KR102073566B1 - Composition for treatment and prevention of hepatitis C - Google Patents

Abstract

The present invention relates to a composition for treating and preventing hepatitis C, and more particularly, to a composition for treating and preventing hepatitis C, including 2-O-methylhonokiol. The composition for treating and preventing hepatitis C according to the present invention has been shown to exhibit an effective inhibitory effect on hepatitis C virus, thereby exhibiting an excellent effect on the treatment or prevention of hepatitis C according to various types of HCV. It is expected to be.

Description

Composition for treatment and prevention of hepatitis C}

The present invention relates to a composition for treating and preventing hepatitis C, and more particularly, to a composition for treating and preventing hepatitis C, including 2-O-methylhonokiol.

Hepatitis C virus (HCV) is currently reported as a disease of 2-3% of the world's population. Hepatitis C virus infection promotes the formation of nitric oxide (NO), which causes DNA modification and damage in host cells, reduces the transmenbrane potential of mitochondria, as well as reactive oxygen species. acts as a cause of oxygen species). The reactive oxygen species formed can act as oxidative stress and damage cellular structure. HCV is one of the causes of acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma, but no effective vaccine has been developed to date.

Recently, DNA treatment has been allowed overseas, but it is not allowed in Korea. So far, interferon alpha (IFN-α) and ribavirin (rivavirin) are used to treat hepatitis C. . However, the methods used in Korea are not only impossible to treat all genotypes of HCV, but also require a lot of treatment cost and treatment time, and side effects are pointed out as problems.

International Publication No. 1999-053917. US Patent Publication No. 7915232. International Publication No. 2013-092481

In order to solve the above problems, the present invention has excellent replication inhibitory effect against hepatitis C virus (HCV), and has low cytotoxicity to human cells, and includes 2-O-methylhonokiol. To provide a composition for treating and preventing hepatitis C.

The present invention provides a 2-O-methylhonokiol derivative compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Chemical Formula 1,

R 1 and R 2 are, independently from each other, hydrogen, (C 2 -C 7) alkenyl;

R 3 and R 4 are independently of each other hydrogen or halogen;

m and n are each independently an integer of 1 to 3;

In addition, the present invention is a disease mediated by hepatitis C virus comprising the 2-O-methyl-honokino derivative compound of Formula 1, its prodrug, solvate, stereoisomer or pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for the prophylaxis or treatment of.

In addition, the present invention provides a C comprising a 2-O-methyl honokiol derivative compound of Formula 1, a prodrug thereof, a solvate thereof, a stereoisomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier It provides a pharmaceutical composition for the prevention or treatment of diseases mediated by hepatitis virus.

The present invention also provides a molding agent comprising the pharmaceutical composition.

The composition for treating and preventing hepatitis C according to the present invention has been shown to exhibit an effective inhibitory effect on hepatitis C virus, thereby exhibiting an excellent effect on the treatment or prevention of hepatitis C according to various types of HCV. It is expected to be.

1 is a result of measuring HCV replication inhibition effect by the luciferase test.
2 is a cytotoxicity measurement results by the MTS test.
Figure 3 is a result of the mechanism analysis according to xenobiotic treatment during HCV replication.
(a) AhR, CYP1A1 protein expression, (b) mRNA expression by siRNA test, (c) CYP1A1 mRNA expression changes according to whether or not Compound 155 treatment.
4 shows the results of changes in the expression level of IFN-α mRNA according to Compound 155 treatment.
FIG. 5 shows HCV replication inhibitory effect and cytotoxicity measurement results when Compound 155 and Compound 156, which is a structural isomer thereof, are treated.

Hereinafter, the hepatitis C treatment and prevention composition of the present invention will be described in detail with reference to the accompanying table or drawings.

When the drawings are described, they are provided as examples in order to ensure that features of the present invention to those skilled in the art will fully convey. Accordingly, the present invention is not limited to the drawings presented, but may be embodied in other forms, and the drawings may be exaggerated for clarity.

At this time, if there is no other definition in the technical terms and scientific terms used, it has the meaning that is commonly understood by those of ordinary skill in the art to which the present invention belongs, the gist of the present invention in the following description and the accompanying drawings Descriptions of well-known functions and configurations that may be unnecessarily blurred are omitted.

In the present invention, "sample", "sample", or "sample" refers to a subject for analysis, and is used in the same sense throughout the specification.

The term "alkenyl" of the present invention, independently or identically, refers to alkenyl radicals having 2 to 9 carbon atoms, preferably linear alkenyl radicals having 2 to 7 carbon atoms, but is not limited thereto. .

The term "halo" or "halogen" in the present invention means fluorine, chlorine, bromine or iodine atoms.

Hereinafter, the 2-O-methylhonokiol compound, prodrug thereof, solvate thereof, stereoisomer thereof or pharmaceutically acceptable salt thereof of the present invention will be described in detail.

The present invention provides a 2-O-methylhonokiol derivative compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Chemical Formula 1,

R 1 and R 2 are, independently from each other, hydrogen, (C 2 -C 7) alkenyl;

R 3 and R 4 are independently of each other hydrogen or halogen;

m and n are each independently an integer of 1 to 3;

2-O-methylhonokiol derivative compound according to an embodiment of the present invention may be represented by the following formula (2).

[Formula 2]

In Chemical Formula 2,

R 3 and R 4 are the same as defined in formula 1 of claim 1;

m and n are each independently an integer of 1 to 2;

In the 2-O-methylhonokiol derivative compound according to an embodiment of the present invention, the 2-O-methylhonokiol derivative compound may specifically have the following structure, but is not limited thereto.

The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases mediated by hepatitis C virus, including the 2-O-methylhonokiol derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a prophylaxis or treatment of a disease mediated by hepatitis C virus comprising the 2-O-methyl-honokiol derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. It provides a pharmaceutical composition.

The present invention also provides a molding agent comprising the pharmaceutical composition.

As described above, since the 2-O-methylhonokiol derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof shows high replication inhibitory activity against hepatitis C virus, the pharmaceutical composition comprising them as an active ingredient It can be usefully used for the treatment or prevention of diseases mediated by hepatitis C virus, such as hepatitis C.

The 2-O-methylhonokiol derivative compound according to the present invention may be used in the form of a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt may be prepared by a conventional method in the art. For example, salts with inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, nitric acid, carbonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, Salts with organic acids such as mandelic acid, cinnamic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid tartaric acid, gluconic acid, lactic acid, fumaric acid, lactobionic acid, ascorbic acid, salicylic acid, or acetylsalicylic acid (aspirin), glycine, Salts with amino acids such as alanine, vanillin, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, proline, methanesulfone Include, ethanesulfonic acid, benzenesulfonic acid, a salt of sulfonic acids such as toluene sulfonic acid, sodium salt by reaction with an alkali metal such as potassium, etc. or a salt with an ammonium ion.

The 2-O-methylhonokiol derivative compounds of the present invention may exist in solvated forms, for example hydrated and unsolvated forms, and solvates of the 2-O-methylhonokiol derivative compounds according to the invention It includes all solvated forms having pharmaceutical activity.

The 2-O-methylhonokiol derivative compounds of the present invention may have a chiral center and may exist as racemates, racemic mixtures and as individual enantiomers or diastereomers. Such isomers may be separated or resolved by conventional methods and any given isomer may be obtained by conventional synthesis or by stereospecific or asymmetric synthesis. All such isomeric forms and mixtures thereof are included within the scope of the present invention.

The 2-O-methylhonokiol derivative compounds of the present invention can be administered in the form of prodrugs that degrade in the human or animal body to provide the compounds of the present invention. Prodrugs can be used to alter or improve the physical and / or pharmacokinetic profile of the parent compound and can be formed if the parent compound contains suitable groups or substituents that can be induced to form the prodrug.

In addition, the pharmaceutical composition of the present invention is a non-toxic pharmaceutical agent conventional in the 2-O-methyl-honokiol derivative compound represented by the formula (1), its prodrug, solvate, stereoisomer or pharmaceutically acceptable salt thereof Oral administration such as tablets, pills, hard capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc., which are customary in the pharmaceutical field, with the addition of acceptable carriers and excipients. It may be formulated into a formulation or parenteral formulation of sterile aqueous or oily solvent for intravenous, subcutaneous, sublingual, intramuscular administration.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and / or mineral oil, It is not limited to this.

Excipients that may be used in the pharmaceutical compositions of the present invention include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances, and the like. The proportion and nature of the excipient can be determined by the solubility and chemical properties of the selected tablet, the chosen route of administration and standard pharmaceutical practice. Examples of excipients include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, sterin, magnesium stearate, magnesium aluminum silicate, starch, gelatin, tragacanth rubber, alginine acid, Sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor and the like.

In addition, the pharmaceutical composition of the present invention may be formulated in a parenteral dosage form, in which case, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration may be used, but is not limited thereto. At this time, in order to formulate the formulation for parenteral administration, the pharmaceutical composition is an active ingredient, that is, a 2-O-methylhonokio derivative compound of Formula 1, a prodrug thereof, a solvate thereof, a stereoisomer or a drug thereof. Pharmaceutically acceptable salts thereof can be mixed in water with stabilizers or buffers to form solutions or suspensions, which solutions or suspensions can be prepared in unit dosage forms of ampoules or vials.

In addition, the pharmaceutical composition of the present invention may be sterile, or may further include adjuvants such as preservatives, stabilizers, hydrating or emulsifiers, salts and / or buffers for controlling osmotic pressure, and further include other therapeutically useful substances. Or may be formulated according to conventional methods of mixing, granulating or coating.

In addition, the 2-O-methylhonokiol derivative compound represented by Formula 1, which is an active ingredient in the pharmaceutical composition according to the present invention, prodrug thereof, solvate thereof, stereoisomer or pharmaceutically acceptable salt thereof may be used in humans. Dosages for mammals can vary depending on the age, weight, sex, dosage form, health condition and disease level of the patient. Generally, the pharmaceutical composition may be included in the pharmaceutical composition in an effective amount of 0.001 to 500 mg / kg body weight, preferably 0.01 to 100 mg / kg body weight, and such pharmaceutical composition may be once or twice a day. The biphasic division may be administered via oral or parenteral routes. However, since the dose may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. The examples are only for more specifically describing the present invention, and the scope of the present invention is not limited thereto.

[Experimental Material]

[How to measure]

1. Virus growth inhibition test method-RNA synthesis inhibition: luciferase assay

Hepatitis C virus replication unit cells (HCV replicon cells) using subgenomic-selectable-APP203 CON1 / SG-Neo (1) consisting of the hepatitis C virus genotype 1b replication unit genome.

Substances to be measured were added to each well by concentration in ConI cells containing luciferase reporter plasmid. Thereafter, the reporter gene was analyzed after incubating in 37 ° C., 5%, CO ₂ environment for 24 hours to sufficiently expose the test substance.

The analytical results are shown by standardizing Firefly luciferase activity relative to Renilla luciferase activity. Promoter activity of cells expressing only the reporter and the Renilla plasmid was measured as a control (Con).

2. Cytotoxicity measurement: MTS assay-Promega cell Proliferation assay manual

After dispensing cells at 5 x 10 ⁴ cells / well per well in a 24 well plate, a substance to be measured was added to each well by concentration. Thereafter, the test material was incubated in a 37 ° C., 5%, CO ₂ environment for 24 hours to sufficiently expose the test material.

After removal of the supernatant, 100 uL of cell culture was mixed with 20 uL of MTS solution, and the mixture was aliquoted in 100 uL of each well. Then, after incubation at 37 ℃, 5%, CO ₂ in about 2 hours (0.5 ~ 4 hours), after shaking the plate sufficiently, the absorbance was measured at 490 nm.

3. Measurement of the inhibitory effect of reactive oxygen species increase: flow cytometry

The cells were treated with 50 μM of 155 (2-O-methylhonokiol) and PCTC for 24 hours each, stained at 37 ° C. for 30 minutes using 50 μM DCF-DA to measure free radicals, and then cells were treated with PBS. Washed three times.

DCF-DA freely crosses the cell membrane and is broken down into DCF by intracellular esterase. Thus, DCF fluorescence value refers to the level of free radicals (ROS) in cells. Accumulation of DCF was measured with a reader at absorbance 490 nm and reference absorbance 690 nm. Flow cytometry was observed and analyzed with Beckman Cytomics FC500 flow cytometer.

4. Measurement of protein expression: Western blot measurement

After treatment with reagent or siRNA by concentration and time, the cells were collected using proteolysis buffer (50mM Tris-Cl, 150nM NaCl, 5mM EDTA, 0.1% NP40, protease inhibitor), and 20 minutes at 14000rpm. Centrifuge. Then, SDS-PAGE is performed using the supernatant, and the protein is transferred to nitrocellulose membrane for western blot. And it was observed using a protein specific antibody (AhR, CYP1A1, Actin).

5. Measurement of mRNA changes in cells-Real time PCR

Cells were treated with 50 μM of 155 (2-O-methylhonokiol) and incubated in 37 ° C., 5%, CO ₂ environment for 24 hours. After washing the cells with PBS, RNA was extracted using Trizol. CDNA was synthesized using cDNA Synthesis kit in the amount of 1.0ug RNA. Real time PCR primers are as follows. IFN-α, forward: TTTCTCCTGCCTGAAGGACAG and reverse: GCTCATGATTTCTGCTCTGACA, ACTIN, forward: CATCGAGCACGGCATCGTCA and reverse: TCGAAGTCCAGGGCGACATA, CYP1A1, forward: CACTGCCAACACCTCTGTCTT and reverse: CAAGGAGCTCCATGGCT 1uL of each primer was added, 12.5uL of Power SYBR ^® Green PCR Master Mix and 2uL of template DNA were added, and sterile distilled water was added so that the total reaction solution was 25uL. Genetic amplifier (Realtime PCR) was used for 7500 Real-Time PCR system, 40 seconds at 95 ℃, 40 seconds at 55 ℃, 1 minute cycle was repeated 40 times at 70 ℃.

6. Method of measuring mRNA and protein changes using siRNA

Small interfering RNA (siRNA) was transformed into cells at a concentration of 100 nM, and then the protein expression level measurement method and the intracellular mRNA change experiment were performed.

Example 1 Measurement of Luciferase Activity

For four compounds predicted as antioxidants, compounds 97, 155, 230 and 399 were prepared at concentrations of 0, 25, 50 and 100 uM to determine HCV replication inhibition. Luciferase activity was measured accordingly.

The results are shown in FIG.

From this, luciferase activity was significantly reduced when Compound 155 was treated at 25 uM or more, and when treated at 100 uM concentration, almost no luciferase activity was observed, and HCV replication was almost suppressed.

On the other hand, for compounds 97, 230 and 399, luciferase activity was increased depending on the treatment concentration, or even 50 μM or more treatment showed no decrease in activity as compared to no treatment, it was confirmed that there is no inhibitory effect on HCV replication.

Example 2 Cytotoxicity Measurement

In Example 1, the luciferase activity was significantly reduced, and Compound 155 (2-O-methylhonokiol), which was found to have a significant HCV replication inhibitory effect, a positive control honokiol (Compound 147) having HCV replication inhibitory effect, and its For the derivatives 614 and 615, cytotoxicity was measured according to the MTS test method.

The results are shown in FIG.

This resulted in high cell survival except when ConI cells were treated with Compound 155 at high concentrations, whereas Compound 147, 614 and 615 showed a sharp decrease in cell survival when treated at concentrations of 50 uM or higher, resulting in nearly 100 uM. It did not survive, it was confirmed to exhibit a strong toxicity to the cells.

From this, it was confirmed that the compound 155 may be utilized as a pharmaceutical substance for inhibiting HCV replication.

Example 3 Determination of Reactive Oxygen Species Effect

The flow cytometry method was used to determine the inhibitory effect on increasing free radical species (ROS) upon HCV infection. The experimental group treated Compound 155 with a concentration of 50 uM, and compared the results by treating pyrrolidine dithiocarbamate (PDTC), which is known to have HCV replication effect, as a control, at a concentration of 0.3 uM. As a negative control, a sample without any reagent was used.

As a result, it was confirmed that the compound 155 exhibits a significantly superior active oxygen species removal effect compared to the PDTC which is a positive control.

Example 4 HCV Replication Inhibition Mechanism

Upon treatment of compound 155 CYP1A1  Of enzyme protein expression AhR  Dependencies

To confirm the HCV inhibition mechanism of Compound 155, ConI cells were treated with Compound 155 to analyze the expression levels of AhR (aryl hydrocarbon receptor) protein and CYP1A1 (Cytochrome P450 1A1) enzyme, which is a target gene of AhR gene, through SDS-PAGE.

The results are shown in FIG.

From this, it was confirmed that the amount of expression of AhR protein significantly reduced after Compound 155 treatment, from which it was again confirmed that Compound 155 is a substance that does not cause toxicity to cells.

On the other hand, the amount of CYP1A1 protein induced by AhR expression was found to increase, from which siRNA analysis was performed to determine whether the induction of CYP1A1 is caused by the expression of the AhR gene.

As a result of measuring mRNA expression of CYP1A1 after siAhR treatment, it was confirmed that induction of CYP1A1 upon compound 155 treatment was independent of the AhR gene (FIGS. 3B and 3C).

Upon treatment of compound 155 IFN of -α expression AhR  Dependencies

In order to determine the dependence of AhR on the treatment of Compound 155 and IFN-α expression, IFN-α mRNA expression levels were measured by treating Compound 155 with ConI cells.

The result is shown in FIG.

From this, it was confirmed that the expression level of the IFN-α gene was significantly increased by 7 times or more after the compound 155 treatment, and inhibited the HCV replication by inhibiting the AhR signal in the cell after the compound treatment, thereby increasing the IFN-α expression. It was newly confirmed.

Example 5 Comparison of HCV Replication Inhibitory Activity of Structural Isomers of Compound 155

Compound 155, Compound 156, and Compound 147 (Honokiol) were each 0, 50, for comparison of the HCV replication inhibitory activity of the 2-O-methylhonokiol with the structural isomer of 4-O-methylhonokiol (Compound 156). And after 100 uM treatment, the luciferase test and cytotoxicity were measured in the same manner as in Examples 1 and 2.

The results are shown in FIG.

From this, when the compound 155 of the present invention is treated with 50 uM, it was confirmed that there is a significant difference in effect because it is three times more efficient than the compound 156 which is a structural isomer.

The survival rate of the cells at 50 uM, which could confirm the effect of inhibiting HCV replication, showed similar survival rates between the two isomers. It was confirmed.

From the above results, even if it is a structural isomer having the same chemical formula, a new result was confirmed that the compound 155 of the present invention has a low cytotoxicity with a significantly superior HCV inhibitory effect compared to the compound 156, and completed the present invention.

Claims (6)

A pharmaceutical composition for preventing or treating hepatitis C, which comprises a 2-O-methylhonokiol derivative compound represented by Formula 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. .
[Formula 2]

In Chemical Formula 2,
R 3 and R 4 are independently of each other hydrogen or halogen;
m and n are each independently an integer of 1 to 2; delete The method of claim 1,
The 2-O-methyl-honokiol derivative compound is characterized in that hepatitis C, characterized in that it comprises a 2-O-methyl-honokino derivative compound having a structure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier Pharmaceutical composition for the prophylaxis or treatment of.

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