KR20050093091A - Gentisyl alcohol showing apoptosis-inhibitive effect - Google Patents

Abstract

The present invention relates to a gentiyl alcohol having apoptosis inhibitory activity, and more particularly, by confirming that the gentiyl alcohol of Formula 1 has apoptosis inhibitory activity, the gentiyl alcohol or a pharmaceutically acceptable salt thereof It relates to a liver disease therapeutic composition and health food caused by inflammatory diseases, neurodegenerative diseases or toxic substances due to increased apoptosis containing as an active ingredient.

Description

Gentisyl alcohol showing apoptosis-inhibitive effect

The present invention relates to a gentiyl alcohol having an apoptosis inhibitory activity, and more particularly, by confirming that the gentiyl alcohol of Formula 1 has apoptosis inhibitory activity, a gentiyl alcohol or a pharmaceutically acceptable salt thereof is identified. The present invention relates to a composition for treating liver disease caused by an inflammatory disease, a neurodegenerative disease or a toxic substance due to an increase in apoptosis, which is contained as an active ingredient, and a health food.

Apoptosis or programmed cell death (PCD) is the basic intracellular process for spontaneous self-elimination of damaged or unwanted cells in a wide range of biological systems. Apoptosis is an essential mechanism for organ development, tissue remodeling, maintenance of intracellular homeostasis and removal of abnormal and damaged cells because the contents of cells that have died through the process of apoptosis are not released extracellularly and do not damage other cells.

As mentioned above, the apoptosis process, which plays an important role in living organisms, must be precisely regulated according to a predetermined program. If apoptosis is inhibited, diseases such as cancer, autoimmune disease and viral infectious diseases are induced. Kerr et al., Br. J. Cancer, 26, 239-257, 1972], Inappropriately increasing apoptosis leads to diseases such as acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and liver diseases caused by toxic substances such as alcohol.

Early cells undergoing apoptosis develop chromatin aggregation, cytoplasm, and the like, which are induced during late stages of damage such as cell junctions, blebbing of cell membranes, and cell shrinkage. Nuclear enrichment (cytoplasm and nuclei condensation), loss of mitochondrial membrane potential, changes in plasma membrane composition, and apoptotic body formation are induced to undergo morphological and biochemical changes throughout. DNA fragmentation in the form of oligonucleosomes, the final result of apoptosis, is a typical biochemical feature of cells undergoing apoptosis [Green DR, and Reed, JC, Science, 281, 1309-1312, 1998].

Many apoptosis-related studies have now revealed key players and mechanisms involved in apoptosis in a variety of organisms. Representatively, in mammalian cells, caspase, a type of cellular cysteine protease, is known to play an important role in regulating apoptosis mechanism, which is estimated by homology of amino acid sequence. The evolutionary phylogenetic tree can be classified into three types: interleukin 1 converting enzyme (ICE), CPP32 and ICH-1.

Caspases may be receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage and cell-cells. ) Or activated by a variety of stimuli, including disruption of cell-matrix interactions, such as poly (ADP-ribose) polymerase (RARP), lamine, Induces apoptosis formation by breaking down cellular proteins such as cytokeratins and caspase-activated DNase inhibitors (ICADs) [Cryns, V., and Yuan, J., Genes Dev., 12, 1551-1570, 1998].

As a result of active research on apoptosis, so far, caspases belonging to at least 14 different classes have been identified in mammals, all of which are peptide substrates after aspartic acid residues that play a pivotal role in the initiation and execution of apoptosis. Has been reported to induce apoptosis by cleaving (Nunez et al., Oncogene, 17, 3237-3245, 1998).

Gentiyl alcohol is already known as a secondary metabolite produced by microorganisms, but it was first confirmed by the inventors that it has apoptosis inhibitory activity.

Accordingly, the present inventors have tried to search for apoptosis inhibitors in order to effectively treat diseases caused by excessive apoptosis. As a result, the present inventors have completed the present invention by confirming that gentiyl alcohol shows an excellent apoptosis inhibitory effect in apoptosis-induced cell lines.

Accordingly, the present invention provides a composition for treating liver disease caused by inflammatory diseases, neurodegenerative diseases and toxic substances due to increased apoptosis containing gentiyl alcohol represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. The purpose is.

It is another object of the present invention to provide a health food containing the gentiyl alcohol or a pharmaceutically acceptable salt thereof.

[Formula 1]

The present invention is characterized by a composition for treating liver disease caused by an inflammatory disease, a neurodegenerative disease, and a toxic substance containing the gentiyl alcohol represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Also included are health foods containing the gentiyl alcohol or a pharmaceutically acceptable salt thereof:

The present invention will be described in more detail as follows.

The present invention relates to a therapeutic agent composition for inflammatory diseases, neurodegenerative diseases and toxic substances caused by an increase in apoptosis containing gentiyl alcohol as an active ingredient by confirming that gentiyl alcohol of Formula 1 has apoptosis inhibitory activity. .

In the present invention, the apoptosis inhibitory effect of the blood cancer cell line of gentiyl alcohol having the following physicochemical properties was tested, and it was confirmed that the apoptosis inhibitory activity was higher than that of the reference substance.

i) Material Properties: Powder ii) Molecular Weight: 140

iii) Molecular formula: C ₇ H ₈ O ₃ iv) Mass Spectrometry (MH) ^- : 139 ( m / z )

Meanwhile, the active ingredient of gentisil alcohol of the present invention may 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. Compounds of formula (1) can form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

Accordingly, the present invention provides a pharmaceutical composition containing gentiyl alcohol or a pharmaceutically acceptable salt thereof as an active ingredient, which inhibits abnormally causing apoptosis mechanisms, thereby causing inflammatory diseases, cancers, acquired immunodeficiency syndrome, and various neurodegenerative diseases. It can be usefully used to treat or prevent diseases, ischemic diseases (stroke) and liver diseases caused by toxic substances such as alcohol.

The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration, and may be used in the form of general medicines and health foods.

Pharmaceutical compositions of the invention may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs ( formulated into elixirs). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the pharmaceutical composition of the present invention may be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection. To formulate into parenteral formulations, gentiyl alcohol is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

In addition, the dosage of the active ingredient is generally 1 to 50 mg / day per kg body weight of an adult patient, preferably 5 to 20 mg / day, 1 to several times at regular intervals according to the judgment of the doctor or pharmacist Preferably, it can be divided into 2-3 times a day.

In addition, the composition according to the present invention includes a health food containing an active ingredient of gentiyl alcohol or a pharmaceutically acceptable salt thereof.

A health food is a food prepared by adding gentisyl alcohol or a pharmaceutically acceptable salt thereof to food materials such as beverages, teas, spices, gums, confectionery, or by encapsulating, powdering, or suspensioning. Means bringing a specific effect on health, but unlike the general medicine has the advantage that there is no side effect that can occur when using the drug as a raw material for long-term use.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Physicochemical Characterization of Gentiyl Alcohol

In order to analyze the physicochemical properties of the compound represented by Formula 1, the present inventors used methods such as electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer (USA), hydrogen and carbon nuclear magnetic resonance spectra. It was. In the NMR experiment, each sample was dissolved in methanol (CD ₃ CD) solvent and measured in a 5 mm NMR tube. The chemical shift was measured based on the peak of each solvent or the peak of TMS (tetramethylsilane). . As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, the compound of the present invention was found to have the following physical and chemical properties.

As a result, the material property of the compound of Formula 1 was powder, the molecular weight was 140, the molecular formula was confirmed as C ₇ H ₈ O ₃ , and the mass spectrometry (MH) ⁻ of the compound was 139 ( m / z ). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol (CD ₃ CD) as a solvent in combination with the above physicochemical properties showed that the compounds of the present invention represented by the formula (1) have not been reported until now. It turned out to be a compound which produces an effect.

<NMR data>

¹³ C NMR (CD ₃ CD) δ 58.6, 115.9, 116.0, 117.3, 129.5, 148.7, 150.0

Example 2 Determination of Apoptosis Inhibitory Effect of Gentiyl Alcohol

In order to measure the apoptosis inhibitory effect of gentiyl alcohol, the present inventors performed the following in vitro experiment using U937 cells, a human blood cancer cell line.

U937 cells were cultured in 96-well microplates until they were 5 × 10 ⁶ cells / well, and then 10 μg / ml of apoptotic inducer etoposide and 1 to 100 μg / of the compound of the present invention. Treatment with the U937 cells at a concentration of ㎖ and incubated for 5 hours at 37 ℃ under 5% CO ₂ -95% air conditions to compare the apoptosis inhibitory effect. At this time, cells treated with only etoposide were used as the positive control group, and cells not treated with both etoposide and the compound of the present invention were used as the negative control group. After 5 hours, the shape of U937 cells was observed under a microscope to determine apoptosis. The cells obtained by centrifugation were subjected to DEVD-AFC (Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin). ) Was used to measure the kease phase-3 activity. Determination of the apoptosis inhibitory effect exhibited by the compounds of the present invention is based on the kease phase-3 activity of the positive control (only treated with etoposide) and the keasephase-3 activity of the negative control (untreated with etoposide and compounds). It is shown in Table 1 below.

Comparison of Apoptosis Inhibitory Effects in U937 Cell Lines compound Inhibition rate (IC ₅₀ , μg / ml) Gentisyl alcohol 2.9 Pyrrolidine dithiocarbonate (PDTC) 15.0

As shown in Table 1, the inhibitory activity concentration (IC ₅₀ , concentration required to exhibit 50% inhibitory effect) of the compound represented by Formula 1 on apoptosis of etoposide-induced U937 cells is 2.9 μg / ml. The inhibitory concentration of 15.0 μg / ml of PDTC (pyrrolidine dithiocarbonate), which is a standard comparator, was found to indicate that the compound of Formula 1 had better apoptosis inhibitory effect than PDTC, a standard comparator. From the above results, it was confirmed that the compound of Formula 1 of the present invention is a compound capable of inhibiting apoptosis in vitro.

Example 3: Acute Toxicity of Oral Administration in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally in doses of 1 g / kg / 1 ml in suspension of gentiyl alcohol in 0.5% methylcellulose solution each. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the tested compounds did not show toxic changes up to 500 mg / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 500 mg / kg or more.

As confirmed above, gentiyl alcohol shows an excellent apoptosis inhibitory effect to treat inflammatory diseases caused by increased apoptosis, acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and liver diseases caused by toxic substances such as alcohol. It can be formulated into a pharmaceutical composition.

Preparation Example 1 Preparation of Syrup

A syrup containing 2% (weight / volume) of the active ingredient of gentiyl alcohol of the present invention and a pharmaceutically acceptable salt thereof was prepared by the following method.

Acid addition salts of saccharin, saccharin and sugars were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Gentiyl alcohol, hydrochloride, 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

Sorbic acid0.4 g

Distilled water ·····················

Preparation Example 2 Preparation of Tablet

A tablet containing 15 mg of active ingredient was prepared by the following method.

250 g of gentiyl alcohol hydrochloride was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Gentiyl alcohol, hydrochloride, 250 g

Lactose ... 175.9 g

Potato starch ·············· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch: 160 g

Talc · 50 g

Magnesium stearate 5 g

Preparation Example 3 Preparation of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of gentiyl alcohol hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Gentiyl alcohol, hydrochloride, 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

Formulation Example 4: Beverage Preparation

After dissolving 500 mg of gentiyl alcohol in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharides as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by adding water to make 100 ml. To prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As described above, since gentiyl alcohol according to the present invention exhibits an excellent apoptosis inhibitory effect, liver caused by toxic substances such as inflammatory diseases, acquired immunodeficiency diseases, various neurodegenerative diseases, ischemic diseases (strokes) and alcohols due to increased apoptosis It can be usefully used to treat or prevent diseases.

Claims (4)

The inflammatory disease therapeutic composition containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1] A neurodegenerative disease therapeutic composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1] The liver disease composition according to the toxic substance containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1] A health food containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]