CN110623968A

CN110623968A - Application of Evans blue in preparation of medicine and medicine composition

Info

Publication number: CN110623968A
Application number: CN201911014475.5A
Authority: CN
Inventors: 陈绪林; 肖宇
Original assignee: WUHAN WEILIDE BIOLOGICAL PHARMACEUTICAL Co Ltd
Current assignee: WUHAN WEILIDE BIOLOGICAL PHARMACEUTICAL Co Ltd
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2019-12-31

Abstract

The invention provides an application of Evans blue in preparing a medicine for treating HBV infection and a medicine composition for treating or preventing hepatitis B virus infection. The medicine or the pharmaceutical composition can effectively inhibit the entry of hepatitis B virus and the assembly of virus particles, and can be effectively used for treating or preventing hepatitis caused by hepatitis B virus infection.

Description

Application of Evans blue in preparation of medicine and medicine composition

Technical Field

The invention relates to the field of biomedicine, in particular to application of Evans blue in preparation of a medicine and a pharmaceutical composition, and more particularly relates to application of Evans blue in preparation of a medicine and a pharmaceutical composition for treating or preventing hepatitis B virus infection.

Background

Hepatitis B Virus (HBV) infection is the most prevalent chronic viral infection in the world. HBV is transmitted mainly by blood transmission, sexual behavior transmission, mother-infant transmission and the like. Hepatitis b virus infection is a serious health problem worldwide, with about 2.2 billion HBV carriers worldwide, causing 768000 deaths each year, which is the tenth leading cause of death worldwide.

However, the current drugs for treating hepatitis B virus infection are only applied to clinic, and are divided into two forms of Interferon (IFN) and polyethylene glycol interferon (PEG-IFN), which are approved by FDA in 1990 and 2005; and five nucleoside analogue viral polymerase inhibitor drugs, namely Lamivudine (Lamivudine,3TC or LMV), Adefovir (Adefovir, ADV), Entecavir (Entecavir, ETV), Telbivudine (Telbivudine, LdT) and Tenofovir (Tenofovir disoproxil fumarate, TDF). However, interferon has human tolerance and has large side effect; polymerase inhibitors, however, do not clear cccDNA, and viral replication rebounds rapidly after drug withdrawal, and long-term administration also causes viral mutation and drug resistance.

Therefore, the screening of new anti-HBV infection drugs is very urgent and important.

Evans blue (Evans blue) has been a common biological dye commonly used for tissue section staining, Blood Brain Barrier (BBB) permeability detection and anti-hypersensitivity mediator assay. At the same time, evans blue has been reported in the literature to inhibit HIV infection.

However, the use of evans blue has yet to be further developed.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.

The present application is based on the discovery and recognition by the inventors of the following facts:

evans blue can significantly inhibit HBV infection, and simultaneously, Evans blue can significantly inhibit the assembly of HBV virus particles. This suggests that evans blue has a dual anti-HBV effect of inhibiting virus entry into cells and inhibiting viral particle assembly.

Therefore, the invention provides a new application of Evans blue in preparing a medicine which targets HBV virus invasion and assembly of virus particles and is used for resisting hepatitis B virus infection.

In a first aspect of the invention, the invention proposes the use of evans blue for the preparation of a medicament. According to an embodiment of the present invention, the medicament is for inhibiting entry of hepatitis b virus into a cell; or treating or preventing hepatitis B virus infection. According to the embodiment of the present invention, evans blue can also significantly inhibit nucleocapsid assembly of HBV and HBV viral capsid total DNA within liver cells, and thus, the use of evans blue proposed by the present invention for preparing a medicament which can significantly inhibit hepatitis b virus particle assembly; or significant treatment or prevention of hepatitis B virus infection.

According to an embodiment of the present invention, the use of evans blue for the preparation of a medicament may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the medicament is for treating or preventing hepatitis b virus infection in a human. In some embodiments of the present invention, the inventors found that evans blue can significantly inhibit HBV against human hepatoma cell line Huh7D^hNTCPInfection of human liver primary cells (PHH) and porcine primary hepatocytes (hNTCP-PPH) stably expressing human NTCP protein. Meanwhile, evans blue can also obviously inhibit the assembly of HBV nucleocapsid and the level of nucleocapsid DNA in human hepatoma cell line HepAD38 cells. Therefore, the use of Evans blue proposed by the present invention in the preparation of a medicament for the treatment or prevention of hepatitis B virus infection in humans can be further improved.

According to an embodiment of the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. According to the embodiment of the invention, the pharmaceutically acceptable carrier can improve the stability of the pharmaceutical composition and the availability of the effective ingredients, thereby further improving the treatment or prevention of the hepatitis B virus infection by the pharmaceutical composition.

According to an embodiment of the invention, the pharmaceutical composition further comprises a second agent, which is different from evans blue and is used for treating or preventing hepatitis b virus infection. According to an embodiment of the present invention, the second agent in combination with evans blue further improves the treatment or prevention of hepatitis b virus infection by the pharmaceutical composition.

According to an embodiment of the invention, the second agent comprises at least one selected from the group consisting of: interferon (IFN), interferon-polyethylene glycol (PEG-IFN), Lamivudine (Lamivudine,3TC or LMV), Adefovir (Adefovir, ADV), Entecavir (Entecavir, ETV), Telbivudine (Telbivudine, LdT), Tenofovir (Tenofovir disoproxil fumarate, TDF). Lamivudine, adefovir, entecavir, telbivudine and tenofovir are HBV virus polymerase inhibitor drugs. The target point of Evans blue for inhibiting HBV virus infection is different from the second medicament, and Evans blue takes HBV invasion and nucleocapsid assembly as the target point, so that the risk of drug-resistant mutation of the virus does not exist, and the aim of continuously and effectively eliminating the hepatitis B virus can be fulfilled. According to an embodiment of the present invention, at least one of the above second agents is combined with evans blue, and the therapeutic or prophylactic effect of the pharmaceutical composition on hepatitis b virus infection is more significant.

According to an embodiment of the present invention, the pharmaceutical composition is in the form of a tablet, injection, powder, elixir, capsule, suspension, syrup, pill, sustained-release preparation, controlled-release preparation or nano-preparation. The pharmaceutical compositions in various dosage forms are administered in a predictable administration manner to inhibit HBV invasion and viral particle assembly, thereby further effectively treating or preventing hepatitis B virus infection.

According to some embodiments of the present invention, the pharmaceutical composition having evans blue as an active ingredient may include a pharmaceutically acceptable carrier, and the dosage form and administration manner of the pharmaceutical composition are not particularly limited. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents. For injectable formulations, pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents (isotonics agents) and stabilizers. For formulations for topical administration, pharmaceutically acceptable carriers may include bases, excipients, lubricants and preservatives. The pharmaceutical composition of the present invention may be prepared in various dosage forms in combination with the above pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical compositions may be prepared as tablets, capsules, elixirs, suspensions or syrups. For injectable preparations, the pharmaceutical compositions may be prepared in ampoules, e.g. in single dose dosage form, or in unit dosage forms, e.g. in multidose containers. The pharmaceutical composition can also be prepared into solution, suspension, tablet, pill, capsule, long-acting preparation, sustained-release preparation, controlled-release preparation or nano-preparation.

According to the embodiment of the present invention, the pharmaceutical composition of the present invention, which contains evans blue as an active ingredient, can significantly inhibit the invasion of hepatitis b virus and the assembly of viral particles at a non-cytotoxic concentration, and thus can be administered in the treatment or prevention of hepatitis b virus infection.

The term "administering" as used herein means introducing a predetermined amount of a substance into a patient by some suitable means. The drug or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration. However, because of oral administration, the active ingredients of orally administered compositions should be coated or formulated to prevent degradation in the stomach. Preferably, the composition of the present invention can be administered in an injectable formulation. In addition, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.

The administration frequency and dose of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient and the type of drug as an active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in a suitable form for administration 1, 2 or more times over the entire period, as long as a therapeutically effective amount is achieved.

The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and administration regimen. For example, in the treatment of hepatitis b virus infection, a drug or compound that reduces, prevents, delays, inhibits or retards any symptoms of the disease or disorder should be therapeutically effective. A therapeutically effective amount of a pharmaceutical composition or compound need not cure a disease or condition, but will provide treatment for a disease or condition such that the onset of the disease or condition in an individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or the disease or condition becomes less severe, or recovery is accelerated, for example.

The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses the treatment of disease (primarily hepatitis b virus infection) in mammals, particularly humans, including: (a) preventing the development of a disease (e.g., preventing hepatitis b virus infection) or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administering to an individual in need thereof a pharmaceutical composition comprising evans blue as an active ingredient as described herein.

Drawings

FIG. 1 is a graph showing the result of measuring anti-HBV activity of Evans blue (Evans blue) according to example 1 of the present invention,

wherein FIG. 1A is a structural diagram of Evans blue,

FIG. 1B is Evans blue vs. Huh7D^hNTCPThe cytotoxicity of (a) and the inhibition rate of HBeAg in the supernatant,

FIG. 1C is a graph showing the real-time quantitative PCR results of total HBV DNA in suppressor cells in which Evans blue can be dose-dependently,

FIG. 1D is a dose-dependent inhibition of myr-preS1-FITC binding to Huh7D by Evans blue^hNTCPOn the surface of the cell, the cell surface,

FIG. 1E is a graph of the results of dose-dependent inhibition of HBV infection of human primary hepatocytes by Evans blue,

FIG. 1F is a graph of results of porcine primary hepatocytes stably expressing human NTCP protein by dose-dependent inhibition of HBV infection by Evans blue;

FIG. 2 is a graph showing the results of Evans blue affecting the assembly of HBV viral particles according to example 2 of the present invention;

wherein FIG. 2A shows cytotoxicity and antigen levels of HepAD38 cells treated with Evans blue for 4 days,

FIG. 2B shows the effect of Evans blue on intracellular HBV total DNA,

FIG. 2C shows the effect of Evans blue on intracellular HBV nucleocapsid (HBV capsid) and total DNA of nucleocapsid;

FIG. 2D shows the effect of Evans blue on the expression level of HBV core protein.

Detailed Description

The following examples of the present invention will be described in detail with reference to specific examples, which are provided for illustration of the present invention and should not be construed as limiting the scope of the present invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 evaluation of Evans blue anti-HBV Activity

1. Experimental Material

1.1 cells and drugs

HepAD38 cell line: the human liver cancer stably transfects the cell line, the HBV genome is integrated in the chromosome, the HBV DNA synthesis and the protein expression can be supported, and infectious virus particles can be produced. In this cell line, viral replication is controlled by the tetracycline regulated CMV promoter.

Huh7D^hNTCPCell line: the human liver cancer cell line stably transfected with human NTCP protein can support HBV infection.

Human primary hepatocytes (PHH): purchased from shanghai reed liver for HBV infection.

Porcine Primary Hepatocytes (PPH) and humanNTCP lentiviral expression vectors: purchase free-standing biotechnology.

Evans blue, entecavir was purchased from Sigma.

1.2 reagents

DMEM/F12 medium and Fetal Bovine Serum (FBS) were purchased from GIBCO; the HBV surface antigen (S antigen) and e antigen detection kit is purchased from Shanghai Kehua biotechnology Limited;reagents were purchased from Invitrogen; iTaq Universal SYBR Green supermix reagents were purchased from Bio-rad; DIG High Prime DNA Labeling and Detection Starter Kit II was purchased from Roche.

2. Experimental methods and results

2.1 cytotoxicity assays of Evans blue (Evans blue)

1) Mixing HepAD38 and Huh7D^hNTCPCells were inoculated into 96-well cell culture plates at 8000 cells/well (100. mu.L) and cultured in a 37 ℃ cell incubator until the cells were adherent.

2) Adding medicine to treat cells: the drug was diluted in cell culture medium in 2-fold gradients of 6-10 gradients, 3 replicates per gradient. 200 μ L of the cell culture solution containing the drug was added to the cells in step (1), and the cells were further cultured in a cell culture chamber at 37 ℃ for 72 hours before examination.

3) Discarding the drug-containing cell culture supernatant, adding 10% per wellAfter 100 mu L of culture solution of the reagent is incubated for 0.5h-1h in an incubator at 37 ℃, an all-band microplate reader is used for detecting the light absorption value at the wavelength of 540nm, the correction wavelength is 620nm, and the cell survival rate of each concentration is calculated, or the fluorescence value (the excitation light wavelength: 540nm, the emission light wavelength: 595nm) is detected to calculate the cell survival rate.

The results are shown in FIGS. 1B and 2A.

The results in FIG. 1B show: 3-day Evans blue treatment versus Huh7D^hNTCPCC of cells₅₀(half cytotoxic concentration) greater than 1000 micromoles per liter.

Figure 2A results show: CC of HepAD38 cells treated with Evans blue for 4 days₅₀(half the cytotoxic concentration) was 351.8 micromoles/liter.

2.2 Evans blue (Evans blue) inhibition of HBV infection experiment

1) Mixing Huh7D^hNTCPThe cells of the/PHH and the PPH-hNTCP are inoculated into a 96-well cell culture plate according to 8000 cells/well (100 mu L), cultured in a 37 ℃ cell culture box, and reserved after the cells adhere to the wall.

2) Dosing and virus treatment of cells: diluting the drug with a cell culture solution in 2-fold gradient, 3 multiple wells per gradient, adding 200. mu.L of the cell culture solution containing the drug to the cells in step (1) while adding HBV viral particles to infect the cells, and continuing to culture in a cell culture chamber at 37 ℃ for 5 days for detection.

3) The cell supernatant and the cells were collected separately, and the content of HBeAg (secretory e antigen) and HBsAg (surface antigen) in the cell supernatant was measured by ELISA assay kit.

4) And (3) extracting the cell supernatant and the intracellular HBV total DNA collected in the step (3) respectively, and detecting the cell supernatant and the intracellular HBV total DNA by a real-time quantitative PCR method.

The primers used in the real-time quantitative PCR are shown as SEQ ID NO: 1-4:

primer (HBV S144-forward primer): 5'-TCACCAACCTCTTGTCCT-3' (SEQ ID NO: 1).

The primer (HBV S144-reverse primer): 5'-GACAAACGGGCAACATACCT-3' (SEQ ID NO: 2).

Primer (GAPDH forward primer): 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO: 3).

Primer (GAPDH reverse primer): 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 4).

The results are shown in FIGS. 1B to 1F. Wherein PreS1 in the figure indicates the group with myr-preS1 drug added and Ctrl is the blank control group, i.e. the group without virus infection. The results show that: evans blue can inhibit HBV infection Huh7D in dose-dependent manner^hNTCPSupernatant HBeAg levels of cells and intracellular total HBV DNA, its EC₅₀(half effect concentration) was about 2.1. mu. mol/l and 6.25. mu. mol/l, respectively (as shown in FIG. 1C); meanwhile, evans blue can also inhibit PHH infection by HBV in a dose-dependent manner, and can inhibit 80% of HBV total DNA and cccDNA at a higher concentration of 50 μmol/l (as shown in fig. 1E); in PPH-hNTCP cellsEvans blue also inhibited HBV infection dose-dependently during infection (as shown in panel F).

2.3 Evans blue inhibits the binding of myr-preS1-FITC to Huh7D^hNTCPCells

1) Mixing Huh7D^hNTCPCells were inoculated into 96-well cell culture plates at 8000 cells/well (100. mu.L) and cultured in a 37 ℃ cell incubator until the cells were adherent.

2) Gradient dilutions of evans blue were added to the wells of the cells of step 1, with 0.1. mu.M myr-preS1-FITC per well, and 0.1. mu.M myr-preS1 per well as a positive control.

3) After incubation for 1 hour at 37 ℃, the medium was aspirated and washed 3 times with PBS, fixed with 4% paraformaldehyde and added with DAPI to stain nuclei, and the results were analyzed on a PerkinElmer Operetta CLS high content imaging analysis system.

The results are shown in FIG. 1D.

Evans blue inhibits 99% of the binding of myr-preS1-FITC to Huh7D at 50 micromoles/liter^hNTCPIn the above, the laboratory simulates the binding of LHBs of HBV viruses to cells in vitro, indicating that Evans blue can inhibit the binding of HBV viruses to the cell surface.

Example 2 Evans blue (Evans blue) acts directly on HBV nucleocapsid assembly to exert antiviral effects

1. Experimental Material

1.1 cells, plasmids and drugs

HepAD38 cell line: the human liver cancer stably transfects the cell line, HBV genome is integrated in the chromosome, HBV DNA synthesis can be supported, protein expression can be realized, and infectious virus particles can be produced. In this cell line, viral replication is controlled by the tetracycline regulated CMV promoter.

Evans blue, oxicaine and entecavir were purchased from Sigma.

1.2 reagents

DMEM/F12 medium and Fetal Bovine Serum (FBS) were purchased from GIBCO;reagents were purchased from Invitrogen; iTaq Universal SYBR Green supermix reagent was purchased from Bio-rad; rabbit polyclonal antibody against HBVcore protein, and mouse monoclonal antibody against GAPDH were purchased from Dako and Biyuntian, respectively; HBV surface antigen (S antigen) and e antigen detection kits were purchased from Shanghai Kowa Biotech, Inc.

2. Experimental methods and results

2.1 Evans blue (Evans blue) Studies on the level of hepatitis B Virus antigen in HepAD38 cells

1) HepAD38 cells were seeded 8000 cells/well in a 96-well cell culture plate and cultured in a 37 ℃ cell incubator for 24 h.

2) Cells from step 1 were treated for 4 days with gradient dilutions of evans blue.

3) Cell supernatants were collected and assayed for viral HBeAg and HBsAg levels in the supernatants by the Elisa Kit.

The results are shown in FIG. 2A. The results in FIG. 2A show that Evans blue dose-dependently inhibits the levels of HBeAg and HBsAg secreted by HepAD38 cells.

2.2 Effect of Evans blue (Evans blue) on HBV Total DNA

1) In HepAD38 cells, after tetracycline induces initiation of viral replication, tetracycline is withdrawn and the HBV genome integrated in the cells begins to replicate, producing HBV dsDNA, rcDNA, ssDNA and cccDNA.

2) The HepAD38 cells after induction are inoculated into a 96-well cell culture plate according to 8000 cells/well and cultured for 24h in a 37 ℃ cell culture box.

3) Adding medicine to treat cells: evans blue was diluted to various concentrations with DMEM/F12 medium, 3 replicates per group, different doses of drug were added to the cell culture plates to culture the cells, and the drug was added for 4 days.

4) Collecting cells, respectively extracting total HBV DNA in the cells to obtain total HBV DNA, and analyzing and researching antiviral activity of the medicine by using a quantitative PCR method.

The primers used in the real-time quantitative PCR are shown as SEQ ID NO:

primer (HBV S144-forward primer): 5'-ACTCACCAACCTCTTGTCCT-3' (SEQ ID NO: 1).

Primer (HBV S144-reverse primer): 5'-GACAAACGGGCAACATACCT-3' (SEQ ID NO: 2).

Primer (GAPDH forward primer): 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO: 3).

Primer (GAPDH reverse primer): 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 4).

The results are shown in FIG. 2B. Compared with the control group which is not added with drugs, the Evans blue treatment group has obvious dose-dependent inhibition effect on intracellular HBV total DNA, and 31.3 micromole/liter of drugs can inhibit 50 percent of HBV total DNA.

2.3 study of the effect of Evans blue (Evans blue) on hepatitis B virus nucleocapsid assembly:

evans blue is reported to be an inhibitor of the BK pathway, which reduces intracellular calcium ion concentration, and related studies show that the assembly of surface HBV nucleocapsids is related to intracellular calcium ion concentration. The inventors therefore investigated the effect of Evans blue on HBV nucleocapsid assembly. The experimental procedure was as follows:

1) HepAD38 cells were treated at 2X 10⁵Cells/well were plated in 6-well cell culture plates and incubated overnight in a 37 ℃ cell incubator.

2) Cells were dosed for 72 hours.

3) Adding 300 microliter of mild cell lysate, lysing at room temperature for 30min, collecting the lysate into an EP tube, and centrifuging to remove cell debris. The lysate sample was fractionated into various fractions by 1% agarose gel electrophoresis, and then transferred to a nylon filter by capillary transfer, using 10 XSSC as a transfer buffer, and HBV nucleocapsids on the filter were detected using an anti-HBV core antibody (Dako). And (3) performing alkali denaturation treatment on the detected filter membrane to release the virus DNA in the nucleocapsid, and detecting the level of the virus DNA in the nucleocapsid by using a Southern blot method.

In addition, the method of western blot is used to detect HBV core protein (protein constituting nucleocapsid coat), and antibody detected is anti-HBV core antibody (Dako).

The results are shown in FIGS. 2C and 2D. Evans blue has a significant dose-dependent inhibitory effect on both the nucleocapsid assembly of HBV and on HBV DNA within the nucleocapsid, and this inhibition does not act by affecting HBV core protein levels.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. Use of Evans blue in preparing medicine for treating or preventing hepatitis B virus infection is provided.

2. A pharmaceutical composition for treating or preventing hepatitis b virus infection, comprising:

evans blue as active ingredient; and

a second agent that is different from evans blue and that is used to treat or prevent hepatitis b virus infection.

3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 2, wherein the second agent comprises at least one selected from the group consisting of: interferon, polyethylene glycol interferon, lamivudine, adefovir, entecavir, telbivudine, tenofovir.

5. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in the form of a tablet, injection, powder, elixir, capsule, suspension, syrup, pill, sustained release formulation, controlled release formulation, or nano-formulation.