KR20230018812A - Pharmaceutical composition for preventing or treating SNCG overexpression disease comprising an AZ-628 as an active ingredient - Google Patents

Abstract

The present invention confirms that AZ-628, a synuclein gamma (SNCG) inhibitor, binds to the C-terminus of the SNCG protein to suppress the expression of MMP3, MMP13, and COX2, which are factors related to degenerative diseases regulated by SNCG. Therefore, the compound is provided as a therapeutic agent for degenerative arthritis and Parkinson's disease caused by SNCG.

Description

Pharmaceutical composition for preventing or treating SNCG overexpression disease comprising an AZ-628 as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating SNCG overexpression diseases comprising AZ-628 as an active ingredient.

Synuclein Gamma (SNCG) is a protein encoded by the SNCG gene, and is a synuclein family protein known to cause neurodegenerative diseases. It has been reported that high levels of SNCG are identified in advanced breast cancer, and overexpression of SNCG is associated with breast tumor development. SNCGs are found primarily in the peripheral nervous system (primary sensory neurons, sympathetic neurons, and motor neurons) and in the retina, as well as in the brain, ovaries, tumors, and olfactory epithelium. SNCG is known as a tumor marker and has been found in the retina of Alzheimer's patients. However, until now, it has not been known what function SNCG plays in normal cells, but many studies are being conducted based on its association with neurodegenerative diseases. .

Degenerative neurological diseases are diseases that cause degenerative changes in the nerve cells of the central nervous system and cause various symptoms. The disease progresses continuously over the years. Representative neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, degenerative arthritis or amyotrophic lateral sclerosis (ALS).

Degenerative arthritis is a type of arthritis, also referred to as osteoarthritis, and refers to arthritis caused by degenerative changes in cartilage and surrounding bones in synovial joints. That is, degenerative arthritis is a disease characterized by gradual loss of articular cartilage, hypertrophy of bones located below the cartilage, bone production at the edge of the joint, and non-specific synovial inflammation. Osteoarthritis is a disease caused by damage to cartilage due to aging or excessive physical pressure (eg, obesity, trauma, etc.). Therefore, degenerative arthritis shows severe pain and movement disorders in joints that receive a lot of weight, that is, knee (knee) joints and hip (hip) joints.

Parkinson's disease is a degenerative disease of the central nervous system mainly caused by degeneration or arteriosclerotic changes of the diencephalon, and movement disorder is the main symptom. It is caused by a lack of the transmitter dopamine. Parkinson's disease is a chronic, progressive motor neurological disease that occurs when dopamine-producing nerve cells are damaged and dopamine is lacking, resulting in acetylcholine, which was maintained in balance, predominance. This is manifested by trembling tremors, stiffness of the limbs or body, bradykinesia that slows movement, and postural instability that prevents balance.

Korean Patent Publication No. 10-2017-0025681 (Announced on March 8, 2017) Korean Patent Publication No. 10-2017-0048989 (Announced on May 10, 2017)

An object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative diseases caused by synuclein gamma (SNCG) overexpression.

The present invention provides a pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases comprising AZ-628 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving Synuclein Gamma (SNCG) overexpression diseases, which contains AZ-628 as an active ingredient.

According to the present invention, AZ-628, a Synuclein Gamma (SNCG) inhibitor, binds to the C-terminus of the Synuclein Gamma (SNCG) protein to suppress the expression of MMP3, MMP13 and COX2, which are factors related to degenerative diseases regulated by SNCG. By confirming that, the compound can be provided as a therapeutic agent for degenerative arthritis and Parkinson's disease caused by Synuclein Gamma (SNCG).

1 is a result of evaluating the expression level of synuclein gamma (SNCG) in the cartilage tissue of a degenerative arthritis patient and in an animal model of degenerative arthritis.
2 is a result of evaluating the correlation between synuclein gamma (SNCG) expression and degenerative arthritis.
3 is a result of selecting Synuclein Gamma (SNCG) inhibitors by in silico compound screening using the CMap approach.
4 is a result of evaluating the treatment effect of AZ-628, a selected Synuclein Gamma (SNCG) inhibitor, on degenerative arthritis caused by overexpression of SNCG (Synuclein Gamma).
5 is a result of measuring in silico binding affinity using Discovery studio to see whether AZ-628, a selected Synuclein Gamma (SNCG) inhibitor, affects Synuclein Gamma (SNCG) activity. 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) (yellow color) indicate

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The 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 For example, an inorganic ion salt made of calcium, potassium, sodium or magnesium, an inorganic acid salt made of hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid or sulfuric acid; 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 organic acid salts made of acid, ascorbic acid, carbonic acid or vanillic acid; sulfonic acid salts prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid; amino acid salts made of glycine, arginine, lysine, and the like; or amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts meant in the present invention are not limited by these listed salts.

The compound is AZ-628, specifically 3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3,4-dihydroquina Zoline-6-yl)amino)phenol)benzamide(3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3,4-dihydroquinazolin- 6-yl)amino)phenyl)benzamide). The compound inhibits the expression of MMP3, MMP13 and COX2 by binding to the C-terminus of Synuclein Gamma (SNCG) protein.

The pharmaceutical composition is to be administered to a patient with a disease caused by overexpression of Synuclein Gamma (SNCG). The synuclein gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease.

The pharmaceutical composition of the present invention is prepared in unit dosage form or multi-dose by formulating using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art. It can be prepared by incorporating into a container.

The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

In the present invention, the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the range of content used in conventional formulations.

The pharmaceutical composition may be formulated as an aqueous solution, suspension, emulsion, etc. for injection, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, and cataplasma agents. It may be formulated in the form of one or more external preparations selected from the group consisting of agents.

The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginates, and polyvinyl fibres. binders such as rolidone, etc., lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol; don't The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method. For oral administration, it may be formulated into tablets, troches, lozenges, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs. In the case of parenteral administration, it may be formulated as an injection solution, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.

The dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, and the excretion rate. , And the range may vary depending on the type of 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 divided and administered once or several times a day.

The pharmaceutical composition may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method. The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or administration route of the pharmaceutical composition, and those skilled in the art can use the purpose Dosages effective for treatment can be easily determined and prescribed. Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided into several administrations.

In addition, the present invention is a health functional food composition for preventing or improving SNCG (Synuclein Gamma) overexpression disease comprising a compound represented by Formula 1 and food additives acceptable in food science

[Formula 1]

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

The food supplement additives include conventional food additives in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like, and are exemplified below.

The health drink composition of the present invention has no particular limitations on the liquid component except for containing the extract as an essential component in the indicated ratio, and may contain various flavors or natural carbohydrates as additional components like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; polysaccharides such as dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. there is.

The composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids , a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination.

The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and "functional" refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.

The food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules of the food additive code approved by the Ministry of Food and Drug Safety and general test methods, etc., unless otherwise specified. It is judged according to the specifications and standards for the item.

Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.

The food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.

For example, among the health functional foods in the form of capsules, hard capsules can be prepared by mixing and filling the composition according to the present invention with additives such as excipients in conventional hard capsules, and soft capsules can be prepared by filling the composition according to the present invention. It can be prepared by mixing with additives such as excipients and filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

Definitions of terms for the excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and include those having the same or similar functions. There is no particular limitation on the type of food, and it includes all health functional foods in the usual sense.

In the present invention, the term "prevention" refers to all activities that inhibit or delay a disease by administering the composition according to the present invention. In the present invention, the term "treatment" refers to all activities that improve or beneficially change the symptoms of a disease by administering the composition according to the present invention. In the present invention, "improvement" means any action that improves the bad condition of a disease by administering or ingesting the composition of the present invention to a subject.

Hereinafter, experimental examples and examples will be described in detail to aid understanding of the present invention. However, the following experimental examples and examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental Example> Experimental Materials and Methods

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

1. Preparation of human osteoarthritis samples and experimental osteoarthritis mouse models

Hypotonic cartilage samples were collected from patients aged 63 to 80 years who were treated with 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).

The degenerative arthritis animal model was performed with male C57BL/6 and SNCG-/- mice (008843-B6.129P2- SNCG ^tm1vlb /J, Jackson laboratory, USA), and all animal experiments were performed using Institutional approved by Ajou University Laboratory Animal Research Center. It was performed according to the guidelines of the Animal Care and Use Committee. To prepare an animal model for degenerative arthritis, 12-week-old male mice were subjected to Destabilization of Medial Meniscus (DMM) surgery and sacrificed 10 weeks after surgery. In the case of female mice, degenerative arthritis caused by female hormones was excluded from the experiment. Adenoviruses for intra-articular injection were purchased from Vector Biolabs (Malvern, USA): Ad-C (1060), Ad-Synuclein Gamma (Ad-SNCG) (ADV-223826). Wild-type mice were injected twice weekly with adenovirus (1 x 10 ⁹ PFUs/10 μL) into the knee joint, and the mice were sacrificed 3 weeks after the first injection.

2. Isolation and cell culture of mouse joint primary chondrocytes

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

3. Reagents

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.

4. Western blot

Cells were lysed in M2 buffer, and tissue from mice was lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% Tween 20, 0.2% NP-40 and protease inhibitors. . Equal amounts of cell extracts were separated by SDS-PAGE (6% stacking gel and 10% running gel) and analyzed by immunoblot.

5. CMap database analysis and Discovery studio analysis

From the drug-induced transcriptome data for about 20,000 small molecules in the CMap database, genes that are up-regulated and then down-regulated by SNCG (Synuclein Gamma) overexpression in chondrocytes are prioritized, and SNCG (Synuclein Gamma) Compounds with an expression inhibition signature for overexpression were selected. Among the compounds with the potential to inhibit the expression of Synuclein Gamma (SNCG), the compound with the highest affinity was selected among the compounds analyzed by SNCG and CMap in Discovery studio.

6. Statistical analysis

All experiments were performed independently at least 4 times. Two independent groups were compared using Shapiro-Wilk normality test, Levene's homogeneity of variance test, and two-tailed independent t-test. Multiple comparisons were performed using the Shapiro-Wilk test, Levene's test, and one-way analysis of variance with Bonferroni's post-hoc test. Data based on an ordinal grading system were analyzed using non-parametric Mann-Whitney U tests. A P value of less than 0.05 was considered statistically significant.

Example 1. Synuclein Gamma (SNCG) Overexpression Analysis of Gene Expression Patterns and Correlation with Degenerative Arthritis

To explore the role of SNCG in osteoarthritis, the expression level of SNCG was investigated using intact and damaged cartilage samples in human and mouse degenerative arthritis. As shown in FIG. 1, the expression of synuclein gamma (SNCG) was significantly higher in the damaged degenerative arthritic cartilage than in the undamaged sample.

Example 2. Analysis of the regulation of the development of degenerative arthritis by SNCG

MMP3 and MMP13 are known to play important roles in the pathogenesis of degenerative arthritis, and Cox2 is mainly involved in inflammation and induces cartilage matrix degradation by activating collagenase and aggrecanase. In order to analyze the relationship between synuclein gamma (SNCG) overexpression and degenerative arthritis, chondrocytes were treated with SNCG or overexpression was induced to confirm changes in related factors. As shown in Figure 2A, when recombinant SNCG (Recombinant SNCG) is treated with chondrocytes, the expression of catabolic factors such as Mmp3, Mmp13 and Cox2 is increased, and the expression of anabolic factors such as Col2 and Sox9 is decreased. It was confirmed by Western blot. In addition, as shown in FIG. 2B, when chondrocytes were infected with adenovirus control (Ad-C) or adenovirus SNCG (Ad-SNCG), overexpression of SNCG was induced by adenovirus SNCG (Ad-SNCG). It was confirmed by Western blot that the expression of SNCG, MMP3, MMP13, and COX2 increased, and the expression of Col2 and Sox9 decreased in chondrocytes. In addition, as a result of confirming the degree of cartilage destruction and degenerative arthritis development of the mouse knee joint injected with adenovirus into the joint by Safranin-O staining, OARSI grade, osteophyte formation, and subchondral bone plate thickness, as shown in FIGS. 2C and 2D, SNCG When was overexpressed, it was confirmed that cartilage destruction was promoted and degenerative arthritis symptoms were induced.

Example 3. Selection of drugs related to SNCG (Synuclein Gamma) expression using CMap

Through in silico compound screening using the CMap approach, drugs that inhibit the expression of SNCG and exhibit therapeutic effects on degenerative arthritis were searched for. From the drug-induced transcriptome data for about 20,000 small molecules in the CMap database, genes that are up-regulated and then down-regulated by SNCG (Synuclein Gamma) overexpression in chondrocytes are prioritized, and SNCG (Synuclein Gamma) overexpression A compound having a synuclein gamma (SNCG) expression inhibitory signature was searched for. As shown in FIG. 3, as a result of the search, a compound represented by Formula 1 was selected, and the selected compound was referred to as AZ-628.

[Formula 1]

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

In order to evaluate whether AZ-628 selected in Example 3 is effective as a therapeutic agent for degenerative arthritis, the effect on the expression of MMP3, MMP13 and COX2 overexpressed by Synuclein Gamma (SNCG) was evaluated. As shown in FIG. 4, as a result of treating AZ-628 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 628 was found to decrease with the administered dose.

Example 5. Synuclein Gamma (SNCG) and AZ-628 correlation analysis using Discovery Studio

In order to analyze the relationship between AZ-628 selected in Example 3 and Synuclein Gamma (SNCG), molecular docking analysis was performed using Discovery studio. As shown in FIG. 5, as a result of measuring the in silico binding affinity with SNCG (Synuclein Gamma) and AZ-628, 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 bind to a specific residue of the synuclein gamma (SNCG) amino chain C-terminal to inhibit the activity of synuclein gamma (SNCG).

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

The pharmaceutical composition according to claim 1, which is characterized in that it is administered to a patient suffering from a disease caused by overexpression of Synuclein Gamma (SNCG). The pharmaceutical composition according to claim 1, wherein the compound inhibits the expression of MMP3, MMP13 and COX2. The pharmaceutical composition according to claim 1, wherein the compound inhibits the expression of Synuclein Gamma (SNCG) by binding to the C-terminus of the protein. The pharmaceutical composition according to claim 1, wherein the synuclein gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease. A health functional food composition for preventing or improving Synuclein Gamma (SNCG) overexpression disease, comprising a compound represented by Formula 1 below and a food additive additive.
[Formula 1]

The health functional food composition according to claim 6, wherein the Synuclein Gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease.

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