Health functional food composition for preventing or improving SNCG overexpression disease comprising an AZ-628 as an active ingredient

Abstract

The present invention provides a compound as a treatment for degenerative arthritis and Parkinson's disease caused by SNCG by confirming that AZ-628, an SNCG (Synuclein Gamma) inhibitor, binds to the C-terminus of the SNCG (Synuclein Gamma) protein and inhibits the expression of MMP3, MMP13, and COX2, which are factors related to degenerative diseases regulated by SNCG.

Description

Technology Field

[0001] The present invention relates to a health functional food composition for the prevention or improvement of SNCG overexpression diseases comprising AZ-628 as an active ingredient. Background Technology

[0002] SNCG (Synuclein Gamma) is SNCG SNCG is a gene-encoded protein belonging to the synuclein family, known as a pathogen of neurodegenerative diseases. High levels of SNCG are identified in advanced breast cancer, and it has been reported that SNCG overexpression is associated with breast tumor development. SNCG is primarily found in the peripheral nervous system (primary sensory neurons, sympathetic neurons, and motor neurons) and the retina, but it is also found in the brain, ovaries, tumors, and olfactory epithelium. While SNCG is known as a tumor marker and has been found in the retinas of Alzheimer's patients, its function in normal cells is currently unknown; however, many studies are being conducted based on its association with neurodegenerative diseases.

[0003] Neurodegenerative diseases are conditions characterized by degenerative changes in the nerve cells of the central nervous system, which cause various symptoms. In most cases, the causes are unknown; the onset of the disease is gradual, and once it develops, it progresses continuously over several years or decades until death. Representative neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, osteoarthritis, and amyotrophic lateral sclerosis (ALS).

[0004] Degenerative arthritis, also referred to as osteoarthritis, is a type of arthritis characterized by degenerative changes in the cartilage and surrounding bone within synovial joints. Specifically, degenerative arthritis is a disease characterized by the gradual loss of articular cartilage, along with hypertrophy of the bone located beneath the cartilage, bone formation at the joint margins, and non-specific synovial inflammation. It is a condition caused by cartilage damage resulting from aging or excessive physical pressure (e.g., obesity, trauma). Consequently, degenerative arthritis manifests as severe pain and limited movement in weight-bearing joints, such as the knee and hip, and if left untreated for a long period, it can even lead to joint deformation.

[0005] Parkinson's disease is a degenerative disease of the central nervous system primarily characterized by degeneration or arteriosclerotic changes in the diencephalon. Its main symptom is motor dysfunction, and it is caused by the degeneration of nerve cells in the substantia nigra region of the brain in normal individuals, resulting in a deficiency of dopamine, a neurotransmitter produced there. Parkinson's disease is a chronic, progressive motor neuron disorder that occurs when dopamine-producing nerve cells are damaged, leading to a dopamine deficiency and the dominance of acetylcholine, which previously maintained balance. The main symptoms of this disease manifest as tremors (shaking of the hands, arms, legs, and face), rigidity (stiffness of the limbs or body), bradykinesia (slowness of movement), and postural instability (inability to maintain balance). Prior art literature

[0006] Korean Published Patent Application No. 10-2017-0025681 (Published Mar. 08, 2017) Korean Published Patent Application No. 10-2017-0048989 (Published May 10, 2017) The problem to be solved

[0007] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative diseases caused by the overexpression of SNCG (Synuclein Gamma). means of solving the problem

[0008] The present invention provides a pharmaceutical composition for the prevention or treatment of SNCG (Synuclein Gamma) overexpression diseases comprising AZ-628 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for the prevention or improvement of SNCG (Synuclein Gamma) overexpression diseases comprising AZ-628 as an active ingredient. Effects of the invention

[0010] According to the present invention, AZ-628, an SNCG (Synuclein Gamma) inhibitor, binds to the C-terminus of the SNCG (Synuclein Gamma) protein and inhibits the expression of MMP3, MMP13, and COX2, which are factors related to degenerative diseases regulated by SNCG, thereby providing the compound as a treatment for degenerative arthritis and Parkinson's disease caused by SNCG (Synuclein Gamma). Brief explanation of the drawing

[0011] Figure 1 shows the results of evaluating the expression level of SNCG (Synuclein Gamma) in cartilage tissue of patients with degenerative arthritis and in an animal model of degenerative arthritis. Figure 2 shows the results of evaluating the correlation between changes in SNCG (Synuclein Gamma) expression and degenerative arthritis. Figure 3 shows the results of screening SNCG (Synuclein Gamma) inhibitors using in silico compound screening with the CMap approach. Figure 4 shows the results of evaluating the therapeutic effect of AZ-628, a selected SNCG (Synuclein Gamma) inhibitor, on degenerative arthritis caused by SNCG (Synuclein Gamma) overexpression. Figure 5 shows the results of measuring in silico binding affinity using Discovery Studio to determine whether AZ-628, a selected SNCG (Synuclein Gamma) inhibitor, affects SNCG (Synuclein Gamma) activity. It indicates that AZ-628 binds to the C-terminal of the AZ-628 amino acid sequence treated with glutamine (Q), glutamic acid (E), glycine (G), alanine (A), and lysine (K) (indicated in yellow). Specific details for implementing the invention

[0012] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0013] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0014] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0015] The present invention will be described in more detail below.

[0016] The present invention provides a pharmaceutical composition for the prevention or treatment of SNCG (Synuclein Gamma) overexpression diseases comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] [Chemical Formula 1]

[0018]

[0019] The above pharmaceutically acceptable salt refers to an acid addition salt formed by a pharmaceutically acceptable free acid, and the pharmaceutically acceptable salt refers to a salt commonly used in the pharmaceutical industry, for example, an inorganic ionic salt prepared with calcium, potassium, sodium, or magnesium, etc., or an inorganic salt prepared with hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, or sulfuric acid, etc.; an organic salt prepared with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, or vanillic acid, etc.; Sulfonates prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or naphthalenesulfonic acid; amino acid salts prepared from glycine, arginine, lysine, etc.; or amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts referred to in the present invention are not limited by these listed salts.

[0020] The above compound is AZ-628, and specifically can be named 3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)amino)phenol)benzamide. The above compound binds to the C-terminus of the SNCG (Synuclein Gamma) protein and inhibits the expression of MMP3, MMP13, and COX2.

[0021] The above pharmaceutical composition is intended to be administered to patients with diseases caused by the overexpression of SNCG (Synuclein Gamma). The diseases with the overexpression of SNCG (Synuclein Gamma) are degenerative arthritis or Parkinson's disease.

[0022] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0023] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0024] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0025] The above pharmaceutical composition may be formulated into one or more external forms selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms.

[0026] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and a diluent for formulation. The pharmaceutically acceptable carrier and diluent include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically affinity to the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0027] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. For oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. For parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0028] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0029] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration and / or route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0030] In addition, the present invention relates to a health functional food composition for the prevention or improvement of SNCG (Synuclein Gamma) overexpression diseases, comprising a compound represented by the following Chemical Formula 1 and a food-grade acceptable food additive.

[0031] [Chemical Formula 1]

[0032]

[0033] The present invention can be generally used as a commonly used food.

[0034] The above food additives include food additives common in the industry, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, etc., and are exemplified below.

[0035] The health beverage composition of the present invention has no particular limitations on the liquid components other than containing the extract as an essential component in the indicated proportion, and may contain various flavoring agents or natural carbohydrates as additional components, as in conventional beverages. Examples of the natural carbohydrates mentioned above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin; and other conventional sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

[0036] The composition of the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the compositions of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0037] The food composition of the present invention may be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term "functionality" refers to consuming the food for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0038] The food composition of the present invention may include conventional food additives, and unless otherwise specified, suitability as a "food additive" is determined in accordance with the specifications and standards for the relevant item, in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety.

[0039] Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0040] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc.

[0041] For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with an additive such as an excipient, and soft capsules can be manufactured by mixing the composition according to the present invention with an additive such as an excipient and filling it into a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0042] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, synergists, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0043] In this invention, the term “prevention” refers to any act of suppressing or delaying a disease through the administration of a composition according to this invention. In this invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of a disease through the administration of a composition according to this invention. In this invention, “improvement” refers to any act of improving a poor condition of a disease by administering or ingesting a composition of this invention to an individual.

[0044] Hereinafter, experimental examples and embodiments will be described in detail to aid in understanding the present invention. However, the following experimental examples and embodiments are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following experimental examples and embodiments. The experimental examples and embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0045] <Experimental Example> Experimental Materials and Methods

[0046] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0047] 1. Preparation of Human Degenerative Arthritis Samples and Experimental Degenerative Arthritis Mouse Models

[0048] Pelvic cartilage samples were collected from patients aged 63 to 80 who underwent total knee arthroplasty as shown in Table 1 below. Written informed consent was obtained from all patients, and sample collection was approved by the IRB of the Catholic University of Korea (UC14CNSI0150).

[0049]

[0050] The animal model for degenerative arthritis was male C57BL / 6 and SNCG- / - mice (008843-B6.129P2- SNCG tm1vlbThe study was conducted using / J, Jackson Laboratory, USA, and all animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee approved by the Ajou University Laboratory Animal Research Center. To prepare an animal model of degenerative arthritis, 12-week-old male mice were subjected to Destabilization of Medial Meniscus (DMM) surgery and sacrificed 10 weeks post-surgery. In female mice, degenerative arthritis induced by female hormones was excluded from the experiment. Adenoviruses for intra-articular injection were purchased from Vector Biolabs (Malvern, USA): Ad-C (1060), Ad-SNCG (Synuclein Gamma) (ADV-223826). Adenovirus (1 x 10⁶) was administered to wild-type mice. 9 PFUs (10 μL) were injected into the knee joint twice a week, and mice were sacrificed 3 weeks after the first injection.

[0051] 2. Isolation and cell culture of mouse articular primary chondrocytes

[0052] Mouse articular chondrocytes were isolated from the cartilage of ICR mice on day 5 after birth. The isolated cartilage was digested with proteolytic enzymes and collagenase and maintained in DMEM medium (Capricorn science GmbH; Hessen, Germany) containing 10% FBS, 100 units / mL penicillin, and 100 µg / mL streptomycin. Chondrocytes cultured on day 3 (4.25 x 10⁶ 5 Cells / wells were infected with adenovirus or treated with recombinant protein.

[0053] 3. Reagents

[0054] Antibodies were purchased from Abcam (Cambridge, UK; SNCG, COX2, MMP3, MMP13), Col2a1 (Merck, USA), Sox9 (Novus, Littleton, USA), and Erk (610408, Beckton Dickinson, New Jersey, USA). Recombinant SNCG was purchased from Abcam.

[0055] 4. Western Blot

[0056] Cells were lysed in M2 buffer, and mouse tissues were lysed in a lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% Tween 20, 0.2% NP-40, and a protease inhibitor. Equal amounts of cell extracts were separated by SDS-PAGE (6% stacking gel and 10% running gel) and analyzed by immunoblotting.

[0057] 5. CMap Database Analysis and Discovery Studio Analysis

[0058] From drug-induced transcriptome data for approximately 20,000 small molecules in the CMap database, genes that are upregulated and then downregulated by SNCG (Synuclein Gamma) overexpression in chondrocytes were prioritized, and compounds having expression-inhibiting signatures for SNCG (Synuclein Gamma) overexpression were selected. Among the compounds with the potential to inhibit SNCG (Synuclein Gamma) expression, the compound with the highest affinity among those analyzed by SNCG and CMap in Discovery Studio was selected.

[0059] 6. Statistical Analysis

[0060] All experiments were performed independently at least four times. Two independent groups were compared using the Shapiro-Wilk normality test, Levene's homogeneity of variance test, and the two-tailed independent t-test. Multiple comparisons were performed using the Shapiro-Wilk test, Levene's test, and the one-way analysis of variance with Bonferroni's post-hoc test. Data based on the ordinal grading system were analyzed using non-parametric Mann-Whitney U tests. A P-value of less than 0.05 was considered statistically significant.

[0061] Example 1. Analysis of Gene Expression Patterns Caused by SNCG (Synuclein Gamma) Overexpression and Association with Degenerative Arthritis

[0062] To investigate the role of SNCG in osteoarthritis, the expression levels of SNCG were examined using non-damaged and damaged cartilage samples from human and mouse osteoarthritis. As shown in Figure 1, SNCG (Synuclein Gamma) expression was significantly higher in damaged osteoarthritis cartilage compared to non-damaged samples.

[0063] Example 2. Analysis of the control of degenerative arthritis development by SNCG

[0064] MMP3 and MMP13 are known to play important roles in the development of osteoarthritis, and Cox2 is primarily involved in inflammation and induces cartilage matrix degradation through the activation of collagenase and aggrecanase. To analyze the relationship between SNCG (Synuclein Gamma) overexpression and osteoarthritis, changes in related factors were confirmed by treating chondrocytes with SNCG or inducing overexpression. As shown in Figure 2A, Western blot analysis confirmed that when chondrocytes were treated with recombinant SNCG, the expression of catabolic factors such as Mmp3, Mmp13, and Cox2 increased, while the expression of anabolic factors such as Col2 and Sox9 decreased. In addition, as shown in Figure 2B, when chondrocytes were infected with an adenovirus control (Ad-C) or adenovirus SNCG (Ad-SNCG), Western blot confirmed that in chondrocytes where SNCG overexpression was induced by adenovirus SNCG (Ad-SNCG), the expression of SNCG, MMP3, MMP13, and COX2 increased, while the expression of Col2 and Sox9 decreased. Furthermore, the degree of cartilage destruction and the development of degenerative arthritis in mouse knee joints injected with adenovirus into the joint was assessed using Safranin-O staining, OARSI grade, osteophyte formation, and subchondral bone plate thickness. As shown in Figures 2C and 2D, it was confirmed that when SNCG was overexpressed, cartilage destruction was promoted and symptoms of degenerative arthritis were induced.

[0065] Example 3. Drug screening related to SNCG (Synuclein Gamma) expression using CMap

[0066] Drugs exhibiting therapeutic effects for degenerative arthritis by inhibiting SNCG expression were searched through in silico compound screening using the CMap approach. From drug-induced transcriptome data for approximately 20,000 small molecules in the CMap database, genes that are upregulated and then downregulated by SNCG (Synuclein Gamma) overexpression in chondrocytes were prioritized, and compounds having SNCG (Synuclein Gamma) expression inhibitory signatures were searched for in response to SNCG (Synuclein Gamma) overexpression. As shown in Figure 3, the search results selected a compound represented by the following chemical formula 1, and the selected compound was hereinafter referred to as AZ-628.

[0067] [Chemical Formula 1]

[0068]

[0069] Example 4. Evaluation of SNCG (Synuclein Gamma)-mediated inhibition of MMP3, MMP13, and COX2 expression by AZ-628

[0070] To evaluate whether AZ-628 selected in Example 3 is effective as a treatment for degenerative arthritis, its effect on the expression of MMP3, MMP13, and COX2, which are overexpressed by SNCG (Synuclein Gamma), was evaluated. As shown in Figure 4, when AZ-628 was treated to chondrocytes in which SNCG (Synuclein Gamma) overexpression was induced by adenovirus, the expression of MMP3, MMP13, and COX2 induced by SNCG (synuclein gamma) overexpression was found to decrease depending on the dose of AZ-628 administered.

[0071] Example 5. Association analysis of SNCG (Synuclein Gamma) and AZ-628 using Discovery Studio

[0072] To analyze the relationship between AZ-628 selected in Example 3 above and SNCG (Synuclein Gamma), molecular docking analysis was performed using Discovery Studio. As shown in Figure 5, the in silico binding affinity between SNCG (Synuclein Gamma) and AZ-628 was measured, and it was confirmed that AZ-628 binds to the C-terminal residue in the amino acid sequence of SNCG. The above results demonstrate that AZ-628 can inhibit the activity of SNCG (Synuclein Gamma) by binding to a specific residue of the amino acid C-terminal of SNCG (Synuclein Gamma).

[0073] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A health functional food composition for the prevention or improvement of degenerative arthritis comprising a compound represented by the following Chemical Formula 1 and a food-grade acceptable dietary additive. [Chemical Formula 1]

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