KR101674541B1

KR101674541B1 - Pharmaceutical composition for preventing or treating pancreatitis including proliferation inhibitory composition of Coxsackie virus B4 inducing pancreatitis

Info

Publication number: KR101674541B1
Application number: KR1020150063682A
Authority: KR
Inventors: 임병관; 김진희
Original assignee: 중원대학교 산학협력단
Priority date: 2015-05-07
Filing date: 2015-05-07
Publication date: 2016-11-09

Abstract

The present invention relates to a coxsackievirus B4 proliferation inhibitor, and more particularly, to an inhibitor of coxsackievirus B4, which comprises a compound 3- [3,4-dihydroxyphenyl] acrylic acid 1- [3,4-dihydroxyphenyl] -2- methoxycarbonylethyl ester It is related to the antiviral inhibitory effect.
The substance of the present invention has an antiviral efficacy against Coxsackie virus B4 that causes pancreatitis and can be effectively used by treating pancreatitis caused by coxsackievirus B4.

Description

[0001] The present invention relates to a pharmaceutical composition for the treatment and prevention of pancreatitis containing pancreatitis-inducing cocksaccin B4 proliferation inhibitor,

The present invention relates to a pharmaceutical composition containing a proliferation inhibitor of pancreatitis-induced Coxsackievirus B4, particularly a compound 3- [3,4-dihydroxyphenyl] acrylic acid 1- [3,4-dihydroxyphenyl ] -2-methoxycarbonylethyl ester (hereinafter referred to as 'the subject matter of the present invention' or 'compound of formula 1' hereinafter) for inhibiting the growth of Coxsackie virus B4.

Treatment of diseases caused by viral infections is usually an indirect treatment that relieves the inflammation caused by viruses or releases heat caused by them. Currently, most antiviral agents that are commercially available include proteins such as nucleoside derivatives and interferon (IFN), but these substances exhibit various side effects such as cytotoxicity, hepatotoxicity and over-tolerance. Therefore, there is a continuing need for the development of new antiviral substances with low side effects, low cost and safety. Or by administering a vaccine before the virus infection, the antibody against the virus is formed to prevent the virus from causing the disease.

Recently, there are many viral diseases, such as influenza virus, vesicular stomatitis, severe respiratory disease virus (SARS), and food poisoning induced norovirus. In this situation, natural product-based antiviral inhibitors can offer an alternative to non-toxic treatment of new viruses, and research is currently underway in various fields.

Coxsackievirus B4 (CVB4) is a non-enveloped RNA virus with a single-stranded positve sense RNA genome (about 7.4 kb) have. These CVB4s belong to the family Picornaviridae and belong to Enterovirus together with poliovirus.

These Coxsackie viruses B4 (CVB4) are known to specifically infect digestive cells of the pancreas and have been reported to cause chronic pancreatitis and cause diabetes in later adult life. In particular, it is important to prevent the onset of chronic pancreatitis and prevent it from being recognized because it causes acute dyspepsia and cold-like symptoms.

In this respect, the proliferation inhibitory effect of the cockscomb virus B4 (CVB4) of the duckweed extract is very remarkable. The inventors of the present invention have disclosed the effect of inhibiting the proliferation of Coxsackie virus causing dysentery myocarditis and cold in the previous papers and patents, but the present invention was differentiated in terms of the treatment of pancreatitis.

Korean Journal of Pharmacology 45 (4) 282-287 0253-3073 KCI

It is an object of the present invention to provide a substance which can be safely used without side effects while having solved the problems of conventional antiviral agents, and also has excellent antiproliferative effect of Coxsackie virus B4 (CVB4).

It is a further object of the present invention to provide a pharmaceutical composition for treating and preventing pancreatitis, which contains the above-mentioned substance as an active ingredient and contains a proliferation inhibitor of pancreatitis-causing cockscomb virus B4.

To achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of pancreatitis comprising a proliferation inhibitor of pancreatitis-inducing cockscomb virus B4, wherein the proliferation inhibitor is represented by the following formula The present invention provides a pharmaceutical composition for the treatment and prevention of pancreatitis, which comprises as an active ingredient at least one compound selected from the group consisting of a compound and a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

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The duckweed extract compound according to the present invention exhibits an effect of inhibiting the proliferation of Coxsackie virus B4 (CVB4), which is a non-toxic and destructive pancreatic cell and induces chronic pancreatitis as a cause of diabetes, as an antiviral therapeutic agent or a preventive health supplement Can be usefully used.

FIG. 1 shows the results of confirming the effect of inhibiting the proliferation and cytotoxicity of Coxsackie virus B4 (CVB4)
FIG. 2 shows the results of Western blot analysis of the protein expression inhibition effect of the membrane protein VP1 of CVB4 in cells extracted from cells by the compound of Chemical Formula 1,
Fig. 3 shows the results of inhibiting the virus growth of the compound of formula (I) in Hela cells infected with CVB4 by the compound of formula (I).
Fig. 4 shows the results of confirming the inhibitory effect of the compound of formula (I) on the CVB4 viral gene amplification.

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

The present inventors have conducted intensive studies for a long period of time in order to achieve the above object. As a result, it has been found that 3- [3,4-dihydroxyphenyl] acrylic acid 1- [3,4-dihydroxyphenyl] -2- methoxycarbonylethyl ester compound has an excellent effect on the inhibition of the growth of coxsackievirus B4, leading to the subject matter of the present invention.

The subject matter of the present invention inhibits the proliferation of Coxsackie virus infected to a cell through modulation of the cell's growth signal when treated with infection of the Coxsackie virus. In addition, the substance of the present invention has antiviral efficacy against Coxsackie virus B4, which causes pancreatitis, and can be effectively used by treating pancreatic inflammation caused by coxsackievirus B4.

The present inventors have found that a compound represented by the following formula (1) isolated from Isodon excisus (Max.) Kudo has an inhibitory effect on the proliferation of coxsackievirus B4, And the possibility of the treatment and prevention of pancreatitis due to the increase of virus B4 and the possibility of health food.

It is known that Coxsakievirus B4 (CVB4) binds to the coxsakievirus and adenovirus receptor (CAR) of host cells and infects cells. It is known to infect mainly the pancreas. CVB4 belongs to the picornaviruses and belongs to the intestinal viruses like polioviruses and rhinoviruses. Although most intestinal virus infections are not symptomatic, CVB4 infection is predicted to cause acute or chronic pancreatitis and a chronic illness, such as diabetes, one of the most serious modern diseases. Chronic pancreatitis can be caused by genetic and environmental factors, and severe acute pancreatitis is predicted to be caused by environmental factors such as viruses.

To elucidate the pathogenesis of acute and chronic pancreatitis, a pancreatitis model induced by CVB4 infection in mice was developed and used for the study of various clinical diseases. The virus used to make the pancreatitis model was classified as CVB4-P and CVB4-V according to the severity of inflammation. CVB4-P causes weak pancreatitis and CVB4-V causes severe pancreatitis. Induced cytokine IL-12 and IFN-γ.

Hereinafter, the compound 1 which is an inhibitory activity effectively acting on CVB4-P and CVB4-V according to the present invention will be described.

First, a method for separating and extracting the growth inhibitory active material of the cockscomb virus B4 from duckweed will be described in detail.

The ducklings were collected and dried, followed by extraction with methanol (MeOH) for 2 weeks, followed by filtration and concentration to obtain a methanol extract. The ethyl acetate (EtOAc) extract obtained by suspending such methanol extract in a predetermined amount of distilled water was subjected to silica gel column chromatography (silica gel column chromatography) using chloroform (CHCl ₃ ): methanol (MeOH) = 20: 1, silica gel column chromatography to concentrate the active fractions. Here, in the present embodiment, the silica gel column chromatography can use a mixed solvent of CHCl ₃ : MeOH in a volume ratio of 100 to 10: 1 as a developing solvent.

The active fraction is purified through high pressure liquid chromatography (HPLC), and then the solvent is dried to extract the compound of formula (1).

The compound represented by Formula 1 may be used in the form of a pharmaceutically acceptable salt. For example, the acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. The compounds of formula (I) may form acid addition salts which are pharmaceutically acceptable according to methods conventional in the art. As the free acid, a free acid and an inorganic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used. As the organic acid, citric acid, tartaric acid, acetic acid, lactic acid, fumaric acid, propionic acid, formic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid or aspartic acid Can be used.

As a result of measuring the inhibitory effect and the VP1 expression inhibiting effect of the coxsackievirus B4 on the thus obtained compound of the formula (1) or a pharmaceutically acceptable salt thereof, it was confirmed that the coxsackievirus B4 had an excellent proliferation inhibitory activity. In addition, it has been confirmed that the treatment of these compounds with Coxsackie virus B4-infected cells significantly inhibits the replication of new viruses by inhibiting the protein production and gene amplification of viruses infected with host cells. These results confirm the potential of the compound to be developed as an agent for treating acute, chronic pancreatitis caused by infection with coxsackievirus B4.

Accordingly, the present invention relates to a therapeutic composition for the inhibitory effect on the proliferation of the abnormally induced cockscomycin B4 containing the compound of the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, have.

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

The compositions may also be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, pharmaceutical powders or granules, emulsions, hard or soft capsules, syrups or elixirs .

Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation in tablets, capsules and the like; Excipients such as dicalcium phosphate; Disintegrating agents such as corn starch or sweet potato starch; Lubricating oil such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intramuscular injection. For formulation into parenteral administration formulations, the compound of formula (I) is mixed with a stabilizer or buffer in water to prepare a solution or suspension, which is then formulated into unit dosage forms of ampoules or vials. In addition, the active ingredient of the present invention generally has an effective dose of 1 to 50 mg / day, preferably 5 to 20 mg / day, per kg body weight of an adult patient and is administered at a predetermined time interval It may be administered several times a day, preferably two to three times.

The above-mentioned health food is a food prepared by adding the active ingredient to a food material such as a beverage, a tea, a spice, a gum or a confection, or encapsulated, powdered, or a suspension, and the health food has a certain health effect However, unlike general medicine, there is an advantage that there is no side effect that can occur when a long-term use of the medicine is performed using the food as a raw material.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It is to be understood, however, that the scope of the present invention is not limited by the examples.

<Example 1> cock extraction and separation of Saki virus B4 proliferation inhibitory substance

2.5 kg of Isodon excisus (Max.) Kudo, which was collected and dried in a member of Jiri Mountain, was extracted with 100% methanol (MeOH) for 2 weeks, filtered and concentrated to obtain a methanol extract (39 g). This methanol extract (39 g) was suspended in distilled water (1 L) and fractionated in the order of n-hexane, EtOAc n-BuOH and H ₂ O. The ethyl acetate (EtOAc) fraction (5 g) showing the most excellent effect was subjected to silica gel column chromatography using CHCl ₃ : MeOH = 20: 1 using a developing solvent with a stepwise increased polarity To obtain an active fraction (42 mg). The active fractions thus obtained were subjected to high performance liquid chromatography (Shisheido-CapCell Pak C ₁₈ UG120, 250 x 100 mm, UV 220 nm, CH ₃ CN gradient 20-100%, flow 1.5 ml / min) After separating the active fractions and removing the solvent with a vacuum dryer, the residue was freeze-dried to obtain 3- [3,4-dihydroxyphenyl] acrylic acid 1- [3,4-dihydroxyphenyl] -2-methoxycarbonylethyl ester compound (5.8 mg).

<Experimental Example 1> Coxsackie B4 virus growth inhibition measured effect of the compound

In order to confirm the effect of the compound represented by the formula (1) on the inhibition of the coxsackievirus B4 proliferation and cytotoxicity, experiments were carried out using HeLa cells as follows.

The compound of Chemical Formula 1 isolated from duckling of Example 1 was added to the cell culture medium diluted 1 mM to HeLa cells cultured in a cell culture dish, treated with 10 ⁴ PFU / ml of Coxsackievirus B4 (CVB4) Ml for 30 min and the compound diluted sequentially with 5% FBS DMEM. After 18 hours, 7 ㎕ of Cell Counting Kit 8 (CCK-8), which is a cell proliferation detection reagent, was added and further cultured for 2 hours. Then, the absorbance at 450 nm was measured using a microplate reader (Molecular device, USA) to confirm the viability of the cells and to confirm the antiviral efficacy.

As shown in FIG. 1, the survival rate of HeLa cells was significantly increased in the treatment of the compound of formula (1) after infection with the coxsackievirus B4, especially in the concentration of the compound. Further, as can be seen from FIG. 1, cell survival was 50% at a relatively low concentration of 0.001 μM, indicating that the compound has strong antiviral effect against coxsackievirus B4. From this, it can be confirmed that the compound of the formula (I) is excellent in the inhibition of the proliferation of Coxsackievirus B4 and the cytotoxicity.

OK <Experimental Example 2> inhibiting expression of VP1 of compounds of formula (I) effect

The inhibitory effect of VP1 on the expression of the compound of formula (1) was tested as follows in order to confirm it by Western blot analysis. 10 ⁴ PFU / ml of Coxsackievirus B4 (CVB4) was added to a 12-well plate incubated with monolayer HeLa cells and infected for 30 min before treatment with the compound at a concentration of 0.1 mM at 1 mM. After 18 hours at 37 ° C and 5% CO ₂ , whole proteins were extracted and subjected to Western blotting. As a result, as shown in FIG. 2, the membrane protein VP1 of the cocksack virus B4 showed a significant decrease according to the concentration of the compound administered. In particular, the transcription factor eIF4GI, which is cleaved by the protease 2A enzyme of the virus produced when the virus grows, is a very important protein for the production and survival of the cell. 2A. According to the treatment concentration of the compound, the destruction of eIF4GI abruptly decreased to leave much of the normal protein on the upper part. When the compound was not treated, the normal protein was destroyed by the production of protease 2A due to virus multiplication, Only partially confirmed. These results show that the compound, a phenylpropanoid component contained in duckweed, is effective in inhibiting CVB4 replication in Hela cells infected with CVB4

FIG. 2 shows Western blot analysis of the inhibitory effect on the protein expression of the membrane protein VP1 of coxsackievirus B4 in the cells extracted from the cells by the compound of formula (1), showing the effect of inhibiting the proliferation of coxsackievirus B4 And it was confirmed that the growth of coxsackievirus B4 was controlled by the concentration of the compound.

& Lt; Experimental Example 3 > The action of the above compound on coxsakiovirus B4 in Hela cells

The concentration of live virus in the supernatant of the cultured cells was confirmed by PFU assay in the coagulation of Coxsackie virus B4 (CVB4) in Hela cells. After the CVB4 infection, the cells were treated with the above compound, cultured, and the cells were treated with HeLa cells, and the concentration of live virus was measured by PFU assay. As shown in Fig. 3, treatment of the compound strongly inhibited the replication and proliferation of CVB4 according to the concentration (Fig. 3). These results indicate that the compounds are very effective in inhibiting the production of virus proteins in infected cells and inhibiting the generation of new viruses. Therefore, as described above, the compound of the present invention represented by the formula (I) can be exhibited excellent effects by being formulated with an antiviral therapeutic agent or composition.

<Experimental Example 4> Confirmation of the effect of suppressing the amplification of Coxsackie virus B4 (CVB4) gene

The positive strain concentration of the membrane protein VP1 was measured by performing rt-PCR on the viral gene amplification inhibitory effect of the compound in HeLa cells infected with CVB4. The positive strain of the virus can be detected by amplifying a small amount of viral gene as a very strong confirmation method that can confirm the viral gene amplification and existence of the gene rather than the direct viral propagation. As shown in Fig. 4, the treatment of the compound strongly inhibited gene amplification of the CVB4 virus (Fig. 4). It was confirmed that the direct viral proliferation was inhibited and the gene of virus was reduced, and that the compound could inhibit the overall virus proliferation in infected cells.

As described above, the results shown in the above-mentioned several experimental examples suggest the possibility of developing an antiviral active ingredient as a natural ingredient contained in domestic native plants. Recently, intestinal viruses have become the cause of many common diseases and cause various infectious diseases especially for children. In this experiment, the phenylpropanoid compound extracted from the domestic native plant Duckweed, was used to test the viability of CVB4-infected HeLa cells, and the compound treatment strongly inhibited the replication of CVB4. In particular, the production of viral membrane protein was significantly reduced in HeLa cells, and the proliferation of new live viruses was significantly inhibited. It was also found that the replication and amplification of the CVB4 gene, which is increased by the virus multiplication, is significantly reduced by the above compound treatment. From these results, it is expected that the compound can be developed as a therapeutic drug for intestinal viral pancreatitis.

& Lt; Formulation Example 1 &gt; Preparation of syrup agent

A syrup containing the compound of formula (I) of the present invention and a pharmaceutically acceptable salt thereof in an amount of 2% (weight / volume) of the active ingredient can be produced by the following method.

The acid addition salt, saccharin and sugar of the compound of formula (1) were dissolved in 80 g of hot water. After the solution was cooled, a solution composed of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to the mixture to make 100 ml.

The addition salt may be replaced with another salt according to the examples, and the addition salt may be replaced with another salt according to the examples.

The components of the syrup are as follows.

2 g of the hydrochloride salt of the compound of formula

0.8 g of saccharine

25.4 g per

Glycerin 8.0 g

Spices 0.04 g

4.0 g of ethanol

Sorbic acid 0.4 g

Determination of distilled water

& Lt; Formulation Example 2 &gt; Preparation of tablet

Tablets containing 15 mg of active ingredient are prepared as follows.

250 g of the hydrochloride salt of the compound of formula (1) was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

The components of the tablet are as follows.

250 g of the hydrochloride salt of the compound of formula

Lactose 175.9 g

Potato starter 180 g

32 g of colloidal silicic acid

10% gelatin solution

Potato starch 160 g

50 g of talc

Magnesium stearate 5 g

& Lt; Formulation Example 3 & gt; Preparation of injection solution

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

1 g of the hydrochloride of the compound of the formula (1), 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

The components of the injection solution are as follows.

1 g of the hydrochloride of the compound of formula (1)

0.6 g of sodium chloride

Ascorbic acid 0.1 g

Claims

A pharmaceutical composition for treating and preventing pancreatitis comprising a proliferation inhibitor of pancreatitis-induced cocksack virus B4,
The above-
A pharmaceutical composition for the treatment and prevention of pancreatitis, which comprises as an active ingredient at least one compound selected from the group consisting of compounds represented by the following formula (1) extracted from duckweed and pharmaceutically acceptable salts thereof.
[Chemical Formula 1]

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