KR102101342B1 - Pharmaceutical composition for prevention or treatment of non-alcoholic steatohepatitis containing tazemetostat or derivative thereof as an active ingredient - Google Patents

Abstract

To a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis containing tazemethostat or a derivative thereof as an active ingredient, the tazemethostat compound exhibits excellent therapeutic effect against non-alcoholic steatohepatitis, non-alcoholic It can be useful as a treatment for sex fatty hepatitis.

Description

Pharmaceutical composition for prevention or treatment of non-alcoholic steatohepatitis containing tazemetostat or derivative thereof as an active ingredient}

It relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis containing tazemethostat or a derivative thereof as an active ingredient.

Non-alcoholic steatohepatitis (NASH) is a form of chronic liver disease often characterized by fibrosis.

NASH sometimes progresses to cirrhosis and hepatocellular carcinoma, and some patients may require liver transplantation. Patients with NASH suffer from conditions such as fat, tissue degeneration, inflammation, cell degeneration, cirrhosis, and increased free fatty acids. NASH is also commonly associated with hyperlipidemia, hyperglycemia, obesity, and type 2 diabetes.

Therefore, it is expected that suppressing progression by finding the mechanism of development and development of NASH may reduce cirrhosis and liver cancer. Unlike simple fatty liver, NASH is activated by a large amount of leukocytes (macrophage) penetrating into the liver tissue, causing inflammation and causing hepatic cell volume to increase compared to the normal size, resulting in liver tissue fibrosis. do. The most important cells contributing to fibrosis of liver tissue are hepatic stellate cells (HSC), which account for 15% of liver cells, and balloon-swollen liver cells. Damaged liver cells or immune cells secrete platelet derived growth factor (PDGF) or TGF-β1 (transforming growth factor-beta1) to activate HSC, and eventually HSC degenerates into myofibroblast-like cells It secretes and accumulates a lot of extracellular matrix components, such as type I and III collagen, which are important for liver fibrosis. HSC is also a major cell that secretes tissue inhibitors of metalloproteinases (TIMP), which inhibit matrix metalloproteinase (MMP), which promotes cell substrate degradation. In addition, balloon-swollen liver cells also secrete a protein that activates a hedgehog signal that promotes fibrosis, thereby promoting fibrosis of liver tissue. Proteins that activate the hedgehog signal are secreted from damaged liver cells, transforming certain cells into scar cells. In addition to the increase in collagen, the scar tissue gradually increases and the fibrosis phenomenon increases, resulting in a decrease in liver cells that function normally, leading to liver failure, and fibrosis further leading to cirrhosis, a prognostic symptom of liver cancer. do.

Compared to the rapid increase in NASH patients, there are no outstanding treatments for NASH yet, and doctors recommend ways to limit diet and strengthen exercise, and in patients with excessive liver function destruction, there is no specific treatment. In the end, the liver must be transplanted. However, as the number of NASH patients increases along with the increase in the obese population, the NASH treatment market has developed into an enormous market size of $ 35 billion per year, and as the causes and mechanisms of NASH have recently been revealed, much attention has been paid to NASH treatment development. (Patent Document 1, Korean Patent Registration No. 10-1665846).

Typical NASH candidate drugs are divided into previously used vitamin E, pioklitazone, and new drug candidates. Aramchol, a mixture of fatty acids and bile acids, and cenicriviroc, which blocks the receptors of immune cells, are currently in Phase 2 clinical trials, and their function is known to inhibit inflammation and fibrosis of liver tissue. GFT505 and obeticholic acid are also drugs that are expected to enter the phase 3 clinical trials. Among them, GFT505 is known to improve insulin sensitivity as well as to promote fatty acid breakdown, but it did not provide good results in the clinical phase 2 study of NASH patients. Obeticholic acid is a modified component of bile acid known to promote sugar and fat metabolism, and is known to have the effect of activating receptors on the cell membrane, increasing insulin sensitivity, and reducing triglycerides. However, obeticholic acid has a serious side effect of causing an increase in LDL cholesterol, which is known to cause cardiovascular disease, and thus its use is limited.

Therefore, the demand for the development of a new NASH therapeutic agent that is safer and can be taken for a long time and suitable for the treatment of chronic diseases is increasing. In the United States, NASH treatment is expected to be approved by the health authorities within 5 years, but drug development or patents targeting NASH in Korea are still insignificant.

In this situation, the present researchers demonstrated that tazemetostat compounds, which were known only for anticancer drugs, show excellent therapeutic effects on animal models that induced non-alcoholic steatohepatitis. The present invention was completed by confirming that it can be developed as a therapeutic agent for non-alcoholic steatohepatitis.

Republic of Korea Registered Patent 10-1665846

An object in one aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis (NASH), which contains tazemethostat or a derivative thereof as an active ingredient.

An object in another aspect of the present invention is to provide a health functional food composition for preventing or improving non-alcoholic steatohepatitis (NASH), which contains tazemethostat or a derivative thereof as an active ingredient.

In order to achieve the above object,

An aspect of the present invention is a pharmaceutical composition for the prevention or treatment of a non-alcoholic steatohepatitis (NASH) containing a compound represented by the following formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient. to provide.

[Formula 1]

(In the formula 1,

R ¹ is -H, or C _1-10 straight or branched chain alkyl;

R ² , R ³ and R ⁴ are independently -H, -OH, halogen, amine, nitro, C _1-10 straight or branched chain alkyl, or C _1-10 straight or branched chain alkoxy).

In addition, another aspect of the present invention is a compound represented by the following formula (1), or a non-alcoholic steatohepatitis (NASH) containing a pharmaceutically acceptable salt thereof as an active ingredient for preventing or improving health Provide a functional food composition.

[Formula 1]

(In the formula 1,

R ¹ is -H, or C _1-10 straight or branched chain alkyl;

R ² , R ³ and R ⁴ are independently -H, -OH, halogen, amine, nitro, C _1-10 straight or branched chain alkyl, or C _1-10 straight or branched chain alkoxy).

The tazemetostat compound shows excellent therapeutic effect on animal models inducing non-alcoholic steatohepatitis, and thus can be usefully used as a therapeutic agent for non-alcoholic steatohepatitis.

1 is an image showing the results of evaluating whether the Preparation Example 1 compound shows therapeutic activity against non-alcoholic steatohepatitis.

Hereinafter, the present invention will be described in detail.

An aspect of the present invention is a pharmaceutical composition for the prevention or treatment of a non-alcoholic steatohepatitis (NASH) containing a compound represented by the following formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient. to provide.

[Formula 1]

(In the formula 1,

R ¹ is -H, or C _1-10 straight or branched chain alkyl;

R ² , R ³ and R ⁴ are independently -H, -OH, halogen, amine, nitro, C _1-10 straight or branched chain alkyl, or C _1-10 straight or branched chain alkoxy).

On the other side,

R ¹ is -H, or C _1-5 linear or branched alkyl;

R ² , R ³ and R ⁴ can independently be -H, -OH, halogen, C _1-5 straight or branched chain alkyl, or C _1-5 straight or branched alkoxy.

In another aspect,

R ¹ is C _1-3 straight or branched chain alkyl;

R ² , R ³ and R ⁴ may independently be -H, halogen, or C _1-3 straight or branched chain alkyl.

On the other side,

The compound represented by Formula 1 may be a compound represented by Formula 2:

[Formula 2]

.

.

The compound represented by Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid and phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes Non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. Get from Examples of these pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulphite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and eye Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Late, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobene Sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1 -Sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, a derivative of Formula 1 is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and a precipitate formed by adding an organic acid or inorganic acid It can be prepared by filtration and drying, or by distilling the solvent and excess acid under reduced pressure and drying to crystallize under an organic solvent.

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

Furthermore, the present invention includes all of the compounds represented by Formula 1 and pharmaceutically acceptable salts thereof, as well as solvates, optical isomers, and hydrates that can be prepared therefrom.

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be administered in various dosage forms, oral and parenteral, during clinical administration. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition, lubricants such as magnesium stearate, talc and the like are used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are common diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Non-aqueous solvents, suspension solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition using the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration may be administered by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. How to do.

At this time, in order to formulate a formulation for parenteral administration, a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, and it is administered in ampoules or vials. Can be produced. The composition may be sterile and / or contain preservatives, stabilizers, hydrating or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances. It can be formulated according to the chemical or coating method.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc., and these formulations include diluents (e.g., lactose) , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycol). Tablets may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and, if desired, a boron such as starch, agar, alginic acid or its sodium salt, etc. It can contain an releasing or boiling mixture and / or absorbent, colorant, flavoring agent, and sweetening agent.

Another aspect of the present invention is a health functional food composition for the prevention or improvement of a non-alcoholic steatohepatitis (NASH) containing a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient Gives

[Formula 1]

(In the formula 1,

R ¹ is -H, or C _1-10 straight or branched chain alkyl;

R ² , R ³ and R ⁴ are independently -H, -OH, halogen, amine, nitro, C _1-10 straight or branched chain alkyl, or C _1-10 straight or branched chain alkoxy).

The compound represented by Formula 1 according to the present invention may be added to food as it is or used with other foods or food ingredients, and may be suitably used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). In general, the amount of the compound in the health food can be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

In addition, the health functional beverage composition of the present invention has no particular limitation on other components except for containing the compound as an essential component in the indicated proportions, and may contain various flavoring agents or natural carbohydrates, etc. as additional components as in conventional beverages. have. Examples of the natural carbohydrates described above include monosaccharides, for example, glucose, fructose, etc .; Disaccharides such as maltose, sucrose, etc .; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.

Furthermore, in addition to the above, the compound represented by Chemical Formula 1 according to the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents, and flavoring agents such as natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pect Acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the compound represented by Chemical Formula 1 of the present invention may contain natural fruit juice and flesh for the production of fruit juice drinks and vegetable drinks.

Another aspect of the present invention provides a method for treating non-alcoholic steatohepatitis, comprising administering a compound represented by Formula 1 to a subject in need thereof. In addition, one aspect of the present invention provides the pharmaceutical composition for use in the prevention or treatment of non-alcoholic steatohepatitis. Furthermore, one aspect of the present invention provides the use of a compound represented by Formula 1 for preparing a medicament used for the prevention or treatment of non-alcoholic steatohepatitis.

The tazemetostat compound exhibits excellent therapeutic effects against non-alcoholic steatohepatitis, and can be usefully used as a therapeutic agent for non-alcoholic steatohepatitis, which is supported by examples and experimental examples described below.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the Examples and Experimental Examples described below are only intended to specifically illustrate the present invention in one aspect, but the present invention is not limited thereto.

< Manufacturing example  1> Tazemeto Start ( tazemetostat ) Preparation of compounds

Preparation Example 1 compound was prepared with reference to US 8,410,088 B2.

< Experimental example  1> Non-alcoholic Hepatitis  Treatment activity evaluation (in vivo )

Preparation Example 1 The following experiment was performed to evaluate whether the compound exhibits therapeutic activity against non-alcoholic steatohepatitis.

More specifically, the therapeutic effect of the hepatitis of the compound on non-alcoholic fatty liver induced by pancreatic destruction and high-fat diet in the insulin-dependent diabetes-like disease mouse (NASH-STAM mouse model) was confirmed as follows.

First, a pregnant female mouse was stabilized by pre-sale from Daehan Biolink Co., Ltd., and then induced childbirth, and male mice were sorted into 3 groups within 48 hours after birth. The general feed group (n = 8) was separated after 3 weeks of life and fed with a normal diet (Research diets, trade name: D12450B) having 10% fat for an additional 6 weeks. 50 mg / kg body weight capacity (0.5 mL / 100 g body weight) by dissolving streptozotocin (STZ, Sigma, USA) in a 0.4 M citrate buffer solution to make an insulin-dependent diabetes-like non-alcoholic fatty liver disease laboratory animal By intramuscular injection.

The high-fat feed group (n = 8) was separated after 3 weeks of age and fed a high-fat diet (Research diets, trade name: D12492) with 60% fat for an additional 6 weeks. The compound administration group (n = 8) was orally administered at a concentration of 10 mg / kg once a day from the 6th week of life with high fat feed intake and supplied for 3 weeks. Diabetes-inducing confirmation was considered to be caused by diabetes if the blood glucose level was 250 mg / dL or more by collecting blood from the tail vein 4 weeks after STZ injection. Subsequently, a portion of the liver extracted by autopsy was fixed in 10 (v / v)% formalin solution to observe histological distribution of fat, and then tissue samples were prepared and oil red O staining was performed. In FIG. 1, the light green stained portion represents normal liver tissue, and the red stained portion is a fat sphere.

As shown in Figure 1,

Oral administration of the compound in Oil Red O staining significantly reduced the fat content in liver tissue compared to the high-fat feed group.

In addition, a portion of the extracted liver was evaluated for expression of DGAT1, CTGF, and TGF-β1 mRNA, respectively. The liver tissue stored in liquid nitrogen was washed with PBS, and then homogenized with 500 μl of Trizol. Add 200 μl of chloroform and shake. After denaturation at room temperature for 15 minutes, centrifugation was performed at 4 ° C for 13000 rpm for 15 minutes. Separate the water layer, add 500 μl of iso-propanol and shake. After standing at room temperature for 10 minutes, centrifugation is performed at 4 ° C for 13000 rpm for 15 minutes. The supernatant is removed and a pellet is obtained. Add 500 μl of 75% ethanol to the RNA pellet and wash. After removing 75% ethanol and drying the pellet for 30 minutes, add 40μl of nuclease-free water and dissolve the RNA pellet. RNA is quantified. Quantified RNA is synthesized by cDNA. For cDNA synthesis, add RT Buffer, dNTP, random primer, reverse transcriptase, nuclease-free water, 25 ° C 10 minutes, 37 ° C 2 hours, 85 ° C 5 minutes It is synthesized by cDNA. cDNA and primers DGAT1, CTGF, TGF-β, SYBR Green PCR master mix, nuclease-free water (Nuclease-free water) was adjusted to 20 μl and analyzed by PCR. do. Real-time PCR activates Uracil-N-glycosylase at 50 ° C for 2 minutes, completely transforms the template DNA at 95 ° C for 2 minutes, then denatures again for 15 seconds, binds at 60 ° C for 15 seconds, The DNA was amplified by repeating the stretching step 40 times at 75 ° C for 1 minute, and finally, a melting curve was analyzed from 60 ° C to 95 ° C. The expression of each of the target genes DGAT1, CTGF, and TGF-β1 was normalized using GAPDH expression. The results are shown in FIG. 1.

As shown in Figure 1,

It can be seen that the preparation example 1 compound has a gene DGAT1 inhibitory effect related to the production of triglyceride that induces fatty liver and an inhibitory effect of hepatic fibrosis markers CTGF and TGF-beta. From this, it can be seen that the tazemetostat compound exhibits an excellent therapeutic effect against non-alcoholic steatohepatitis.

<Formulation Example 1> Preparation of pharmaceutical formulation

1-1. Preparation of powder

500 mg of compound of formula 1

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight fabric to prepare a powder.

1-2. Preparation of tablets

500 mg of compound of formula 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

1-3. Preparation of capsules

500 mg of compound of formula 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into a gelatin capsule to prepare a capsule.

1-4. Preparation of injection

500 mg of compound of formula 1

Suitable amount of sterile distilled water for injection

pH Adjuster

It is prepared with the above-mentioned ingredient content per ampoule (2 ml) according to the preparation method of a conventional injection.

1-5. Preparation of liquid

100 mg of compound of formula 1

Isomerized sugar 10 g

5 g mannitol

Purified water

Each component was added to the purified water to dissolve it according to the method of preparing a conventional liquid, and the amount of lemon was added in a proper amount. Then, the above components were mixed, and then purified water was added to adjust the total to 100 ml, followed by filling into a brown bottle. Sterilize to prepare a liquid.

Claims (5)

A pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis (NASH), which contains the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

(In the formula 1,
R ¹ , R ² , R ³ and R ⁴ are independently C _1-3 straight or branched chain alkyl).
delete delete According to claim 1,
The compound represented by Formula 1 is a pharmaceutical composition, characterized in that the compound represented by Formula 2:
[Formula 2]

.
Health functional food composition for preventing or improving non-alcoholic steatohepatitis (NASH) containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

(In the formula 1,
R ¹ , R ² , R ³ and R ⁴ are independently C _1-3 straight or branched chain alkyl).

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