsin, chymotrypsin and elastase inhibitory activities were measured using 0.05 M Tris-HCl (pH 8.2) in 0.02 M CaCl ₂ buffer and substrates were 1.25 mM z-Arg-pNA, 1.25 mM zL-Tyr-pNA, 3.6 mM N- Suc- (Ala) 3-pNA was used. OD values were measured at 410 nm using a 96-micro well plate. In addition, 50 μL of recombinant human TACE (0.1 ppm in 25 mM Tris buffer), sample 48 μL, Mca-PLAQAV-Dpa-RSSR-NH2 to determine the activity of non-amylodogenic pathway by measuring the degree of α-secretase (TACE) inhibition (substrate) was put into a 96-black micro well plate and reacted at 25 ° C. for 60 minutes, and the fluorescence intensity was measured at fluorescence ELISA Ex 320 nm and Em 405 nm, and calculated in the same manner as in BACE1.

<Example 2>

Experiment result

<2-1> Measurement of β-Secretase (BACE1) Inhibitory Effect and Identification of Inhibition Pattern

As a result of examining the inhibitory effect of BACE1, cardamonin showed a concentration-dependent inhibition pattern, and showed an excellent inhibitory effect at 35, 68 ± 0.18%, 62.33 ± 9.11%, and 95.07 ± 5.19% at 1, 10, and 25μ, respectively. The IC ₅₀ value of cardamonine was 4.35 × 10 ⁻⁶ M, which was 3 times more potent than the IC ₅₀ value (1.5 × 10 ⁻⁵ M) of the positive control resveratrol.

Dixon and Lineweaver Burk plots showed that K _i 1.6 × 10 ^-5 M and K _i values were χ fragments, and biochain A was BACE1 substrate. It was found that the material has a pattern of inhibiting BACE1 in a non-competitive manner (see FIGS. 4 and 5).

<2-2> Selectivity and Specificity Study Results for BACE1

In order to observe the specificity for BACE1, the results of the inhibition of cardamonine against α-secretase (TACE) and serine proteases (trypsin, chymotrypsin, elastase) involved in the nonamyloidogenic process are shown in FIG.

Cardamonine did not show inhibitory effects on trypsin, chymotrypsin, elastase, and TACE. It was confirmed that cardamonine was a specific inhibitor of BACE1.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (6)

A pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient;
[Formula 1]

. The method of claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that contained in a concentration of 0.01 to 70μM relative to the composition. The method of claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that by inhibiting free radicals (ROS) production or apoptosis due to Aβ induced toxicity. The method of claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that by inhibiting or inhibiting the activity of BACE1 by Aβ induced toxicity. The method according to any one of claims 1 to 4,
The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Amyotrophic lateral sclerosis, Multiple sclerosis, Immune dysfunctional brain dysfunction, Progressive neurodegenerative disease, Metabolic brain disease, Niemann-Pick disease, Brain ischemia and cerebral hemorrhage A pharmaceutical composition for preventing or treating degenerative neurological disease, characterized in that selected from the group consisting of dementia. Health functional foods for the prevention or improvement of degenerative neurological diseases comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient;
[Formula 1]

.

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