KR101924299B1 - Use of cetylpyridinium chloride for anti-Hepatitis B virus - Google Patents

Abstract

According to the present invention, disclosed is a pharmaceutical composition capable of preventing production of hepatitis B virus (HBV), containing cetylpyridinium chloride (CPC). The pharmaceutical composition, according to the present invention, is capable of inhibiting capsid assembly essential for HBV production particularly through interactions with capsid dimers, and exhibits synergistic effects when used in combination with conventional HBV polymerase inhibitors. Therefore, the pharmaceutical composition can be effectively used for the treatment and prevention of various diseases requiring inhibition of HBV production.

Description

Use of Cetylpyridinium Chloride for Anti-HBV Virus {Use of cetylpyridinium chloride for anti-Hepatitis B virus}

It is related to hepatitis B virus suppression technology.

Hepatitis B virus (HBV) is a virus that causes chronic hepatitis by infecting hepatocytes. It is estimated that about 350 million people worldwide are infected with HBV, and 5 to 10% (Ahn et al., 2005. J. Hepatol 42 (2): 188-94; Chung et al., 2005). In addition, chronic hepatitis is most likely to develop into hepatocellular carcinoma (HCC) through liver cirrhosis and hepatic failure. Montalto, G. et al. 2002. Ann NY Acad Sci 963: 13-20).

Therefore, it is important to treat carriers infected with hepatitis B virus to prevent further development of liver disease.

Stray et al. Have reported that heteroaryldihydropyrimidine (HAP) specifically reacts with methyl 4- (2-chloro-4-fluorophenyl) -6-methyl-2- (pyridin-2-yl) -1,4-dihydropyrimidine-5-carboxylate (HAP-1) (Proc Natl Acad Sci US A. 2005; 102 (23): 8138-8143).

United States Patent RE39155 E1 discloses drugs for treating hepatitis B with nucleoside analogs.

U.S. Patent No. 6,544,520 relates to HBV virus inhibitors and discloses peptides with specific sequences that bind to core antigens and inhibit the assembly of HBV.

Development of a stable and effective therapeutic agent focused on inhibiting the proliferation of various HBV strains is required. Particularly, in the formation and survival of viruses, capsids are essential. Therefore, substances capable of blocking or inhibiting the formation of these capsids can function as powerful antiviral agents, and development of such therapeutic agents is required.

It is intended to provide a material having anti-HBV efficacy.

In one aspect, the invention provides an anti-HBV (Hepatitis B Virus) composition comprising cetylpyridinium chloride or a pharmaceutically acceptable salt thereof.

In one embodiment, the composition according to the present disclosure may be provided in the form of a pharmaceutical composition.

In one embodiment, the anti-HBV composition according to the present invention is a hepatitis B therapeutic agent with patent HBV infection.

In another embodiment, the anti-HBV pharmaceutical composition according to the present invention is a chronic hepatitis B therapeutic agent.

All of the above compositions and compositions disclosed herein inhibit the capsid assembly of HBV viruses, thereby inhibiting the production of particles of the HBV virus.

All of the above compositions and compositions disclosed herein may further comprise one or more of an HBV polymerase inhibitor or an HBV entry inhibitor. Thus, one or more of the HBV polymerase inhibitors, or one or more of the HBV entry inhibitors, or one or more of the HBV polymerase inhibitors and one or more of the HBV entry inhibitors may be used.

In one embodiment, the HBV polymerase inhibitor is selected from the group consisting of Lamivudin, Adefovir dipivoxil, Telbivudine, Clevudine, Entecavir or Tenofovir, Entry inhibitors are not limited to, but are not limited to, Myrcludex, Ezetimibe, Suramine, Epigallocatechin-3-gallate (EGCG), or Irbesartan.

In one embodiment, the HBV polymerase inhibitor is lamivudine.

In other embodiments, the concentration ratio of cetylpyridinium chloride to lamivudine may be in the range of 12 to 0.5: 1.

In another embodiment, the concentration ratio of cetylpyridinium chloride to lamivudine is 12: 1, 4: 1, 3: 1, 2: 1, 1.5: 1, 1: 1, 0.67: 1.

In another aspect, the present invention also provides a method of inhibiting the capsid assembly of HBV in vitro, comprising treating cetylpyridinium chloride or a salt thereof, or a composition comprising the same, with a cell that produces HBV or a cell infected with HBV, or Lt; RTI ID = 0.0 > HBV < / RTI > production.

A pharmaceutical composition comprising a CPC according to the present disclosure may inhibit the production of HBV.

In particular, the interaction with the capsid dimer can inhibit the capsid assembly essential for HBV production, and exhibits a synergistic effect when used in combination with the conventional HBV polymerase inhibitor. Therefore, it can be effectively used for the treatment and prevention of various diseases requiring suppression of HBV production.

Figure 1 shows that cetylpyridinium chloride (CPC) inhibits hepatitis B virus capsid formation in vitro. A is a capsid-forming assay between a core protein and a candidate substance for inhibition of hepatitis B virus capsid formation, and the abbreviations are as follows: dimethylsulfoxide (DMSO); Benzenesulfonamide (BS, benzenesulfonamide); Idoxuridine (IDO); Cetylpyridinium chloride (CPC); Amoxicillin (AMO, Amoxicillin); Triphenylamine (TPH, Tripelennamine HCl); Fluorocinonide (FLU, Fluocinonide); Gliquidone (GLI, Gliquidone); Ropecoxib (RFC, Rofecoxib); Amlodipine besylate (AML); Clozapine (CLZ, Clozapine); Tetracycline (TET, Tetracycline HCl). B is the result of treating the group of inhibition of hepatitis B capsid formation with a concentration range of 0-20 μM and measuring the IC ₅₀ value of cetylpyridinium chloride (CPC) by immunoblotting. Values are expressed as mean ± standard deviation. * p <0.05, ** p <0.01, *** p <0.001, t test.
FIG. 2 shows the results of analyzing the binding and structural changes of the hepatitis B virus core protein and cetylpyridinium chloride (CPC). A, B and C were the results of biomolecule changes between core protein and cetylpyridinium chloride (CPC, A), HBcAg antibody (B) and dimethyl sulfoxide (DMSO, Power 20, 40, 60, and 80%, and FIG. 2 shows results for 40%. D is the result of the analysis of the ellipticity of the core which changes after treatment with 1% dimethyl sulfoxide (DMSO, negative control), 50 μM benzenesulfonamide (BS, positive control) and 5 μM cetylpyridinium chloride (CPC).
Fig. 3 shows the results of modeling and numerical analysis of capsid formation inhibition. Side view (top) and top view (bottom) of the core protein duplex model and binding site structure. The common site of the core protein dimer interacting with cetylpyridinium chloride (CPC) in A was represented by a network of green dots and a transparent red color. Cetylpyridinium chloride (CPC) molecules were labeled with the Corey-Pauling-Kortum model (CPK model). B is the cetylpyridinium chloride (CPC) bond simulation result. Adjacent amino acid residues are represented by elemental colored line structures, and the interaction between the core protein duplex and cetylpyridinium chloride (CPC) is depicted by the pink line. C is the result of analysis of capsid formation using hepatitis B virus core protein under a series of cetylpyridinium chloride (CPC) concentration conditions. Capsid versus mass was quantified via band intensity obtained from immunoblotting. The capsulated formation inhibition curves and the predicted curves were obtained by numerical analysis. The association constants, R squared, and correlation test p values of cetylpyridinium chloride (CPC) were indicated. D is the result of thermal diffusion biomolecule analysis using a hepatitis B viral core protein under a series of cetylpyridinium chloride (CPC) concentration conditions. The normalized fluorescence signal intensity was interpreted according to the overlapping thermal diffusion equation of capsid and core protein dimer. The thermal diffusivity curves and the predicted curves calculated by numerical analysis are shown. The association constants, R squared, and correlation test p values of cetylpyridinium chloride (CPC) were indicated.
Figure 4 is a numerical result obtained from Compusyn results of cetylpyridinium chloride (CPC) and lamivudine (LAM). A shows the change of virus concentration by lamivudine treatment (0-0.1 μM), and shows relative virus concentration change by combination of cetylpyridinium chloride (CPC) and lamivudine (LAM). C is the dose-effect curve. D is a combination index plot. E is Isoblograms by combination.
Figure 5 shows the results of inhibition of hepatitis B virus in vivo by cetylpyridinium chloride (CPC). A is the result of changes in the capsid formation induced by the core protein over time (0-30 min) at 37 ℃. B is the result of confirmation of the formation of capsids after treatment with DMSO, 200 μM sulfanilamide (SA), 200 μM benzenesulfonamide (BS) and 20 μM cetylpyridinium chloride (CPC) by the analysis of the change in the Skruz density. C was a 1 μM concentration of dimethylsulfoxide (DMSO); Benzenesulfonamide (BS, benzenesulfonamide); (SA, Sulfanilamide) treatment of hepatitis B virus. The figures were expressed as mean ± standard deviation. * p <0.05, ** p <0.01, *** p <0.001, t test. D. Quantitative changes of hepatitis B virus DNA by treatment with cetylpyridinium chloride (CPC) (0-1 μM) in HepG2.2.15 cells.
FIG. 6 is a transmission electron microscopic image of core protein capsid with or without cetylpyridinium chloride (CPC) treatment. A is a core protein that does not undergo a capsid formation reaction, and B is a core protein that has undergone a complete reaction in a 37 ° C reaction buffer. C is an enlarged image of capsized particles that have undergone complete formation reaction. D is a core protein that has been reacted with capsid formation after treatment with 20 μM cetylpyridinium chloride (CPC). E is an enlarged image of broken capsid particles after treatment with cetylpyridinium chloride (CPC). F is a schematic diagram of capsid formation inhibition. The core protein dimer is depicted as a solid solid surface structure and cetylpyridinium chloride (CPC) is highlighted in red. Cetylpyridinium chloride (CPC) binds to the binding site of the core protein duplex, and the formation of the capsid has a negative correlation with the concentration of cetylpyridinium chloride (CPC).

Herein, cetylpyridinium chloride (CPC) interacts with a viral nucleocapsid protein (also known as a core protein or HBcAg), which constitutes an HBV capside, to inhibit capsid assembly and thereby inhibit virus production It is based on the discovery that it can.

In one aspect herein, this invention relates to an anti-HBV agent, pharmaceutical or pharmaceutical composition comprising CPC or a pharmaceutically acceptable salt thereof.

Cetylpyridinium chloride according to the present invention can be prepared using conventional knowledge known in the art of organic chemistry and can also be obtained commercially.

The pharmaceutically acceptable salts of the compounds according to the invention, or solvates thereof, may be suitably prepared or selected using knowledge known to those skilled in the art in the field of organic chemistry.

Salts are those which are physiologically tolerated and do not cause an allergic reaction or similar reaction that is common in humans. The salts may be organic or inorganic salts, including, but not limited to, sodium, calcium, and potassium salts. Can be prepared through a process or process known to those skilled in the art. As the salt, an acid addition salt formed by free acid may be used. The free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutaric acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

In one embodiment according to the present disclosure, the pharmaceutically acceptable salt may be present as an acid addition salt wherein the CPC compound forms a salt with the free acid. In addition, the CPC according to the present invention may include not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates which can be prepared by conventional methods.

The CPC compound according to the present invention may be stabilized by an anion mating with an ammonium cation in the compound, and the anion may be any anion that can be paired with the ammonium cation while being pharmaceutically acceptable, for example, (I ^- ), sulfonate (SO ₃ ^2- ), chloride (Cl ^- ), and the like.

The composition according to the present invention may inhibit or block capsid assemblies that play a significant role in the forefront of HBV proliferation. In particular, the compounds according to the present invention can block capsid assembly through interaction with capsid dimers to effectively block the production of hepatitis B virus.

Hepatitis B virus (HBV) or HBV (Hepatitis B Virus) is a 3.2-kb circular DNA virus with a partially double-stranded genome. There are four overlapping frames in the genome, from which surface proteins (HBs) ), Polymerase (HBV pol), and X protein (HBx) are expressed (Seeger, C., Mason, WS, 2000. Hepatitis B virus biology. Microbiol Mol Biol Rev 64, 51-68). Core proteins are involved in packaging HBV pol, free genomic RNA (pgRNA) and other proteins, and are known to play an important role in the HBV life cycle ( ibid .). Capsid assembly by a core protein is an important step in HBV replication, as it requires the assembly of core proteins, viral genomes, and other proteins to generate viruses.

The core protein is composed of 183-185 amino acids (ibid.) With two regions consisting of the N-terminal region required for core assembly and the C-terminus regulating viral replication. The core protein forms the capsid of the virus, and in the process the core protein forms a dimer, which in turn interacts with the dimer to ultimately form capsids of 180 or 240 core proteins. Capsid is essential for the protection of packaged viruses and host factors. Therefore, when inhibiting the formation of capsid, it can be usefully used for the prevention or treatment of diseases caused by HBV proliferation.

Thus, the composition according to the present invention can be used as an anti-HBV agent.

The anti-HBV composition herein is capable of inhibiting or blocking the production of HBV. In one embodiment, the mechanism of anti-HBV according to the present disclosure is to interfere with, inhibit or block the capsid assembly through interaction with the capsid dimer. As mentioned earlier, the production of viruses requires the assemble of core proteins, viral genomes and other proteins. In other embodiments, the anti-HBV composition according to the present invention is capable of inhibiting or blocking HBV production in cells infected with HBV, such as hepatocytes.

Thus, inhibiting / blocking / interfering with capsid assemble by the core protein can be used for the treatment or prevention of various diseases caused by HBV infection.

In addition, the pharmaceutical compositions according to the present invention can be effectively used for the treatment, alleviation, improvement and / or prevention of various diseases requiring inhibition of HBV production through anti-HBV efficacy.

Various diseases in which a disease due to HBV infection or an inhibition of HBV production is required herein include, but are not limited to, hepatitis B virus. Hepatitis B is an inflammation of the liver caused by infection with the hepatitis B virus, including acute and chronic hepatitis. Acute hepatitis B is acute, but it usually recovers within 6 months, although it may have jaundice, blackness, anorexia, nausea, myalgia, severe fatigue, right upper abdominal tenderness and asymptomatic infection. Chronic hepatitis B transitions to chronic hepatitis if clinical symptoms persist for more than 6 months and HBsAg positive. Although acute hepatitis is treated conservatively, chronic hepatitis requires administration of an antiviral agent, and inhibition of virus production is an essential treatment. Chronic hepatitis can develop into liver cirrhosis, liver failure, and hepatocellular carcinoma, especially when not treated.

Therefore, the anti-HBV agent or pharmaceutical composition according to the present invention can inhibit HBV production of HBV carriers because it can inhibit the generation of hepatitis B virus, and thus can prevent the hepatitis B, especially chronic hepatitis B treatment Liver cirrhosis, liver failure, and complications such as hepatocellular carcinoma.

&Quot; Treatment ", " mitigation " or " improvement " as used herein refers to any act that improves or alleviates the symptoms of a disease caused by HBV infection upon administration of a composition according to the invention. Those skilled in the art will be able to ascertain the precise criteria of the disease by referring to the data provided by the Korean Medical Association, and to judge the degree of improvement, improvement, and treatment of the disease.

As used herein, " prophylactic " means any act that inhibits or delays the onset of a disease caused by HBV infection upon administration of a composition according to the present disclosure. It will be clear to a person skilled in the art that the pharmaceutical composition according to the present invention can prevent virus-related diseases or complications caused by viruses at the early stage of infection or before symptoms appear.

The pharmaceutical composition according to the present invention may further contain, in addition to the above-mentioned active ingredients, one or more active ingredients exhibiting the same or similar functions, or a compound that maintains / increases the solubility and / or absorbency of the active ingredient. The composition of the present invention can also be used alone or in combination with other methods for the treatment or prevention of liver diseases or using surgery, other drug therapy and biological response modifiers.

For example, one or more DNA polymerase inhibitors and / or entry inhibitors which act on known antiviral agents, particularly DNA polymerases, to inhibit proliferation and can induce synergy.

In one embodiment, the DNA polymerase inhibitor acts in a manner that interferes with, inhibits, or blocks viral genome replication, including but not limited to lamivudine (2'-3 'dideoxy-3'- thiacytidine, enantiomer, adefovir dipivoxil (ADV), telbivudine (L-nucleoside analogue of L-dT, thymidine), Clevudine (1-deoxy-2fluoro-beta-L-arabinofuranosyl ) thymidine, L-FMAU), entecavir (carbocyclic analogue of 2-deoxyguanosine) or tenofovir (tenofovir disoproxil fumarate).

In other embodiments, the entry inhibitor is a drug that interferes with, inhibits or blocks the entry of HBV into the cell via receptors on the cell surface and has recently been identified as a sodium taurocholated cotransporting polypeptide (NTCP) H, et al., Elife 2012 Nov 13; 1. doi: 10.7554 / eLife.00049), and drugs are being developed (Kim, The entry inhibitor may be selected from the group consisting of, but not limited to, Myrcludex (MYR Pharama GmbH), Tvibe Ezetimibe [(3R, 4S) -1- (4-fluorophenyl) -3- [ (4-fluorophenyl) -3-hydroxypropyl] -4- (4-hydroxyphenyl) azetidin-2-one], Suramine, N- (4,8- disulfo-6-sulfonatonaphthalen- methyl-3 - [[[3 - [[N- [3- [N- 2 -methyl-5- C-oxido-N- (4,6,8-trisulfonaphthalen- 1- yl) carbonimidoyl] 2-butyl-3 - [[4- (4-fluorophenyl) -5-oxocarbonylamino] phenyl] -oxidomethylidene] amino] benzenecarboximidate, EGCG, Irbesartan, [2- (2H-tetrazol-5-yl) phenyl] phenyl] methyl] -1,3-diazaspiro [4.4] non-1-ene-4-one.

The composition according to the present invention can inhibit virus proliferation more effectively by the combination therapy with the above-mentioned drugs, that is, cocktail therapy, and is very useful for the treatment of related diseases. The ratio of each component can be selected in consideration of a therapeutic purpose and a specific ingredient,

In one embodiment, lamivudine is used. CPC according to the present application shows a synergistic effect when co-administered with lamivudine. The concentration ratio of cetylpyridinium chloride to lamivudine showing synergy is 12 to 0.5: 1. In another embodiment, the concentration ratio of cetylpyridinium chloride to lamivudine is 12: 1, 4: 1, 3: 1, 2: 1, 1.5: 1, 1: 1, 0.67: 1.

In addition to the above-mentioned active ingredients, the composition of the present invention may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The method of administering the composition of the present invention is not particularly limited thereto, and the known method of administering the inhibitor may be applied. The composition may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) In case of parenteral administration, it can be administered through a patch type nasal / respiratory patch attached to the skin. In order to obtain a rapid therapeutic effect, administration by intravenous injection is preferable. The dosage varies widely depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. For typical drugs, the dosage unit comprises, for example, from about 0.01 mg to 100 mg, but does not exclude the following ranges and ranges above. The daily dose may be about 1 μg to 10 g, and may be administered once a day or divided into several times a day.

In another aspect, the present invention is directed to a method of inhibiting capsid assembly of HBV virus by treating a composition comprising CPC or CPC herein with HBV-infected cells in vitro or in an animal other than a human.

In another aspect, the present invention provides a method of treating HBV-infected cells, comprising administering a composition comprising CPC or a salt thereof or a CPC or a salt thereof according to the present invention to an HBV-infected cell, To a method for inhibiting a tumor.

In one embodiment, the HBV-infected cells are cells capable of replication and transcription of the HBV virus-producing cell, HBV, by infection with the HBV.

In another embodiment, the HBV-infected cells are hepatoma-derived cells, such as HepG2, Huh-6 and Huh-7, or primary human hepatocytes.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

1. Experimental Method

1-1. Analysis of hepatitis B virus capsid formation and sucrose density change

The hepatitis B virus capsid formation reaction was carried out at 37 ° C for 1 hour at a concentration of the candidate drug (in the range of 1-20 μM) under reaction buffer (50 mM HEPES, 15 mM NaCl, 10 mM CaCl ₂ (pH 7.5) And cultured. Capsid formed particles were observed by electrophoresis and immunoblotting experiments on 1% agarose gel. The sucrose density change analysis was carried out for three and a half hours at a rate of 250,000 g using an ultracentrifuge (HITACHI, Tokyo, Japan). And 20 μM cetylpyridinium chloride (Sigma-Aldrich) and 200 μM sulfanilamide (SA), respectively. Distribution of sucrose concentration from 0 to 50% was confirmed by using HBcAg antibody (Dako, Japan) on a 12% SDS-PAGE gel.

1-2. Cell viability measurement

Cell viability was measured by MTT assay. HepG2.2.15 cells were cultured in DMEM (Wellgene, Gyeongsan-si, South Korea) until they showed 80% fusibility in 96-well plates. Cetylpyridinium chloride (CPC) was treated with cells for 24 hours at a concentration of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 μM and washed with PBS. The MTT solution was then reacted at 37 ° C for 4 hours. The supernatant was removed and the remaining cells were lysed using dimethyl sulfoxide (DMSO). Finally, the absorbance was measured at a wavelength of 570 nm using a micrograph (BioTek, Vermont, USA).

1-3. Cadmium chloride cetylpyridinium (CPC) bond modeling

Discovery studio 2.5 (Accelrys, San Diego, USA) was used to construct a silico-core protein dimer structure model from the X-ray crystal structure results. The simulation was based on the CHARMM force field and the Momany-Rone partial charge method. LibDock, LigandFit, and CDOCKER algorithms were used to select candidates that bind to the core structure of the core protein duplex. The optimal binding structure showing the highest receptor-ligand binding score was also investigated. The energy level was minimized by the Adopted Basis-set Newton-Raphson algorithm and the structure obtained was equilibrated to 100,000 cycles at 37 ° C.

1-4. Measurement of binding force between proteins and molecules by thermal diffusion biomolecule reaction analysis

Hepatitis B virus core protein was labeled with NT.115 protein labeling kit RED (Nanotemper, Munich, Germany), and MST buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, and 0.05% Tween-20). Cetylpyridinium chloride (CPC) was then serially diluted in a solution containing core proteins (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.5, 25, 50, 100 μM). Samples were incubated at 37 ° C for 10 minutes and then analyzed using the Monolith NT.115 program (Nanotemper, Munich, Germany). Dimethyl sulfoxide (DMSO) and HBcAg antibodies were selected as negative and positive controls, respectively, and all experiments were repeated three times.

1-5. Quantification of viral DNA in and out of cells by real-time PCR analysis

978 FDA-approved chemicals (Cat. L1300, Selleckchem, Houston, USA) were treated with HepG2.2.15 cells at a concentration of 1 μM and incubated for 24 hours. The hepatitis B virus DNA was extracted from the cells and quantitated using a real-time PCR analyzer (Qiagen Real-Time polymerase chain reaction, Rotor-Gene Q). Real-time PCR primer information was used in the RCP forward (5'-TCCTCTTCATCCTGCTGCTATG-3 ') and reverse direction (5'-CGTGCTGGTAGTTGATGTTCCT-3').

1-6. Synergy analysis of cetylpyridinium chloride (CPC) and lamivudine using Compusyn program

To confirm the synergistic effect between cetylpyridinium chloride (CPC) and lamivudine, each material was mixed in various ratios (12: 1, 4: 1, 3: 1, 2: 1: 1, 0.67: 1, 0.5: 1, 0.33: 1) to HepG2.2.15 cells in 96 well plates. After incubation for 24 hours, the culture broth was collected and the hepatitis B virus DNA was extracted. The IC ₅₀ value and the combination index (CI) were calculated according to the manufacturer's method using Compusyn (Molecular Pharmacology and Chemistry program, New York, USA) The values were analyzed.

1-7. Numerical analysis of hepatitis B virus capsid formation inhibition

Based on the molecular simulation, we confirmed the binding sites of adjacent protomeric subunits and cetylpyridinium chloride (CPC) in core protein duplexes. The outline of capsid formation inhibition was summarized in the competitive equilibrium between cetylpyridinium (CPC) bonds and capped formation (Formulas 1 and 2 below). Calculation of association constants was based on the law of chemical equilibrium and conservation of mass.

(1)

(One)

(2)

(2)

(3)

(3)

(4)

(4)

The equilibrium between capsid and core protein dimers was described by K _capsid (Eq. 5). The formulas were developed in terms of the concentration of core protein duplex bound to capsid and cetylpyridinium chloride (CPC) (Equations 6 and 7). [ Cp 149 ₂ ] _Total and [CPC] _Total values were confirmed prior to the experiment, and [ Capsid ] and K _capsid values were determined experimentally.

(5)

(5)

(6)

(6)

(7)

(7)

Detection and analysis of hepatitis B virus capsid formation by immunoblotting was interpreted based on the assumption that no protomeric polymer other than capsid was present. The association constant of cetylpyridinium chloride (CPC) was calculated and the inhibition curve was interpolated. The results of the thermal diffusion biomolecule reaction analysis were analyzed using the normalized fluorescence intensities (Equation 8). The Soret coefficients of the capsids were approximated based on geometric symmetry and solvent approximation calculations from the coefficients of the core protein dimer. The thermal diffusivity curve was calculated and the association constant of cetylpyridinium chloride (CPC) was obtained.

(8)

(8)

1-8. Circular dichroism analysis

Circular polarization analysis was carried out using a J-815 (Jasco, Oklahoma city, USA) instrument under three repeating conditions with a bandwidth of 1 nm spectrum, a scan rate of 50 nm / min, and a reaction time of 1 second. The final 0.1 mg / ml protein was analyzed in a 1 cm cuvette. Experiments were carried out at 25 ° C and the values were measured by Spectra manager and Spectra analysis version 2.01A program.

1-9. Transmission electron microscope (TEM)

At each reaction condition, a 10 μl volume of core protein sample was reacted for 1 min on a separate carbon-coated lattice chip for negative staining, and the lattice chip was stained with 2% uranyl acetate. Transmission electron microscope (TEM) was analyzed by LIBRA 120 (Carl Zeiss, Oberkochen, Germany) at 80 KV at NICEM (National Instrumentation Center for Environmental Management, Seoul, South Korea). The capsid particles were classified into three categories, normal (noraml), broken, and aberrant, respectively, and counted. The broken capsids are spherical and partially broken, and the mutated capsids are defined as particles of identifiable size.

2. Experimental results

2-1. Analysis of efficacy of CPC as an HBV-removing drug through inhibition analysis of capsid assembly

HepG2.2.15 cells were treated with 978 FDA-approved compounds, and a substance that reduced 100 viral particles by quantitative PCR was selected as the first candidate. Subsequently, a further analysis was performed on the top 10 materials by performing a capsid assembly inhibition assay on 100 candidate groups (FIG. 1, A, Table 1). The HBV core protein Cp149 was treated with 6 of 10 substances (0-20 μM) including CPC (FIG. 1B). Among the six substances, CPC showed the strongest inhibitory effect of the IC50 of 2.5 ± 0.5 μM and IC ₅₀ of the other substances (AMO, TPH, FLU, GLI, TET) was larger than 5 μM (B in FIG. These results indicate that CPC is an effective substance blocking the capsid assembly of HBV.

The ADMET analysis was conducted according to six criteria. Log S solubility values were predicted to be an important criterion for determining absorbency. Plasma protein binding affinity (PPB) and cerebrovascular wall penetration (BBB) reliability were suggested as the circulation index of the drug. As a criterion for evaluating drug metabolism and emissions, the probability of inhibition against the cytochrome P450 2D6 (CYP2D6) enzyme involved in biohydrogen metabolism was presented. As a measure of toxicity, the probability of hepatotoxicity was predicted. Molecular partition coefficients (AlogP98) and polar surface area (PSA2D) values were plotted on the graph and a confidence ellipse for absorbance and penetration into the blood vessel wall was created.

[Table 1] ADMET characteristics of candidates of hepatitis B virus capsid inhibitor

2-2. Interaction with Cp149 dimer by CPC and induction of structural change

Monolithic analysis is based on thermodynamic changes that show the interaction between proteins and small molecules. Microscale thermal diffusivity analysis revealed that CPC interacted with the HBV Cp149 dimer in a concentration-dependent manner (FIG. 2A). Anti-HBcAg antibody and DMSO were used as positive and negative controls, respectively (Fig. 2B, 2C). CPC showed an interaction tendency unlike the negative control DMSO (Fig. 2). The automatically calculated dissociation constants for the 1: 1 stoichiometric primary reaction were 9.9776 and 2.2998 for the CPC and anti-HBcAg antibodies, respectively. The dissociation constant for DMSO is undefined, indicating that DMSO does not interact with the Cp149 dimer. CD was measured for the core protein. The core protein treated with DMSO showed no significant change in molar ellipticity, and the key protein treated with the positive control BS was. In addition, the CPC-treated core protein exhibits a similar tendency to change, indicating that a similar structure change is induced in the secondary structure (Fig. 2D).

2-3. Docking modeling between HBV Cp149 dimer and CPC using In silico computer simulation

CPC is a saturated hydrocarbon consisting of a ring with 16 carbon chains and finally a cationic quaternary ammonium. The single bond of the carbon chain is rotatable and has some energy barrier from the saturated hydrocarbon eclipse. Among the candidate binding sites calculated from the position finding algorithm, structures such as the groove between residues 23-29 and 106-119 and 96 cubic Angstroms for receptor-ligand docking have several possible structures (Fig. 3A). The flexible carbon chains were reconstituted to Cp149 dimer cavities. The interaction energy stabilized the Cp149 dimer-CPC complex (Fig. 3B). The groove structure of the dimer and tumor Cp149 individually defined from the search algorithm was different in space occupancy and ligand accessibility. The surface-to-volume ratio of the grooved structure was found to be larger in the hematocyte model and induce a more divided bond structure than the dimer Cp149. In addition, the two-fold rotational symmetry in the carcass Cp149 required structural compaction along the dimeric interactions residues. The C-terminal residues compressed by the adjacent dimer surround the groove structure and interfere with ligand access from the extra molecular space. The groove structure of dimer Cp149 was found to be more relaxed than ligand Cp149 and ligand binding was possible. We have found that the bond strength between Cp149 dimer and CPC is 8.5 kcal / mol. CPC was predicted to bind dimer CP149 (Figure 3). The results confirmed that the CPC directly binds to the HBV Cp149 dimer in agreement with the monolith analysis (A-C in FIG. 2).

2-4. Restriction of capsid assembly by CPC binding

Based on the difference in solvent accessibility between the protomer and the free dimer core protein, the binding site of CPC was different from that of the protomer adjacent to the dimeric core protein. The capped structure was modeled as spherical symmetry geometry for area calculation and the virtual mole fraction coefficient of the capsid was derived to be 25. Thus, we have determined that CPC is a noncompetitive inhibitor and set a competitive equilibrium between the binding of CPC and capsid assembly (FIG. 1C). This approach, calculated using a 120th order multivariable function, yielded consistent results for dimer concentration. As a result, the capsid and CPC-bound dimer concentrations were linearly inversely proportional to each other. The inhibitor binding constants for CPC and two other positive control compounds, SA and BCM-599, were calculated from the capsid assembly results. At a series of gradient CPC concentrations, the capsid assembly inhibition was extrapolated to the CPC binding constants obtained by plotting the predicted curves (Figure 3C). Microscale thermal diffusivity measurement data were also analyzed numerically. Optimal soret coefficients and assembly inhibitor association constants were calculated using two significant digits. The calculated thermal diffusive curve is shown in the same way (Fig. 3 D). The CPC binding constant was calculated as 1.0 ± 0.5 μM ^-1 in the capsid assembly in monolith analysis and the optimal binding constant was 1.2 ± 0.9 μM ^-1 . SA and BCM-599 showed a weak association with CPC (Table 2).

[Table 2] Calculation of Inhibitor Conjugation Constants by Numerical Analysis

Hepatitis B virus capsid formation and thermal diffusion biomolecule response analysis results were interpreted based on a noncompetitive inhibitor model. The association constant constants of cetylpyridinium chloride (CPC), sulfanilamide (SA), and BCM-599 inhibitor were determined by arithmetic operations. The R-squared value with the fitting curve was noted.

Examples 2-5. Thinner effect by combination of CPC and lamivudine (LAM)

Lamivudine (LAM) reduced the secreted viral concentration of HepG2.2.15 cells (Fig. 4A). Also, it was decreased by the synergy effect between relative virus concentration CPC (0, 0.025, 0.05 μM) and lamivudine (0, 0.01, 0.02 μM) (FIG. The synergistic effect was high in the high concentration ratio of CPC to lamivudine and the CI value was low (Table 3). (Fig. 4C), combination index plot (Fig. 4D) and isobologogram (E of Fig. 4), denoted CPC, lamivudine (LAM) and mixed (CPC + LAM) Respectively. Single drug therapy and drug combination therapy with CPC and lamivudine showed a logarithmic dose curve converging towards the upper limit in the partial effect value. The dose reduction index (DRI) was calculated as the ratio of the single treatment dose due to the effect value equivalent to the input dose in the combined treatment. CI was calculated by summing the titers of each DRI to show synergistic effects. The concentration ratio 12 showed a CI value of less than 1, indicating synergistic effects at several fractional effect points. According to isovolgograms, a concentration ratio of 12 showed that CPC and lamivudine concentrations were below the additive line at 0.5, 0.75, and 0.9 partial effect points, indicating that synergistic effects reduced CPC and lamivudine requirements. In addition, a moderate synergy effect was observed from a concentration ratio of 12 to 1.5, but not below 0.5 (CI Instruction Manual, Table 3-2). HBV biosynthesis inhibition synergy increased as the CPC to lamivudine concentration ratio increased from 12 to 0.33.

[Table 3-1] Cetylpyridinium chloride (CPC) and lamivudine inhibitor mixed IC ₅₀ value and combination constant

In Table 3, all combination ratios were expressed as the amount of CPC: lamivudine calculated with Compusyn software. IC50 is a measure of the efficacy of a substance that interferes with a particular biological action, which is one-half of the maximum inhibitory concentration. The CI-Index is a measure of synergy, additive or antagonistic action.

The effects of multiple different cetylpyridinium chloride (CPC) and lamivudine combination treatments were analyzed by the Compusyn program. 2: 1, 1.5: 1, 1: 1, 0.67: 1, 0.5: 1) at a ratio of the concentration of cetylpyridinium chloride (CPC) to the concentration of lamivudine , 0.33: 1) was treated for fixed lamivudine IC ₅₀ . Extracellular hepatitis B virus DNA was extracted from HepG2.2.15 cells and quantitated by real-time PCR. The inhibitory effect of the drug was calculated as the ratio of the reduced amount of hepatitis B virus DNA compared to the negative control. IC ₅₀ values and combination constants were determined for each drug combination ratio. Synergy effects were determined based on Table 3-2 below.

[Table 3-2] Synergy / antagonistic degree, symbol and combination index value using Chou-Talalay method

Examples 2-6. Suppression of HBV biosynthesis by blocking CPC capsid assembly

In this example, the effect of CPC on HBV biosynthesis in invivo was analyzed. First, the effective activity of CPC on the decrease of capsid formation was analyzed. A sucrose density analysis was performed using Cp149 and CPC, based on data (A in Figure 5) showing that Cp149 performed the capsid assembly in a time dependent manner. As a result, CPC significantly reduced the amount of capsid (FIG. 5B).

Next, since HepG2.2.15 cells release HBV virus particles, the in vivo activity of CPC was analyzed using HepG2.2.15 cells. The results showed that 1 μM CPC was more effective at inhibiting HBV replication than the other five drug candidates (FIG. 1 A, FIG. 5 C). CPC inhibited HBV biosynthesis in HepG2.2.15 cells; HBV particles were significantly decreased in both intracellular and extracellular (Fig. 5D). On the other hand, when HepG2.2.15 cells and CPC treated cells were treated with the same amount of free nucleotide RNA, HepG2.2.15 cells showed consistent cell viability when treated with CPC concentration of 0 to 1 μM.

Observations by transmission electron microscopy also showed that capsid assembly inhibition was induced by CPC (FIG. 6A, FIG. 6B). Capsid formation was performed at 37 [deg.] C in the reaction buffer. As a result of the detection, circular particles having a diameter of 30 to 35 nm with a dark center and a bright annulus were detected (FIG. 6B, FIG. 6C). However, CPC reduced capsid formation in the reaction buffer at 37 ° C and the capsid particles showed rupture and asymmetric deformation (FIG. 6D, FIG. 6E, Table 4). These results indicate that CPC inhibits HBV biosynthesis by inhibiting the conversion of dimeric Cp149 to a capsid structure through interaction between the free dimeric subunit and the receptor ligand (Fig. 6F). Taken together, these results indicate that CPC can act as a potent drug for HBV removal.

[Table 4] Classification of capsid particles

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (10)

An anti-HBV (Hepatitis B Virus) composition comprising cetylpyridinium chloride or a pharmaceutically acceptable salt thereof.
The method according to claim 1,
Wherein the anti-HBV composition is a hepatitis B therapeutic.
3. The method according to claim 1 or 2,
Wherein the anti-HBV composition is a chronic hepatitis B therapeutic.
3. The method according to claim 1 or 2,
Wherein said composition inhibits capsid assembly of HBV virus to inhibit said HBV virus production.
3. The method according to claim 1 or 2,
Wherein said composition further comprises at least one of an HBV polymerase inhibitor or an HBV entry inhibitor.
6. The method of claim 5,
Wherein said HBV polymerase inhibitor is selected from the group consisting of Lamivudin, Adefovir dipivoxil, Telbivudine, Clevudine, Entecavir or Tenofovir,
Wherein the entry inhibitor is Myrcludex, Ezetimibe, Suramine, Epigallocatechin-3-gallate (EGCG), or Irbesartan.
The method according to claim 6,
Wherein the HBV polymerase inhibitor is lamivudine.
8. The method of claim 7,
Wherein the mole ratio of the cetylpyridinium chloride to the lamivudine is 12 to 0.5: 1.
A method for inhibiting HBV production in Invitro, comprising treating cetylpyridinium chloride with cells infected with HBV.
A method of inhibiting the capsid assembly of HBV in Invitro, comprising treating cetylpyridinium chloride with cells infected with HBV.

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