KR101329575B1 - Pharmaceutical composition comprising Guggulsterone derivatives for gastric mucosal protection - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for gastric mucosa protection comprising a novel gugulsterone derivative as an active ingredient.
Gugulsterone derivatives according to the present invention inhibit the cell damage increased by ethanol in a concentration-dependent manner, promote cell migration for the repair of destroyed gastric mucosal epithelial cells, and the degree of gastric mucosal damage in ethanol-induced acute gastritis mouse model. Significantly reduced. Therefore, it can be usefully used as a pharmaceutical composition for the prevention and treatment of gastric mucosa protective agent, gastrointestinal inflammation and ulcer.

Description

Pharmaceutical composition comprising guggulsterone derivatives for gastric mucosal protection

The present invention relates to a pharmaceutical composition for gastric mucosa protection comprising a novel gugulsterone derivative as an active ingredient.

Gastric mucosa is an organ that is irritated by a number of endless irritations and substances, and gastric mucosa damage is commonly found. In other words, gastric mucosal damage is caused by the imbalance of various attack and defense factors. Causes include Helicobacter pylori (H.P) infection, non-steroid anti-inflammatory drugs (NSAIDs), stress, and alcohol.

A representative example of gastric mucosal injury is peptic ulcer, and until now, treatment for peptic ulcer has been mainly suppressed by gastric acid. Alcohol is one of the main causes of gastritis and gastric ulcer by damaging gastric mucosa by various mechanisms. However, unlike peptic ulcers, alcohol is not an attacker associated with gastric acid, so even if a gastric acid secretion inhibitor is used, the effect of alcohol gastritis is lowered. In addition, a number of complications have recently emerged for the long-term use of proton pump inhibitors. Therefore, there is a need for a new therapeutic or preventive agent that can protect gastric mucosal cells from damage to the mucosa by alcohol or other attack factors.

Meanwhile, guggulsterone (4,17 (20) -pregnadiene-3,16-dione) is a Commiphora , a kind of herb tree. Phytosterol extracted from mukul has been known to have anti-inflammatory effects and has been used for the treatment of obesity, diabetes, hyperlipidemia, arteriosclerosis and degenerative arthritis.

However, in reality, when applying gugulsterone to clinical treatment, not only a large dose is required, but also when administered, it is likely to cause side effects due to absorption in vivo. In order to minimize these side effects, the development of derivatives is required. In other words, if a derivative having higher fat solubility than the existing gugulsterone is developed, it may not be absorbed at the time of actually taking it, and it may be used more effectively even at a low dose because it can be directly contacted with the treatment site. In addition, the synthesis of the derivative has the advantage that can solve the capacity shortage phenomenon due to the extraction of natural substances.

Therefore, the present inventors synthesized a new lipid-soluble gugulsterone derivative, and then confirmed the inhibitory effect in the colonic epithelial cell model and at the same time as well as the prevention and treatment of enteritis in the enteritis animal model by DSS. Gugulsterone derivatives not only inhibit the inflammatory response by interfering with the NF-κB signaling system in vitro , but also found to be very effective in preventing and treating inflammatory enteritis in vivo . It was registered as patent registration No. 10-1082331.

The present inventors earnestly researched the novel use of the novel gugulsterone derivatives, and the novel gugulsterone derivatives alleviate the destruction of the epithelial cells by alcohol and promote cell migration for the recovery of the destroyed epithelial cells. By confirming that it can be used for the treatment of gastrointestinal diseases such as gastric mucosa and gastritis, based on this, the present invention was completed.

Republic of Korea Patent Registration No. 10-1082331

An object of the present invention is to provide a pharmaceutical composition for gastric mucosa protection comprising a novel gugulsterone derivative as an active ingredient.

In order to achieve the above object, the present invention is a guggulsterone derivative represented by the following formula (guggulsterone) derivatives, racemates thereof, optical isomers thereof, and pharmaceutically acceptable salts thereof, comprising: Provide a composition:

[Formula 1]

(In Formula 1, R ₁ is a C3 to C8 linear or branched alkyl group)

Gugulsterone derivatives according to the present invention concentration-dependently inhibited cell damage increased by ethanol in vitro , promoted cell migration for repair of destroyed gastric mucosal epithelial cells, and in vivo . In ethanol-induced acute gastritis animal model, gastric mucosal damage was reduced by pretreatment of gugulsterone derivatives. Therefore, it can be usefully used as a pharmaceutical composition for the prevention and treatment of gastric mucosa protective agent, gastrointestinal inflammation and ulcer.

Figure 1 shows the results of MTT analysis and LDH analysis performed to evaluate the effect of the gugulsterone derivative GG-52 on cell damage by ethanol in MKN-45 cells.
Figure 2 shows the results of cell migration analysis performed to evaluate the effect on the migration of gastric mucosal epithelial cells of the gugulsterone derivative GG-52 in HT-29 cells.
Figure 3 shows the results of visual observation and microscopic observation of the gastric mucosal damage of the positive control group and gugulsterone derivative GG-52 administration group in acute gastritis animal model.
Figure 4 shows the gross mucosal damage and histopathologic scores of gastric mucosal damage of the positive control group and gugulsterone derivative GG-52 administration group in acute gastritis animal model.

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

The present invention relates to a pharmaceutical composition for gastric mucosa protection comprising guggulsterone derivative represented by Formula 1, a racemate thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

(In Formula 1, R ₁ is a C3 to C8 linear or branched alkyl group)

Preferably, R ₁ is 1-propyl group, 2-propyl group, 1-butyl group, 2-butyl group, 3-butyl group, 1-pentyl group, 2-pentyl group, 4-pentyl group, 1-hex It is a real group, 2-hexyl group, 5-hexyl group, 1-heptyl group, 2-heptyl group, 4-heptyl group, or 6-heptyl group, and 1-octyl group.

Representative compounds of the invention include compounds as indicated below:

1) (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

2) (10R, 13S) -17-pentylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

3) (10R, 13S) -17-hexylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

4) (10R, 13S) -17-heptylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

5) (10R, 13S) -17-heptylidene-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta [a] Phenanthrene-3 (6H, 9H, 14H) -on

More preferably, the gugulsterone derivative of formula 1 according to the present invention is represented by the following formula (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8,10,11 , 12,13,15, 16,17-decahydro-2H-cyclopenta [a] phenanthrene-3 (6H, 9H, 14H) -one:

[Chemical Formula 5]

The gugulsterone derivative of the present invention represented by Formula 1 may be used in the form of a pharmaceutically acceptable salt, wherein the salt is an acid addition salt formed by a pharmaceutically acceptable free acid. Do.

Organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metalsulfonic acid may be used as the organic acid. , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.

At this time, the addition salt is a conventional method, that is, after dissolving the compound of Formula 1 in a water miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile and adding an equivalent or excess of an organic acid or an aqueous acid solution of an inorganic acid It can be prepared by precipitation or crystallization or by evaporation of the solvent or excess acid followed by suction filtration of the dried or precipitated salt.

Since the compound of formula 1 according to the present invention has an asymmetric carbon center in its structure, it may exist as its optical isomer and also as a mixture thereof including racemates. Accordingly, such isomers or mixtures thereof are also within the scope of the present invention. Preferred are racemates in which E -and Z -are mixed.

Gugulsterone derivatives of Formula 1 according to the present invention is prepared by the Wittig reaction of a compound of Formula 2 and a triphenylphosphonium halide of Formula 3 in the presence of a base and a solvent, as shown in Scheme 1 below. do:

[Reaction Scheme 1]

(In Scheme 1, X is Br, Cl or I, and R ₁ is as described above)

Compounds of the formula (II) used as starting materials can be purchased commercially or prepared directly. When the compound of Formula 2 is reacted with a triphenylphosphonium halide of Formula 3 under a strong base, triphenylphosphine ylide formed as a half-chain of the strong base and the triphenylphosphonium halide is selectively selected from the ketone at the C-17 position. By the Wittig reaction (Guittosterone derivative of Formula 1) is prepared.

The triphenylphosphonium halide of formula (3) is used in 1 to 3 equivalents, preferably 1 to 1.5 equivalents, based on the compound of formula (2).

As the strong base of the reaction, alkali metal hydrides such as lithium hydride, sodium hydride and potassium hydride generally used in the Wittig reaction; organometallic reagents such as n-butyllithium, sec-butyllithium and tert-butyllithium; And alkali metal alcohol base reagents such as sodium methoxide, sodium ethoxide and potassium-t-butoxide. The amount of base used in the reaction is 1 to 1.5 equivalents based on the compound of formula (2).

At this time, the solvent used may be tetrahydrofuran, diethyl ether, hexane, heptane, methanol, ethanol, benzene, toluene, or a mixture thereof.

The reaction is carried out for 30 minutes to 10 hours at -10 ℃ to 120 ℃.

The composition comprising the gugulsterone derivative protects the gastric mucosa from gastritis and ulcers caused by alcohol, drugs, stress, and the like. Therefore, the composition containing the gugulsterone derivative of the present invention can be used for the prevention and treatment of gastrointestinal diseases such as gastritis and gastric ulcer.

These gastrointestinal disorders are caused by a variety of causes and mechanisms, including alcohol, physical and mental stress, oxidative stress, gastric acid, nonsteroidal anti-inflammatory drugs, steroid preparations, tobacco, irritating foods, and infections of Helicobacter pylori. Can be.

According to Experimental Example 1 of the present invention, the gugulsterone derivative of Formula 1 inhibited the cell damage increased by ethanol in a concentration-dependent manner, and also restored the migration of gastric mucosal epithelial cells reduced by treatment with ethanol. Able to know.

According to Experiment 2, the gugulsterone derivative of Formula 1 significantly alleviated gastric mucosal damage in an alcohol-induced acute mouse model.

Through these results, it was confirmed that the gugulsterone derivative of formula 1 according to the present invention is useful as a pharmaceutical composition for protecting the gastric mucosa.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition according to the present invention can be administered orally or parenterally during clinical administration, and can be used in the form of general pharmaceutical preparations, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. Diluents or excipients may be used.

Solid preparations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin in the gugulsterone derivative according to the present invention. . In addition to simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions or syrups, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. can be used in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent or the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used, and the bases of the suppositories may include Utopepsol, macrogol, Tween 61, Cacao butter, laurin butter, glycerol, gelatin and the like can be used.

The composition of the present invention can be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage is in the range of 0.01 to 1000 mg per kg of body weight. Administration may be divided into several times or several times. In this case, the dosage may be adjusted according to the weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the patient.

[Example]

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Experimental Example  1: Inhibition of alcohol toxicity and confirmation of epithelial cell migration

1-1. Epithelial cell line  culture

The basic human phase epithelial cell lines used in the experiment were AGS cells (ATCC CRL 1739) and MKN-45 cells (JCRB), which are human stomach cancer cell lines. These cells were treated with 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah, USA) and antibiotics (100 unit / ml) in Dulbecco's modified Eagle's medium (DMEM, pH 7.4, Sigma, St. Louis, Missouri, USA). Penicillin, 100 μg / ml of streptomycin) was cultured in a 37 ° C. 5% CO ₂ incubator. Gugulsterone derivative GG-52 was stimulated with TNF-α (10 ng / ml) after 24 hours of treatment in this cell line.

1-2. Statistical analysis

All numerical values of the experimental example of this invention were obtained with mean: standard deviation. Differences between the groups were compared by the Mann-Whitney U test and judged to be statistically significant when the P value was less than 0.05.

1-3. Gugulsterone  Derivative GG-52

Synthesis as disclosed in the Republic of Korea Patent Registration No. 10-1082331 and (10R, 13S) -17-butylidene-10,13-dimethyl-1,7,8,10,11,12,13, 15,16, 17-decahydro-2H-cyclopenta [a] phenanthrene-3 (6H, 9H, 14H) -one was obtained and this gugulsterone derivative GG-52 was used in the experiment.

[Chemical Formula 5]

1-4. MTT  analysis, LDH  Analysis and Cell Migration Analysis

High concentrations of alcohol lead to the destruction of phase epithelial cells. To investigate the effect of gugulsterone derivatives (GG-52) on epithelial cell destruction by ethanol, MKN-45 cells were pretreated with gugulsterone derivatives (GG-52) and ethanol (100 mM) Time was processed. Subsequently, the cell destruction effect was measured by MTT (3- [4,5-dimethylthiazol-2-yl] -2,5 diphenyltetrazolium bromide) analysis and lactate dehydrogenase (LDH) analysis.

MTT and LDH tests were performed according to the conventional methods to investigate the cell proliferation inhibition and destruction effects. The MTT test evaluated the ability of mitochondrial succinate dehydrogenase to reduce MTT to MTT formazan. On the other hand, LDH activity in culture supernatant was evaluated based on the reduction of pyruvate to lactate in the presence of LDH and NADH. These experiments were performed using Sigma's X-ray analysis kit.

In vivo, the protective mechanism against mucosal damage is activated. In other words, when a wound on the mucosa, the surrounding epithelial cells migrate to replace the damaged cells. Cell migration assays were performed to investigate the effect of the gugulsterone derivative GG-52 on epithelial cell migration. Cell migration assay was CytoSelect ^T A wound healing assay kit (Cell Biolabs, Inc., San Diego, CA, USA) was used. That is, HT-29 cells (1 × 10 ⁶ cells / ml) were put into 24 wells of the kit and incubated for 24 hours to make a monolayer. 0.9 mm wounded inserts were removed from the wells, exchanged with fresh culture, and ethanol (100 mM) and gugulsterone derivative GG-52 were added. Then, the epithelial cell movement distance from the wound was measured using an alternative lens with cross hairs according to the conventional method.

Figure 1 shows the results of MTT analysis and LDH analysis performed to evaluate the effect of the gugulsterone derivative GG-52 on cell damage by ethanol in MKN-45 cells.

Referring to FIG. 1, it was confirmed that cell damage increased by ethanol was suppressed concentration-dependently by the pretreatment of the gugulsterone derivative GG-52.

Figure 2 shows the results of cell migration analysis performed to evaluate the effect on the migration of gastric mucosal epithelial cells of the gugulsterone derivative GG-52 in HT-29 cells.

Referring to Figure 2, it was observed that the epithelial cell migration reduced by ethanol alone treatment recovered from 12 hours after the treatment of the gugulsterone derivative GG-52. These results suggest that the gugulsterone derivative GG-52 can restore alcohol-induced gastric mucosal epithelial cells.

Experimental Example 2: Mitigating Gastric Mucosal Damage in Acute Gastritis Mouse Model

2-1. Experimental animal

Eight-week-old female specific-pathogen free (SPF) C57BL / 6 mice (20-25 g body weight) were purchased from Orient (Seongnam, Korea), 12 hours light and 12 hours light in SPF environment. Breeding in the absence of. Experiments were carried out up to 9 weeks after birth, ie after 1 week of adaptation. In addition, 12 hours before the experiment was fasted. All animal testing procedures passed the deliberation of the Animal Experiment Ethics Committee of Seoul National University Hospital.

2-2. Acute Gastritis Mouse Model

A total of 20 mice were divided into four groups. That is, negative control group (4 mice), alcohol alone group (positive control group, 4 mice), and gugulsterone derivative GG-52 experimental group [GG-52 50 mg / kg post-treatment group (4 mice), GG-52 200 mg / kg post-treatment (4 mice)]. In the control group, 0.9% physiological saline was administered, and in the experimental group, a solution in which 0.5% methyl cellulose was dissolved in GG-52 was administered through an oral gavage. After 1 hour, only methyl cellulose was administered to the negative control group and 100% ethanol (5 mg / kg) was administered to the other group (alcohol alone group and GG-52 experimental group) through oral gavage to induce acute gastritis. It was. After 1 hour, the animals were sacrificed, the stomach was removed, and the naked eye was checked and photographs were taken at the same time. Half of the tissues were placed in formalin solution and 1/2 were stored in a -70 ℃ freezer.

2-3. Visual and Microscopic Observation of Acute Gastritis Mouse Model

(1) macroscopic finding

The stomach tissues of the extracted mice were excised along the Taiwanese grains and washed with physiological saline, and then examined for gross gastric mucosal damage. Referring to one animal study, scores from 0 to 5 were:

0 points: normal

0.5 point: local weak redness

1 point: overall redness or small (less than 1 mm) bleeding

2 points: large (1 mm or more) bleeding

3 point: small (less than 2mm) ulcer

4 point: large (more than 2mm) ulcer

5 points: perforated ulcer

(2) histopathological score (microscopic finding)

The gastric tissue section cut along the Taiwan valley was cut at 5 mm intervals in the longitudinal direction of the pyloric region and fixed after formalin storage. Tissue sections were subjected to hematoxylin and eosin (H & E) staining. Then, one pathologist compared the findings of inflammation by scoring from 0 to 3 points as in the conventional method.

0 points: normal

1 point: When the surface mucous membrane is slightly damaged or only 2-3 cells in the upper part of the gland cells are damaged

2 points: The damage is more severe than 1 point, but the damage area is less than 50% of the total thickness of the gastric mucosa.

3: When more than 50% of the total thickness of the gastric mucosa is damaged

Figure 3 shows the results of visual observation and microscopic observation of the gastric mucosal damage of the positive control group and gugulsterone derivative GG-52 administration group in acute gastritis animal model.

As shown in FIG. 3, it was confirmed that the degree of damage of the macroscopic gastric mucosa in the group treated with the gugulsterone derivative GG-52 was significantly reduced compared to the alcohol-only group. In addition, the microscopic findings showed that the degree of mucosal destruction was remarkably alleviated in the group treated with the gugulsterone derivative GG-52 compared to the positive control group.

Figure 4 shows the gross mucosal damage and histopathologic scores of gastric mucosal damage of the positive control group and gugulsterone derivative GG-52 administration group in acute gastritis animal model.

Referring to FIG. 4, as a result of quantitatively measuring the degree of gastric mucosal damage, the degree of damage of the gastric mucosa was statistically significantly smaller than that of the positive control group in the high dose experimental group (gross and microscopic). Redness p = 0.020, microscopic findings p = 0.038). These results in the mouse model of acute gastritis suggest that pretreatment of the gugulsterone derivative GG-52 protects the gastric mucosa and mitigates gastric mucosal damage.

Claims (3)

Guggulsterone derivative represented by the following formula (5) or a pharmaceutical composition for gastric mucosa protection comprising a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 5]

delete The pharmaceutical composition according to claim 1, which is for preventing or treating one or more conditions selected from the group consisting of acute gastritis, chronic gastritis, gastric ulcer, gastric perforation, erosion of the gastric mucosa due to the disease, bleeding, redness and edema. .

Images