KR20210007437A - Composition for preventing or treating neurodegenerative diseases comprising fucoxanthin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases containing fucoxanthin as an active component, wherein the fucoxanthin promotes signal transduction by a dopamine D3 receptor or a dopamine D4 receptor, thereby being able to be usefully used for preventing or treating neurodegenerative diseases.

Description

Composition for preventing or treating neurodegenerative diseases comprising fucoxanthin {Composition for preventing or treating neurodegenerative diseases comprising fucoxanthin}

The present invention relates to a composition for preventing or treating neurodegenerative diseases comprising fucoxanthine as an active ingredient.

Neurodegeneration refers to the persistent loss of structural and functional properties of neurons that deteriorate with aging. Neurodegenerative diseases are a huge collection of various neurological disorders that appear pathologically and clinically, including Alzheimer's disease, which is cognitive decline due to nerve damage; Parkinson's disease characterized by dyskinesia caused by selective loss of dopaminergic neurons in substantia nigra pars compacta (SNPC); And Huntington disease characterized by loss of progenitor projection neurons, abnormal energy metabolism and excitatory toxicity (IGI Global, PA, USA, 2019, pp. 1-23). In particular, research on the therapeutic agent for Parkinson's disease has been extensively conducted in animal models, and one of the common characteristics of the therapeutic agent is to improve dopamine, a neurotransmitter in the central nervous system (Evrard et al. , 2002; Finberg and Rabey, 2016).

Dopamine, a catecholamine-based neurotransmitter, is known to act by promoting dopamine receptors, a member of the G-protein-coupled receptors (GPCRs) superfamily involved in a variety of neurological processes including motivation, emotion, and reward. Yes (Newman et al., 2012). Dopamine receptors are classified into D ₁ -like (D ₁ R, D ₅ R) and D ₂ -like (D ₂ R, D ₃ R, D ₄ R) subtypes based on their similarity in signaling (Butini et al., 2016).

D ₂ -like D ₂ R, D ₃ R and D ₄ R stimulate G a _i/o to inhibit adenylate cyclase activity and cAMP (cyclic adenosine monophosphate) production. On the other hand, D ₁ -like D ₁ R is the most dopamine receptor in the forebrain, and is known to stimulate G a _s/olf to enhance adenylate cyclase activity and cAMP production (Butini et al., 2016).

Fucoxanthin is a carotenoid that is abundantly present in edible seaweeds such as seaweed, hathaban, kelp, and tot, and is known as a safe physiologically active substance, and has been studied to have various physiological activities. In particular, fucoxanthin is an antioxidant (J. Agric. Food Chem. 55 (2007) 8516-8522), anticancer (Mar. Drugs 10 (2012) 2055-2068), anti-inflammatory (Food Chem. Toxicol. 50 (2012) 3336- 3342) is known to have such effects.

Korean Patent Registration No. 10-1818736

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising fucoxanthine as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving neurodegenerative diseases comprising fucoxanthin as an active ingredient.

Another object of the present invention is to provide a method for promoting signaling by dopamine D ₃ receptor or dopamine D ₄ receptor comprising administering fucoxanthine to an individual other than human.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative disease (neurodegenerative disease) comprising fucoxanthin (fucoxanthin) as an active ingredient.

In one embodiment of the present invention, the fucoxanthin may be derived from seaweed ( Undaria pinnatifida ), but is not limited thereto, and chemically synthesized or a commercially available material may be used by a method known in the art.

In one embodiment of the present invention, the fucoxanthin may be a dopamine D ₃ receptor or an agonist of a dopamine D ₄ receptor.

In one embodiment of the present invention, the fucoxanthin may be administered at a concentration of 0.1 to 200 μM, preferably at a concentration of 1 to 100 μM, more preferably 10 to 100 μM It may be administered at a concentration of, but is not limited thereto.

In one embodiment of the present invention, the neurodegenerative disease may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, senile dementia, diabetic dementia, alcoholic dementia, and vascular dementia.

In addition, the present invention provides a health functional food for preventing or improving neurodegenerative diseases comprising fucoxanthin as an active ingredient.

In addition, the present invention provides a method for promoting signaling by a dopamine D ₃ receptor or a dopamine D ₄ receptor comprising administering fucoxanthine to an individual other than human.

Fucoxanthin, a compound according to the present invention, has an effect of promoting signal transduction by dopamine D ₃ receptor or dopamine D ₄ receptor, and thus can be usefully used in the prevention or treatment of neurodegenerative diseases.

1 shows the chemical structural formula of fucoxanthin.
Figure 2 is a result showing the agonist effect of fucoxanthin on dopamine D ₃ receptor and dopamine D ₄ receptor.
Figure 3A is a result showing the binding pattern of fucoxanthin docked to the dopamine D ₃ receptor and a positive control (fucoxanthin, yellow; dopamine, red; and eticlopride, black), 3B and 3C are dopamine D ₃ receptors It is an enlarged view showing the interaction of fucoxanthin bound to.
4A is a result showing the binding pattern of fucoxanthin docked to the dopamine D ₄ receptor and a positive control (fucoxanthin, yellow; dopamine, red; and clozapine, black), 4B and 4C are bound to the dopamine D ₄ receptor. It is an enlarged view showing the interaction of fucoxanthin.

Fucoxanthine, a compound of the present invention, refers to a compound represented by the following formula (1). The fucoxanthine of the present invention may be isolated from natural sources such as seaweed, but is not limited thereto, and chemically synthesized or commercially available materials may be used by methods known in the art.

[Formula 1]

The compound of the present invention, fucoxanthin, may be derived from an extract or fraction of seaweed ( Undaria pinnatifida ).

The extract may be extracted and separated from nature using a method known in the art for extraction and separation, and the "extract" as defined in the present invention is extracted from seaweed using an appropriate solvent, for example For example, a crude extract of seaweed, a polar solvent-soluble extract, or a non-polar solvent-soluble extract is included.

As a suitable solvent for extracting the extract from the seaweed, any organic solvent that is pharmaceutically acceptable may be used, and water or an organic solvent may be used, but is not limited thereto, for example, purified water, methanol ( alcohols having 1 to 4 carbon atoms, including methanol), ethanol, propanol, isopropanol, butanol, etc., acetone, ether, benzene, chloroform ( Various solvents such as chloroform), ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. Preferably, ethanol may be used.

As the extraction method, any one of methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation method, ultrasonic extraction method, elution method, and compression method may be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method. The method for preparing the seaweed extract of the present invention is not limited, and any known method may be used.

The term "agonist" of the present invention is a substance that activates a receptor in order to produce a biological response by binding to a receptor, and "antagonist" binds to a receptor like the above agonist, but a biological reaction opposite to the agonist It is a substance that inhibits the receptor to inhibit.

The fucoxanthin of the present invention may be a dopamine D ₃ receptor or an agonist of a dopamine D ₄ receptor, and the fucoxanthin has an effect of promoting signal transduction by a dopamine receptor, so that degenerative neurological diseases, in particular, Parkinson's disease Can be used for treatment.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.

In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. I can. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be performed, but are not limited thereto.

The term "individual" refers to all animals including humans, such as mice, mice, and domestic animals. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.

The food composition of the present invention may include all foods in a conventional sense, and may be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the term "functional" It means ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients for structure and function or for physiological effects.

The term "health functional food" of the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, and mixing specific ingredients contained in food ingredients or using specific ingredients for the purpose of health supplementation. It refers to foods designed and processed to sufficiently exert biological control functions such as biological defense, biological rhythm control, disease prevention and recovery, etc. by ingredients. The health food composition is used to prevent diseases and recover diseases. Can perform functions related to etc.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition of the present invention may be prepared by mixing suitable other auxiliary ingredients and known additives that may be included in food according to the choice of a person skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the extract according to the present invention and a fraction thereof as a main component to juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formulas, infant foods, etc.), processed meat products, fish meat products, tofu, rice cakes, noodles (e.g., ramen, noodles, etc.), health supplement foods. , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc. ), beverages (e.g. fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, like a normal beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Experimental materials and methods

1.1. Chemicals and reagents

Fucoxanthin was isolated from seaweed ( Undaria pinnatifida ) and used (Fish. Sci. 78 (2012) 1321-1329). The chemical structural formula of fucoxanthine is shown in FIG. 1.

1.2. Functional GPCRs (G-protein-coupled receptor) assay

Functional GPCRs cell-based assays include cAMP for G _i /G _s -coupled receptors; And the reading of multiple secondary messengers comprising IP ₁ /IP ₃ and calcium flux to G _{q -linked} receptors. Functional assays were performed in Eurofins Cerep (Le Bois I'Eveque, France) using transfected cells expressing human cloned receptors. The protocol of the in-house assay is shown in www.eurofinsdiscoveryservices.com/cms/cms-content/services/in-vitro-assays/gpcrs/functional, and specific experimental conditions are shown in Table 1 below. In the present invention, a stabilized cell line expressing recombinant GPCRs was used.

1.3. Measurement of cAMP levels

Target GPCR gene (dopamine D ₁ , D ₃ or D ₄ receptor) was introduced into CHO (chinese hamster ovary) cells and transfected. The transfected CHO-GPCR cell line was suspended in HBSS/20 mM HEPES buffer (pH 7.4) to which 500 μM IBMX was added, dispensed on a microplate, and then treated without fucoxanthin (control) or at room temperature for 30 minutes. Cultured. After incubation, the cells were lysed, and a fluorescent acceptor (D2-labeled cAMP) and a fluorescent donor (anti-cAMP antibody with europium cryptate) were added. After 1 hour, fluorescence transfer was measured at λex=337 nm and λem=620 and 665 nm using an Envision™ microplate reader (Perkin Elmer, Waltham, MA, USA). The cAMP concentration was determined by dividing the signal measured at 665 nm by the signal measured at 620 nm. The results of the agonist effect were expressed as the percent of the control response to dopamine, and the results of the antagonist effect were expressed as the percent inhibition of the control response to dopamine.

1.4. Intracellular [Ca ²⁺ ] Level measurement

Cells expressing other receptors were transfected with an expression vector encoding the receptor polypeptide and grown for a time sufficient for the receptor to be expressed. Then, a fluorescent probe (Fluo8 Direct, Invitogen, Carlsbad, CA, USA) mixed with probencid in HBSS/2 M HEPES buffer (pH 7.4) was added to each well and equilibrated at 37° C. for 1 hour. Made it. Thereafter, the assay plate was placed in a CellLux™ microplate reader (PerkinElmer, Waltham, MA, USA), and after adding fucoxanthine, a reference agonist or a buffer (blank), Ca ²⁺ ions of the free ^cytosol The fluorescence intensity which changes in proportion to the concentration was measured. The experimental conditions carried out in each experiment are shown in Table 1 above. Accordingly, a concentration-response curve was generated to calculate the EC ₅₀ value.

The agonist effect was calculated as the percentage of the control response (measured response / control response × 100) to the known reference agonist of each target, and the antagonist effect was calculated as the control reference agonist in each target. ) Was calculated as the percent inhibition (100-(measured reaction / control reaction) × 100).

1.5. Molecular docking study

The docking simulation of target receptor and fucoxanthin was performed using the AutoDock 4.2 program. The X-ray crystal structure of the h D ₃ R-ethiclopride complex (PDB ID: 3PBL) and the h D ₄ R-nemonapride complex (PDB ID: 5WIU) is RCSB Protein Data Bank ( PDB). The 3D structure of fucoxanthin was generated using the Marvin Sketch program (v17,1,30, ChemAxon, Budapest, Hungary). The automatic docking simulation was run using AutoDockTools (ADT) to evaluate the proper mating orientation. For the docking calculation, Gasteiger charges were added by default, the rotatable bond was set to ADT, and all torsions were rotatable. Grid maps were created with AutoGrid. The docking protocol for docking non-bent or bendable ligands consisted of 15 independent genetic algorithms. Other parameters used are ADT defaults. Results were visualized and analyzed using Discovery Studio (v17.2, Accelrys, San Diego, CA, USA) and UCSF Chimera (http://www.cgl.ucsf.edu/chimera/).

1.6. ADME (absorption, distribution, metabolism and excretion) prediction experiment

The pharmacokinetic parameters of fucoxanthin, such as ADME (absorption, distribution, metabolism, and excretion), were analyzed using Web-based software, PreADMET (v2.0, YONSEI University, Seoul, Korea). Used.

Example 2. Functional GPCRs assay results

The present inventors analyzed dopamine D ₁ , D ₃ and D ₄ receptors (D ₁ , D ₃ and D ₄ ) through in vitro cell-based functional assay assays; Muscarinic acetylcholine receptor (M ₅ ); Neurokinin-1 receptor (NK ₁ ); Serotonin 1A receptor (serotonin 1A receptor, 5HT _1A ); And vasopressin V _1A receptor (V _1A ) to confirm the effect of fucoxanthin was performed.

As a result, it was confirmed that fucoxanthin is a potent agonist by activating dopamine D ₃ receptor and dopamine D ₄ receptor in a concentration-dependent manner (FIG. 2). In particular, when 100 μM of fucoxanthin was treated with respect to the dopamine D ₃ receptor, agonist activity of 71.05 ± 4.09% was shown, respectively, and the EC ₅₀ value was 16.87 ± 3.41 μM (FIG. 2A and Table 2). In addition, when 100 μM of fucoxanthin was treated with respect to the dopamine D ₄ receptor, agonist activity of 59.90 ± 2.05% was shown, and the EC ₅₀ value was found to be 81.87 ± 6.11 μM (FIG. 2B ).

On the other hand, in addition to the dopamine D ₃ and D ₄ receptors, other receptors (D ₁ , M ₅ , NK ₁ , 5HT _1A and V _1A receptors) did not exhibit agonist activity at a concentration of 100 μM (Table 2). Also, D ₁ , D ₃ , D ₄ , It did not show antagonist activity against M ₅ , NK ₁ , 5HT _1A and V _1A receptors (Table 3).

From the above results, it was confirmed that fucoxanthin is an agonist of dopamine D ₃ receptor and dopamine D ₄ receptor, and thus it was confirmed that it can be used in the treatment of neurodegenerative diseases.

Example 3. Dopamine D ₃ And D ₄ Simulation results of fucoxanthin docking to receptors

The present inventors performed a docking simulation for the dopamine D ₃ receptor and the fucoxanthin complex bound to the dopamine D ₄ receptor. As a result, as shown in Table 4 and Figure 3, fucoxanthin exhibited an interaction with the dopamine D ₃ receptor. Fucoxanthin had a binding energy of -10.61 kcal/mol to the dopamine D ₃ receptor and formed strong hydrogen bonds with Val111, Ser196, and Ser366 residues. In addition, fucoxanthin formed a hydrophobic interaction with Ile183, His349, Pro362, Phe345, Phe346, Cys114, Val107, Asp110, Ser192 and Thr115 residues. Likewise, hydrogen bonds with Thr115, Ser196, Val111 and Asp110 residues were observed in the dopamine D ₃ receptor-dopamine complex.

In addition, as shown in Table 5 and Figure 4, fucoxanthin showed an interaction with the dopamine D ₄ receptor. Fucoxanthin had a binding energy of -7.15 kcal/mol to the dopamine D ₄ receptor, and formed a strong hydrogen bond with the Asp115 and Ser196 residues. The hydrogen bonding was also observed in the positive control (agonist) dopamine and nimonapride. However, no interaction with the Ser196 residue was observed in clozapine, an antagonist.

From the above results, fucoxanthin was strongly bound to the orthostatic binding site including the major residues of the dopamine D ₃ receptor and the 3rd and 5th transmembrane sites of the dopamine D ₄ receptor, and the binding was prevented by the surrounding hydrophobic residues. It was confirmed that it was stabilized. Thus, it was confirmed that fucoxanthin is an agonist of dopamine D ₃ receptor and dopamine D ₄ receptor, and it was confirmed that it can be used as a therapeutic agent for neurodegenerative diseases through signaling by dopamine receptors.

Example 4. ADME prediction result

During drug development, pharmacokinetics is used as a useful tool in defining the drug's batch characteristics and treatment. Accordingly, the present inventors performed an experiment to measure pharmacokinetic parameters such as ADME (absorption, distribution, metabolism and excretion).

As a result, as shown in Table 6, it was confirmed that fucoxanthin has a high logP _o/w value (8.62). Effects on plasma protein binding (PPB) and human intestinal absorption (HIA) were found to be 90.86% and 96.70%. In addition, the BBB (blood brain barrier) passage rate, which is a major factor in the development of CNS (central nervous system) active drugs, was predicted to pass well through the BBB by having a value of 5.55.

From the above results, it was confirmed that fucoxanthin can be used as a therapeutic agent for neurodegenerative diseases.

Claims (7)

A pharmaceutical composition for the prevention or treatment of neurodegenerative disease (neurodegenerative disease) comprising fucoxanthin as an active ingredient. The method of claim 1,
The fucoxanthin is a composition that is derived from seaweed ( Undaria pinnatifida ). The method of claim 1,
The composition of the fucoxanthin is a dopamine D ₃ receptor or an agonist of a dopamine D ₄ receptor. The method of claim 1,
The composition of the fucoxanthin is administered at a concentration of 0.1 to 200 μM. The method of claim 1,
The degenerative neurological disease is a composition selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, senile dementia, diabetic dementia, alcoholic dementia and vascular dementia. Health functional food for the prevention or improvement of neurodegenerative diseases containing fucoxanthin as an active ingredient. A method of promoting signaling by dopamine D ₃ receptor or dopamine D ₄ receptor, comprising administering fucoxanthine to an individual other than human.

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