KR102132904B1 - Use of ciclopirox to inhibit HBV core assembly - Google Patents

Abstract

The present invention is an anti-HBV (Hepatitis B Virus) composition comprising a cyclopirox or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for the prevention or treatment of HBV virus-induced disease, comprising cyclopyrox or a pharmaceutically acceptable salt thereof; A method for treating HBV virus-induced disease, comprising administering the pharmaceutical composition to an individual; And cyclopyrox or a physiologically acceptable salt thereof, to a health functional food composition for preventing or improving HBV virus-induced disease.
The present invention not only overcomes the problem of not being able to remove cccDNA with monotherapy of previously used drugs, by newly identifying the HBV inhibitory capacity of cyclopyrox, as well as effectively removing HBV by preventing core assembly during the life cycle of the virus It can provide a treatment that can. Furthermore, this can socially reduce the incidence of diseases such as chronic hepatitis B, cirrhosis and hepatocellular carcinoma.

Description

Use of ciclopirox to inhibit HBV core assembly}

The present invention has elucidated the novel drug effects of cyclopyrox, specifically, an anti-HBV (Hepatitis B Virus) composition comprising cyclopirox or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for the prevention or treatment of HBV virus-induced disease, comprising cyclopyrox or a pharmaceutically acceptable salt thereof; A method for treating HBV virus-induced disease, comprising administering the pharmaceutical composition to an individual; And cyclopyrox or a physiologically acceptable salt thereof, to a health functional food composition for preventing or improving HBV virus-induced disease.

Hepatitis B Virus (HBV) infection is a very important health problem because it is not only common in the world, but also 6-10% is likely to develop into chronic liver disease such as cirrhosis or hepatocellular carcinoma. The incidence of hepatocellular carcinoma in Korea is 22.2 per 100,000 people (36.0 men, 10.2 women), and the mortality rate from hepatocellular incidence is 15.4 per 100,000 population (25.8 men, 6.6 women) (Disease Management Headquarters).

In the initial Interferon (IFN) treatment to suppress HBV causing this disease, HBV was treated by activating cytotoxic T lymphocytes by increasing the immune response. It showed a high response rate in case of short prevalence or high serum aminotransferase value and low hepatitis B virus DNA. In addition, HBV replication can be suppressed through transcriptional inhibition of covalently closed circular DNA (cccDNA) by covalent bonding, and NK cell activation can be regulated. However, there is a problem that serious side effects such as fever, chills, general weakness, depression, congestive heart failure, and neutropenia occur.

Lamivudine (3-TC), developed as an AIDS treatment to compensate for the disadvantages of interferon, has been found to be effective in treating HBV. However, although lamivudine is an effective agent for the treatment of chronic hepatitis B, there is a problem that lamivudine-resistant HBV appears when used for a long time.

In the past 10 years, entecavir (ETV) or tenofovir (TDF), which has a high anti-viral effect and has little resistance in the treatment of hepatitis B, has been used. Oral antiviral drugs that block the reverse transcription process of these viruses showed improved treatment performance with fewer side effects. However, these drugs are nucleotide analogs, which inhibit the replication process of the virus, but cannot remove the virus completely and cannot remove cccDNA in the nucleus, so long-term treatment using it is impossible. Therefore, a new therapeutic agent capable of completely removing HBV is needed.

Meanwhile, HBV is a double-stranded DNA virus, and upon infection, RNA polymerase generates polymerase, coat protein, coat protein, and HBx protein from the DNA template. 200~300 new hepatitis B viruses are produced by the genome by infection of one cell, and a large amount of viruses are produced and released based on this. HBx is known as a representative pathogenic protein and does not directly bind to DNA, but is known to affect the role of a transactivator, as well as interactions with proteins associated with immune responses and various signaling in cells.

Cyclopyrox is known as a hydroxypyridinone antifungal agent and is being studied as a treatment for seborrhoeic dermatitis, but its effectiveness in HBV treatment is not known at all.

Under these backgrounds, the present inventors have completed the present invention by newly confirming the HBV inhibitory capacity of cyclopyrox as a result of earnest efforts to solve the above problems.

One object of the present invention is to provide an anti-Hepatitis B Virus (HBV) composition comprising cyclopirox or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of HBV virus-induced disease, comprising cyclopyrox or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for treating HBV virus-induced disease, comprising administering the pharmaceutical composition to an individual other than a human.

Another object of the present invention is to provide a health functional food composition for preventing or improving HBV virus-induced disease, comprising cyclopyrox or a physiologically acceptable salt thereof.

Specifically, it is as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is to provide an anti-HBV (Hepatitis B Virus) composition comprising a cyclopirox or a pharmaceutically acceptable salt thereof.

The cyclopyrox is a compound represented by the following Chemical Formula 1, and is known as an antifungal agent, and is being studied as a treatment for seborrhoeic dermatitis, but there is no known effect of its HBV treatment.

[Formula 1]

In the present invention, it was newly confirmed that cyclopyrox specifically inhibits the core assembly process during the HBV life cycle. Specifically, the present inventors tried to solve the disadvantages that the existing drugs, such as Entecavir and Tenofovir, cannot remove the cccDNA of the HBV virus, and as a result, cyclopyrox effectively removes the cccDNA and inhibits HBV core assembly. It was newly confirmed that it is possible.

Specifically, it was confirmed that cyclopyrox inhibits the assembly of the core protein in the assembly environment of the purified HBV core protein, and it was confirmed that cyclopyrox inhibits its assembly even in cells overexpressing the entire core or HBV DNA. In addition, it was confirmed that when the core protein purified according to the sucrose concentration gradient was separated according to the shape, the core assembled by cyclopyrox decreased and the core in the form of heteroforms increased (FIG. 2 ).

More specifically, as a result of analyzing the structure of the HBV core protein binding to cyclopyrox, it was confirmed that cyclopyrox is binding to the core protein, and particularly, the tyrosine 118 site was proved to be important for binding (FIG. 3). . In addition, it was confirmed from this that cyclopyrox directly binds to the HBV core protein and inhibits core assembly.

The cyclopyrox of the present invention shows that in the cell line expressing HBV and optionally the cell line expressing HBV, HBV is reduced without affecting cell survival according to the concentration gradient of drug treatment (FIG. 5). In addition, it was confirmed that when the cyclopyrox treatment was performed on the cell line expressing HBV, not only the DNA discharged outside but also the DNA inside was reduced according to the drug concentration gradient (FIG. 6).

The cyclopyrox may inhibit the assembly of the HBV core protein, and more specifically, may inhibit core assembly by binding to a tyrosine residue (tyrosine 118) and a tryptophan residue (tryptophan 102), which are essential for the core assembly step.

"Anti-HBV" used in the present invention means an action of specifically inhibiting the proliferation of HBV virus by specifically inhibiting the cytopathic effect by HBV virus.

In the present invention, hepatitis B virus (HBV), that is, hepatitis B virus, is a DNA virus that causes hepatitis B, and is also called HBs. Hepatitis B virus contains DNA, DNA polymerase, HBc antigen, and HBe antigen in the core.

The anti-HBV composition may further include entecavir, tenofovir, or a combination thereof.

It was confirmed that the cyclopyrox of the present invention showed a synergistic effect when compared with Entecavir or Tenofovir compared to the single treatment, and also reduces the DNA inside, as well as the DNA discharged out according to the concentration gradient of the cyclopyrox drug ( Fig. 7).

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating HBV virus-induced disease, comprising cyclopyrox or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a method of treating HBV virus-induced disease, comprising administering the pharmaceutical composition to an individual.

The cyclopyrox, pharmaceutically acceptable salt, and HBV virus are as described above.

In the present invention, HBV virus-induced disease refers to a disease that may occur in vivo due to HBV infection, and may be, but is not limited to, hepatitis, cirrhosis and hepatocellular carcinoma, or a combination thereof.

The pharmaceutical composition may further include Entecavir, Theophore, or a combination thereof.

The term'prevention' as used in the present invention means all actions to suppress or delay HBV virus infection disease by administration of the composition of the present invention. In addition, the term'treatment' used in the present invention means any action in which symptoms of HBV virus-induced disease are improved or beneficially changed by administration of the composition.

As used herein, the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to an individual in any suitable way, the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.

The term'individual' used in the present invention means all animals including humans who have or may have HBV virus-infected disease, and by administering the composition of the present invention to an individual, the disease can be effectively prevented and treated. .

The composition of the present invention is administered in a pharmaceutically effective amount. The term'pharmaceutically effective amount' used in the present invention refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment in medical treatment, and the effective dose level is the individual type and severity, age, It can be determined by gender, the type of viral infection disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of discharge, the duration of treatment, factors including the drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredients described above. The carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, or sterile injectable solution, respectively, according to a conventional method. have. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weights, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. In addition to liquids for oral administration and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be added. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage may be adjusted according to the patient's condition, weight, and disease. Depending on the degree, the type of drug, the route of administration, and the time, generally 50 mg/kg per day, preferably 20 to 100 mg/kg is administered, and at regular intervals according to the judgment of the doctor or pharmacist It may be administered several times a day, preferably 1 to 4 times, which may be appropriately selected by those skilled in the art.

Another aspect of the present invention provides a health functional food composition for preventing or improving HBV virus-induced disease, comprising cyclopyrox or a physiologically acceptable salt thereof.

The cyclopyrox, salt, and HBV virus are as described above.

Cyclopyrox or a physiologically acceptable salt thereof can be added to the dietary supplement composition for the purpose of preventing or improving HBV virus infection. When the above ingredients are used as a dietary supplement, it may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

As used herein, the term "improvement" can mean any action that at least reduces the parameters associated with the condition being treated, such as the severity of symptoms.

The term "health functional food" as used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills, etc. using ingredients or ingredients having useful functionality for the human body. Here, the term "functionality" means obtaining a useful effect for health use, such as adjusting nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method conventionally used in the art, and may be prepared by adding raw materials and ingredients conventionally added in the art. In addition, unlike general medicines, it has the advantage that there is no side effect that can occur when taking medicines for a long time using food as a raw material, and it can be excellent in portability.

When the composition of the present invention is used in a health functional food, the composition may be added as it is or used together with other health functional food or health functional food components, and may be suitably used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the composition of the present invention in the production of food is added in an amount of 1 to 10% by weight, specifically 5 to 10% by weight of the raw material composition. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be used below the above range.

The health functional food composition may further include Entecavir, Theophore, or a combination thereof.

The present invention not only overcomes the problem of not being able to remove cccDNA with monotherapy of previously used drugs, by newly identifying the HBV inhibitory capacity of cyclopyrox, a drug with guaranteed stability, as well as core assembly during the life cycle of the virus Can provide a therapeutic agent that can effectively remove HBV. Furthermore, this can socially reduce the incidence of diseases such as chronic hepatitis B, cirrhosis and hepatocellular carcinoma.

1 is a view showing a process for identifying the HBV inhibitory capacity of cyclopyrox, A of FIG. 1 is a schematic diagram for screening a drug having HBV inhibitory ability in about 1000 drugs; 1B is a graph showing HBV inhibitory capacity of 19 drugs selected through the first screening; 1C is a graph showing the HBV transcript expression levels of the 19 drugs; And D of FIG. 1 is a graph showing the inhibitory ability of 19 drugs to express core and capsid proteins. The results were expressed as mean±standard deviation, and through the Student's t test, * has a confidence value of p <0.05 and ** of p <0.01.
2 is a view showing the ability of cyclopyrox to inhibit core assembly, and FIG. 2A is an immunoblotting image showing the ability of cyclopyrox to inhibit core assembly; C and D of Figure 2 shows the core protein inhibiting ability of cyclopyrox in the case of expressing the core protein in the hepatocyte cell line and treating cyclopyrox. Specifically, the amount of the core protein reduced through the SDS Page does not change, but the native gel probing the assembled core protein shows that the assembled core protein is significantly reduced. FIG. 2B shows that core assembly is reduced in a sucrose concentration gradient using a core protein purified in the same manner as in FIG. 2A.
Specifically, B of FIG. 2 makes a concentration gradient of 10% to 50% sucrose by using a point in which the core protein position varies according to the sucrose concentration gradient after core assembly, and the drug-treated core protein is combined with the assembly reaction. It shows that the amount of protein in the form of a non-assembled dimer increased significantly with the decrease of the assembled core protein when separated by centrifugation. C of FIG. 2 shows the core protein of HBV, and FIG. 2D shows the ability to inhibit core assembly by cyclopyrox when the entire protein of HBV is expressed. This also shows that the amount of individual core proteins is unchanged, but the core assembled by cyclopyrox is significantly reduced. 2E shows electron microscopic observations, showing that the assembled core size is increased by cyclopyrox and the prototype is destroyed. In short, Figure 2 shows that cyclopyrox shows HBV inhibitory ability by inhibiting the combination of HBV core proteins.
Figure 3 shows the results of the analysis of the position where the cyclopyrox is bound to the HBV core protein. Specifically, FIG. 3A shows the overall structure of an asymmetric unit hexagon. The secondary structure of the protein was calculated using STRIDE, which shows that cyclopyrox is bound to the core protein by being expressed in a space-filling model. B and C of FIG. 3 indicate sites where the cyclopyrox and HBV core proteins bind, and particularly, the hydrogen bond at the tyrosine (Y) 118 site is important. 3D shows that the inhibition of core assembly by cyclopyrox is actually reduced through mutation of the Y118 site. E and F in Fig. 3 show a comparison between the positions of the cyclopyrox bonds and non-existent positions of the chains B and C.
Figure 4 shows the results of quantifying the amount of HbsAg protein discharged out of each cell line expressing HBV, and the cell line over-expressing HBV. Specifically, A and B of Figure 4 shows an enzyme-linked immunoprecipitation assay for changes in the amount of HBsAg that is released out during cyclopyrox treatment. As a result, it was confirmed that HBsAg according to the cyclopyrox concentration gradient was not significantly changed. The results were expressed as mean±standard deviation, and through the Student's t test, * has a confidence value of p <0.05 and ** of p <0.01.
5 is a view showing the results of confirming the HBV inhibitory ability according to the concentration gradient of cyclopyrox, it was confirmed using 7 concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10 mM, respectively.
Specifically, A and B of FIG. 5 confirmed the reduction of HBV DNA by concentration gradient in hepatocytes expressing HepG2.2.15 and HBV after treating cyclopyrox for 6 days, in the same manner as the screening method in which HBV inhibitory ability was identified. Results are shown. C and D of FIG. 5 show the result of confirming the ability to inhibit cyclopyrox by extracting HBV DNA in cells, not DNA that is discharged out. Specifically, hepG2.2.15 and HBV-expressing hepatocytes were lysed, respectively, and then the capsidized RNA was removed using micrococcal nuclease, and viral DNA was extracted to confirm the expression level. As a result, it was confirmed that HBV DNA decreased according to the concentration gradient of cyclopyrox.
6 shows the HBV inhibitory ability of cyclopyrox treated after infection of HBV by constructing an HBV infection system. 6A shows the flow for the HBV infection system. Specifically, after treating cyclopyrox for 6 hours in HepG2 and Huh7 cell lines expressing receptors (NTCP) essential for HBV infection, the supernatant of HepG2.2.15 is used to treat cyclopyrox again with viral infection. . After 16 hours, the virus was washed, and after 14 days of cyclopyrox treatment, the cells and the supernatant were collected and analyzed. B and C of FIG. 6 show the results of NTCP expression of the NTCP cell line that enables HBV to be infected using an immunoblot method and a flow cytometer. 6D to F show the HBV inhibitory capacity of cyclopyrox in a virus infection system. Specifically, D of FIG. 6 shows that the HBV DNA discharged from both cell lines is significantly reduced according to the cyclopyrox concentration. E of FIG. 6 shows that cccDNA from which rcDNA present in the cell was removed was extracted to confirm the expression level. As a result, the expression level was significantly decreased according to the concentration of cyclopyrox in both cell lines. F of FIG. 6 shows that the expression amount of rcDNA present in the cell is significantly reduced according to the concentration of cyclopyrox in two cell lines.
FIG. 7 shows the possibility of combined treatment of cyclopyrox, and shows that cyclopyrox treated with entecavir (ETV) and tenofovir (TDF) shows a synergistic effect on HBV inhibitory ability. After treatment according to the cyclopyrox concentration gradient, ETV and TDF were treated in combination with 1 mM.
Specifically, A of FIG. 7 quantified HBV DNA discharged after treating the drug according to conditions for 6 days. As a result, it shows that HBV DNA was significantly reduced when ETV and TDF were used together than when cyclopyrox was treated alone within the logarithmic range. B of FIG. 7 shows the results obtained by extracting and quantifying HBV DNA present in cells under the same conditions, and the HBV DNA was significantly reduced when ETV and TDF were used in combination than when cyclopyrox was treated alone within the logarithmic range. Shows. 7C shows the result of measuring the amount of HBsAg protein discharged out, and shows that there is no HBsAg inhibitory ability when cyclopyrox is treated alone or when ETV and TDF are used together.
8 shows HBV inhibitory ability when cyclopyrox is injected into mice expressing HBV. 8A shows the process of expressing HBV in mice and treating cyclopyrox, TDF or combination of cyclopyrox and TDF daily for 5 days. Specifically, the pAAV HBV 1.2x plasmid expressing HBV was inserted into the tail of the mouse by hydrodynamic injection to express HBV in the mouse, and the drug was treated daily for 5 days. 8B shows that when the expression levels of HBV core protein and surface protein in hepatocytes after drug treatment were confirmed, the amount of HBV core protein was decreased by cyclopyrox, and more effectively decreased when combined with TDF. 8C shows that HBV DNA contained in blood was quantified from blood collected after drug treatment, and HBV DNA was reduced by cyclopyrox and decreased more effectively when combined with TDF. 8D shows the result of quantifying the amount of HBsAg protein contained in blood from blood collected after drug treatment.
9 shows MTS results for confirming cell survival by cyclopyrox. As a result, it was shown that cell survival by cyclopyrox treatment was not affected in HBV-expressing cell lines HepG2.2.15 and HBV-expressing hepatocyte cells.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental Example 1. Cell culture method

Overexpression of HegG2, HepG2.2.15, Huh7, NTCP HepG2, Huh7 cells were cultured in a medium at 37° C. with DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum) and 1% antibiotic.

Experimental Example 2. Manufacturing method of plasmid

HBV1.2×adr subtype ORF (Open Reading Frame) was prepared in pUC19, and Myc marker-CP149 in pCDNA3. ORF (Open Reading Frame) was prepared. An HBV1.2×adr subtype ORF (Open Reading Frame) was prepared in pAAV for mouse hydrodynamic injection.

Experimental Example 3. Quantification method of virus discharged out

Cells were treated with drugs such as cyclopirox, entecavir and/or tenofovir, and then the supernatants of the cells were collected. 1X PBS was added at the same volume to 30 ul of the supernatant, 6 ul of 1N NaOH was added, and after 1 hour of reaction at 37° C., 6 ul of Tris-HCl/HCl was added. Thereafter, the protein was denatured by heat treatment at 98°C for 5 minutes. This was centrifuged to remove the denatured protein, and the virus present in the supernatant was quantified through real-time PCR.

Experimental Example 4. Quantification method of virus present inside

Cells were treated with drugs such as cyclopyrox, entecavir and/or tenofovir, and then the cells were washed with 1X PBS. Then, after lysing the cells, nuclease was treated, and then HBV DNA was extracted from the cells. For this, DNA was extracted according to the manufacturer's instructions.

Experimental Example 5. Analysis method of HBsAg protein

The secreted HBV HBsAg protein was analyzed using an enzyme-linked immunoprecipitation assay. Specifically, HBsAg specific ELISA was used. The supernatant was collected from cells treated with each drug and analyzed using a kit (Hepatitis B surface antigen ab ELISA kit), which was analyzed according to the manufacturer's instructions (abnova).

Experimental Example 6. HBV capsid protein detection method

HBV capsid protein was detected using agarose gel electrophoresis. Specifically, the isolated Cp149 dimer protein was reacted with a core assembly reaction buffer 150mM NaCl and 15mM HEPES at 37°C for 1 hour to separate the protein using an agarose gel, and rabbit polyclonal anti-HBV Immunoblotting was performed using a core polyclonal antiHBV core antibody. In addition, in order to separate the core expressed in the cells, cells were lysed with 1% NP-40 and ultracentrifugation (55000rpm) was performed at 20°C for 8 hours, and then the assembled core that had sunk to the bottom was agarose gel. After separation by using a rabbit polyclonal anti-HBV core antibody was performed by immunoblot.

Example 1. Confirmation of HBV inhibitory activity of cyclopirox

To screen for drugs with inhibitory activity against HBV, an FDA approved drug library with guaranteed stability was used.

Specifically, the first screening was performed by measuring the amount of HBV DNA released outside after treating about 1000 drugs daily at 1 mM for 3 days in the HepG2.2.15 cell line producing HBV, and the selected 19 drugs for 6 days After treatment, 13 drugs were selected through a second screening to measure HBV DNA in the same manner as described above. At this time, Entecavir used as an HBV drug was used as a positive control.

As a result, as can be seen in A and B of Figure 1, 19 drugs were selected that inhibit the release of HBV DNA more effectively than Entecavir, 13 of them (#1, #3, #4, #5, # It was confirmed that 6, #7, #10, #11, #12, #13, #16, #17, #18, #19) continued to be effective for a long period of 6 days.

In addition, as can be seen in FIG. 1C, among the 19 drugs, 4 drugs (#6, #12, #16, #19) were confirmed to decrease the transcript expression of HBV.

On the other hand, as can be seen in D of FIG. 1, it was confirmed that one drug (#7) among the 19 drugs does not affect core protein expression, but significantly inhibits the expression of capsid protein, which is cyclopyrox. Confirmed.

Through the above results, cyclopyrox does not affect the transcript of HBV occurring in the RNA stage during HBV life cycle, and thus does not affect core protein expression, but is finally discharged by inhibiting the assembly of capsid protein, which is a subsequent process. It can be seen that it can be used as a drug showing anti-HBV effect by reducing viral DNA.

Example 2. Identification of the mechanism of HBV inhibitory activity of cyclopyrox

Through the above Example 1, it was confirmed that cyclopyrox has HBV inhibitory activity, and thus, it was intended to identify its inhibitory mechanism. That is, although it did not affect the expression of the core protein, it was confirmed that the expression of the capsid protein is significantly inhibited.

Specifically, after the cyclopyrox was treated on the purified core protein 149 (CP149) variant, the core protein overexpressing cell line, and the whole protein overexpressing cell line of HBV, the assembly reaction of the core was analyzed. As a result, as shown in A to C of FIG. 2, as the concentration of cyclopyrox increases, the assembly of the purified core protein 149 (CP149) heteroforms is inhibited, and the core assembly is also inhibited according to the concentration gradient in each cell line. Confirmed.

In addition, after assembling the CP149 under the assembly reaction conditions to confirm whether the core assembly is actually inhibited and remains as a heteroform, the reactants were separated by sucrose concentration through ultracentrifugation. As a result, as shown in D of FIG. 2, when the cyclopyrox was not treated, the heterozygous CP149 decreased (fraction number 1-3), and the core-assembled CP149 showed a tendency to increase (fraction number 5- 8), it was confirmed that when the cyclopyrox is treated, the heterozygous CP149 increases and the core-assembled CP149 decreases.

Furthermore, as can be seen from A and B of FIG. 4, it was confirmed that both the cell line expressing HBV and the cell line overexpressing HBV had no effect on the amount of HBsAg protein expressed and discharged.

Through the above results, cyclopyrox inhibits only core assembly without affecting the core protein expression of HBV, and inhibition of the core assembly step is currently used as a target of a viral inhibitor. -It was found that it can be used as a drug exhibiting HBV effect.

Example 3. Identification of HBV core protein binding site of cyclopyrox

Through Example 2, it was confirmed that cyclopyrox exhibits an inhibitory effect on HBV proliferation in vitro by inhibiting HBV core assembly, and confirms the location of the cyclopyrox binding in the HBV core protein. Specifically, to confirm this, a core protein crystal was generated, and structural analysis with cyclopyrox was performed.

As a result, as can be seen in Figure 3, the binding site of the cyclopyrox to the hexamer-formed HBV core protein was confirmed. In particular, the tyrosine 118 site is important for hydrogen bonding between the drug and the core protein through mutation. It was confirmed (D in Fig. 3). Through the above results, it was proved that cyclopyrox directly binds to the HBV core protein and inhibits core assembly, thereby inhibiting HBV inhibition.

Example 4. Confirmation of the inhibitory effect of cyclopyrox on HBV proliferation

Through the above Examples 1 and 3, it was confirmed that cyclopyrox inhibits core assembly of HBV, and thus it was intended to confirm whether the proliferation of HBV can actually be suppressed.

First, as shown in A to D of Figure 5, in both the cell line expressing HBV and the cell line overexpressing HBV, the amount of HBV DNA discharged out or remaining in the cell decreases as the concentration of cyclopyrox increases. Confirmed.

Furthermore, the degree of HBV proliferation was analyzed by treating cyclopyrox in the HCP-infected liver cancer cell lines NTCP-HepG2 and NTCP-Huh7. After treating the liver cancer cell line with cyclopyrox in advance for 6 hours, HBV was incubated with cyclopyrox again for 16 hours. Thereafter, after culturing for 14 days, the cells were analyzed. The schematic diagram of this experiment is shown in Fig. 6A. In Figures B and C, the NTCP proteins expressed in the liver cancer cell lines NTCP-HepG2 and NTCP-Huh7 were verified by immunoblot and flow cytometry.

As a result, as can be seen from D to F of FIG. 6, it was confirmed that the HBV DNA discharged out according to the concentration gradient during cyclopyrox treatment decreases, and the HBV cccDNA and rcDNA present inside the cell decrease (FIG. 6). ).

Through the above results, it was found that cyclopyrox can effectively inhibit HBV proliferation, and has an effect not only on HBV DNA, but also on the reduction of cccDNA, which is the biggest problem of currently used drugs. Therefore, it was found that cyclopyrox can be used as an HBV inhibitor for future HBV treatment.

Example 5. Confirmation of the synergistic effect of cyclopyrox with entcavir and/or tenofovir

Through the above Examples 1 to 4, it was confirmed that cyclopyrox can inhibit the core assembly of HBV, thereby inhibiting its proliferation. Conventional anti-HBV drugs, entecavir (ETV) or tenofovir (TDF) It was intended to confirm whether it exhibits a synergistic effect when used in combination with.

On the other hand, Entecavir (ETV) and Tenofovir (TDF) are currently used as drugs that inhibit HBV, but have the disadvantage of not being able to remove the cccDNA of HBV. To complement this, it is used in combination with interferon-based drugs depending on the situation. Accordingly, in order to confirm that cyclopyrox can also exhibit an effect when used in combination with the conventional drugs Entecavir or Tenofovir, these synergistic effects were confirmed.

Specifically, cyclopyrox alone in the liver cancer cell line; Cyclopyrox and entacarvir; Alternatively, after treating cyclopyrox and tenofovir with the method according to Example 3, the proliferative capacity of HBV was confirmed. As a result, as shown in A and B of FIG. 7, when cyclopyrox and entecavir or tenofovir are treated simultaneously, HBV DNA and cells inside the cells are discharged out of the cells rather than cyclopyrox alone. It was confirmed that the amount of HBV DNA was significantly reduced. On the other hand, it was confirmed that the amount of HBsAg protein was not significantly changed (FIG. 6C ).

Through the above results, it was found that although cyclopyrox alone can effectively suppress HBV proliferation, when used in combination with entecavir or tenofovir, it can exhibit a better anti-HBV effect.

Example 6. Confirmation of HBV proliferation inhibitory effect of cyclopyrox in vivo

Through Examples 1 to 5, it was confirmed that cyclopyrox exhibits an excellent HBV proliferation inhibitory effect, and it was intended to confirm whether it shows the same effect in vivo.

Specifically, HBV was generated in vivo by injecting a plasmid expressing HBV into the mouse using a hydrodynamic method on the tail. Thereafter, after daily treatment for 5 days with cyclopyrox, tenofovir alone, or a combination of cyclopyrox and tenofovir, the degree of proliferation of HBV present in mouse serum was confirmed. The schematic diagram of this experiment is shown in Fig. 8A.

As a result, as shown in FIG. 8B, it was confirmed that the HBV core protein was decreased in the hepatocytes. In addition, it was confirmed from FIG. 8C that cyclopyrox alone reduced the amount of HBV DNA present in the mouse in vivo, and almost no HBV DNA was present on the 5th day of treatment. In particular, it was confirmed that even when the cyclopyrox and tenofovir are simultaneously treated, the HBV DNA inhibition inhibitory effect is excellent, and the effect is slightly superior to the cyclopyrox alone treatment. On the other hand, it was confirmed that the amount of HBsAg protein did not change significantly (D in FIG. 8).

Through the above results, it was found that cyclopyrox can effectively inhibit the proliferation of HBV present in vivo, and thus can be usefully used as a drug exhibiting anti-HBV effects.

Example 7. Cyclopyrox cytotoxicity confirmation

Through the above Examples 1 to 6, it was confirmed that cyclopyrox exhibits an excellent HBV proliferation inhibitory effect in vitro and in vitro, and its cytotoxicity was analyzed to confirm whether it can be actually developed as an HBV inhibitory drug.

As a result, as shown in FIG. 9, it was confirmed that the cell viability reduction by cyclopyrox was not affected in both the cell line expressing HBV and the cell line overexpressing HBV. In addition, it was confirmed that cell survival was maintained even at the highest concentration of 10 uM.

Through the above results, it was found that cyclopyrox can be safely used in the human body because it does not affect cell survival, and can be very useful as a drug exhibiting anti-HBV effects.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the claims, which are described later, rather than the above detailed description, and all modified or modified forms derived from equivalent concepts thereof.

Claims (10)

An anti-Hepatitis B Virus (HBV) composition comprising cyclopirox or a pharmaceutically acceptable salt thereof.
The anti-HBV composition of claim 1, wherein the cyclopyrox is represented by Formula 1 below.
[Formula 1]

The anti-HBV composition of claim 1, wherein the cyclopyrox inhibits assembly of the HBV core protein.
The anti-HBV composition of claim 1, wherein the anti-HBV composition further comprises entecavir, tenofovir, or a combination thereof.
A pharmaceutical composition for the prevention or treatment of HBV virus-infected disease, comprising cyclopyrox or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition according to claim 5, wherein the HBV virus-infected disease is hepatitis, cirrhosis and hepatocellular carcinoma, or a combination thereof.
The pharmaceutical composition of claim 5, wherein the pharmaceutical composition further comprises entcavir, tenofovir, or a combination thereof.
A method of treating an HBV virus-infected disease, comprising administering the pharmaceutical composition of any one of claims 5 to 7 to an individual other than a human.
A health functional food composition for preventing or improving HBV virus-infected disease, comprising cyclopyrox or a physiologically acceptable salt thereof.
The dietary supplement composition of claim 9, wherein the dietary supplement composition further comprises Entecavir, Tenofovir, or a combination thereof.

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