Antiviral composition comprising spiperon as cccdna inhibitor and pharmaceutical composition for preventing or treating hepatitis b

Abstract

The present invention relates to: an antiviral composition comprising spiperone as a cccDNA inhibitor; and a pharmaceutical composition which is for preventing or treating viral infections or hepatitis B, and comprises spiperone as a cccDNA inhibitor. The antiviral composition comprising spiperone according to the present invention has antiviral activity against a wide range of viruses in view of the cccDNA inhibitory efficacy and mechanism of action. In addition, the pharmaceutical composition comprising spiperone has excellent cccDNA inhibitory efficacy, antiviral efficacy, and therapeutic efficacy against viruses and thus can be effectively used for preventing or treating viral infections such as hepatitis B. In addition, spiperone has a synergistic effect with existing antiviral agents and is thus effective in treating viral infections.

Description

Antiviral composition containing spiperone as a CCCDNA inhibitor and pharmaceutical composition for preventing or treating hepatitis B

[0001] An antiviral composition comprising spiperone as a cccDNA inhibitor; and a pharmaceutical composition for preventing or treating viral infection or hepatitis B comprising spiperone as a cccDNA inhibitor.

[0002] Approximately 296 million people (3.8%) worldwide are living with chronic hepatitis B, a disease caused by the hepatitis B virus (HBV), and the number of infected people continues to increase with approximately 1.5 million new cases being infected every year, with approximately 820,000 deaths from cirrhosis or liver cancer due to HBV reported in 2019 alone.

[0003] Chronic hepatitis caused by HBV infection is difficult to treat and carries a high risk of developing cirrhosis or liver cancer. HBV infection is responsible for 75% of liver cancer cases, and chronic hepatitis increases the risk of liver cancer by 20 times compared to the general population. In Korea, chronic HBV infection causes more than 50% of liver cancer cases, and liver cancer is the leading cause of death among men in their 50s, significantly impacting public health and making treatment difficult.

[0004] Despite the high prevalence and availability of effective treatments, HBV remains incurable, leaving a significant unmet need for functional cure. Consequently, the development of antiviral agents for chronic HBV is in high demand in the market. The HBV treatment market is expected to expand significantly, with the recently published "Global Hepatitis B Treatment Market: Growth Trends and Competitive Analysis (2022-2033)" (Report for More Insights on the Hepatitis B Tests Market Forecast 2023-2033) projecting explosive growth of 30% per year across the globe.

[0005] When infected with HBV, cccDNA inserted into the patient's genes is difficult to remove, making a cure virtually impossible. Therefore, the most fundamental treatment goal at present for curing HBV is to completely remove cccDNA from the host by interfering with the formation of cccDNA or suppressing its transcription.

[0006] Currently, no matter which treatment agent is used, Nucleotide analogs (NAs) or PEG-IFN, there is an inhibitory effect on HBV DNA, but the reduction rate of HBsAg, which is considered a major functional cure index related to clinical outcomes such as liver cancer, liver function decline, liver transplantation, and death caused by HBV, is insufficient at 1-2% for NA and 4% or less for PEG-IFN, and there is no significant effect on cccDNA removal.

[0007] Therefore, there is a growing need for effective drugs that can provide functional treatment when chronic hepatitis progresses.

[0008] However, developing a new drug typically takes approximately 15 years and over $1 billion, and it takes a significant amount of time to reach the market. Most drugs fail to gain approval in Phase 2 clinical trials, with at least 50% failing due to lack of efficacy and 25% due to toxicity. Therefore, only 1-3% of drugs make it from the target stage to final approval.

[0009] Drug repositioning is a development strategy that repurposes drugs that have already been developed and approved for other purposes, or are undergoing clinical trials but not yet approved. This strategy repurposes drugs that have already demonstrated a certain level of efficacy and safety. Since the COVID-19 pandemic, when the need for urgent treatments and vaccines has emerged, drug repositioning has become a crucial strategy, and drug repositioning is actively being developed within and across therapeutic areas.

[0010] This type of drug repurposing has clear advantages: it reduces overall development costs, reduces development risk because the substance has proven to be safe, and significantly shortens the development schedule because the safety assessment and drug formulation have already been prepared.

[0011] Accordingly, the inventors of this application sought to develop new antiviral uses for existing drugs and for the treatment of viral infections through drug repurposing. Through screening and mechanistic studies, the inventors of this application discovered new antiviral uses for Spiperone and its use in the treatment of viral infections.

[0012] Spiperone is a dopamine antagonist that binds to dopamine and serotonin receptors and is a substance approved for use in treating schizophrenia in Japan.

[0013] The inventors of the present application, through in-depth research, have discovered the mechanism by which spiperone increases the body's antiviral activity through endogenous activation of type 1 interferon via the PERK-eIF2α-ATF4-IFN mechanism, thereby deriving the use of spiperone as a new antiviral agent for various viruses as well as DNA viruses that generate cccDNA in host cells, such as HBV, and for the treatment of viral infections.

[0014] In addition, the inventors of the present application have overcome the limitations of existing HBV treatments through spiperone, which has a different mechanism from various existing known antiviral agents, and have derived a new, innovative and safer treatment use of spiperone for HBV infection to cure HBV by eliminating cccDNA, which is a difficult problem in treating HBV infection.

[0015] [Prior Art Literature]

[0016] [Non-patent literature]

[0017] (Non-patent Document 0001) Xue, M., Fu, F., Ma, Y., Zhang, X., Li, L., Feng, L., & Liu, P. (2018). The PERK arm of the unfolded protein response negatively regulates transmissible gastroenteritis virus replication by suppressing protein translation and promoting type I interferon production. Journal of virology, 92(15), 10-1128.

[0018] (UK 0002) Clavarino , G. , Claudio , N. , Couderc , T. , Dalet , A. , Judith , D. , Camosseto , V. , … & Pierre , P. (2012). Induction of GADD34 is necessary for dsRNA-dependent interferon-βproduction and participates in the control of Chikungunya virus infection. PLoS Pathogens, 8(5), e1002708.

[0019] (Alcohol 0003) Liu, YP, Zeng, L., Tian, ​​A., Bomkamp, ​​A., Rivera, D., Gutman, D., ... & Smith, JA (2012). Endoplasmic reticulum stress regulates the innate immunity critical transcription factor IRF3. The Journal of Immunology, 189(9), 4630-4639.

[0020] (Paper 0004) Tan, G., Song, H., Xu, F., & Cheng, G. (2018). When hepatitis B virus meets interferons. Frontiers in Microbiology , 9 , 1611 .

[0021] (English 0005) Liu , YP , Zeng , L. , Tian , A. , Bomkamp , A. , Rivera , D. , Gutman , D. , … & Smith , JA (2012). Endoplasmic reticulum stress regulates the innate immunity critical transcription factor IRF3. The Journal of Immunology, 189(9), 4630-4639.

[0022] One aspect of the present invention provides an antiviral composition comprising Spiperone.

[0023] Another aspect is that spiperone provides antiviral uses.

[0024] Another aspect provides the use of spiperone for the preparation of an antiviral composition.

[0025] Another aspect is to provide a pharmaceutical composition for preventing or treating viral infections, comprising spiperone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0026] Another aspect provides a method for preventing or treating a viral infection comprising administering spiperone or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0027] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of a viral infection.

[0028] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the prevention or treatment of viral infections.

[0029] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of hepatitis B, comprising spiperone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0030] Another aspect provides a method for preventing or treating hepatitis B, comprising administering spiperone or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0031] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for use in the manufacture of a pharmaceutical composition for the prevention or treatment of hepatitis B.

[0032] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the prevention or treatment of hepatitis B.

[0033] Another aspect is to provide an antiviral health functional food containing spiperone.

[0034] Another aspect provides the use of spiperone for the manufacture of antiviral health functional foods.

[0035] Another aspect is to provide an antiviral feed composition comprising spiperone.

[0036] Another aspect provides the use of spiperone for the preparation of an antiviral feed composition.

[0037] One aspect of the present invention provides an antiviral composition comprising Spiperone.

[0038] Another aspect provides antiviral uses for spiperone.

[0039] Another aspect provides the use of spiperone for the preparation of an antiviral composition.

[0040] Spiperone is a dopamine antagonist that binds to dopamine and serotonin receptors.

[0041] The CAS number of the above spiperone is 749-02-0 and has the structure of a compound represented by the following chemical formula 1:

[0042] [Chemical Formula 1]

[0043] .

[0044] In this specification, the term “antiviral” means the ability to resist a virus, or any mechanism that is performed to resist a virus, suppress reproduction, or protect against the action of a virus.

[0045] In one specific example, the antiviral composition may have antiviral activity against a virus, such as inhibition of viral gene expression, inhibition of protein expression, or inhibition of viral proliferation.

[0046] In one specific example, the virus is Hepatitis B virus, Adenovirus, Herpes virus, Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Varicella-Zoster Virus (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), Kaposi's Sarcoma-associated Herpesvirus (KSHV / HHV-8), Variola virus, Monkeypox virus, Human Papillomavirus (HPV), JC virus, BK virus, Merkel Cell Polyomavirus (MCV), Human Parvovirus B19, Bocavirus, Hepatitis D virus, Coronavirus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Hepatitis A virus, Hepatitis C virus, COVID-19 virus (or Severe Acute Respiratory Syndrome Coronavirus-2: SARS-CoV-2),It may be one or more selected from the group consisting of Human Immunodeficiency Virus (HIV), Severe Fever with Thrombocytopenia Syndrome, Polio Virus, Dengue Virus, Measles Virus, Virus, Influenza Virus, Rotavirus, Herpes Virus, Flavivirus, Toga Virus, Rubivirus, Pestivirus, Marburg Virus, Encephalitis Virus, Japanese Encephalitis Virus, Ebola Virus, Nipah Virus, and Rhinovirus.

[0047] In one specific example, the virus may be one or more selected from the group consisting of DNA viruses, RNA viruses, and retroviruses. Specifically, the virus may be a DNA virus.

[0048] In one specific example, the virus may be a virus that produces cccDNA within the nucleus of a host cell.

[0049] In one specific example, the antiviral composition may have cccDNA inhibitory activity and / or removal activity.

[0050] CccDNA (covalently closed circular DNA) is a circular, closed DNA molecule produced by some viruses during their replication within the host cell nucleus. It plays a crucial role in viral replication and maintaining persistent infection. Therefore, suppressing cccDNA is a key antiviral strategy.

[0051] In one specific example, the antiviral composition may have antiviral activity through endoplasmic reticulum stress (ER stress) and interferon induction mechanisms. Specifically, the antiviral composition may have antiviral activity by activating the PERK-eIF2α-ATF4 signaling pathway, which is one of the ER stress pathways, and inducing the secretion of type 1 interferon.

[0052] Specifically, the composition containing spiperone may exhibit antiviral activity by inducing an ER stress pathway centered on the PERK-eIF2α-ATF4 axis, thereby activating the STING-TBK1-IRF3 / 7 signaling pathway through the leakage of viral DNA into the cytoplasm and activation of IFI16, a DNA sensor, thereby inducing the expression of type I interferon (type I IFN) and interferon-stimulated genes (ISGs).

[0053] More specifically, spiperone significantly activates ER stress response by inducing phosphorylation of PERK, phosphorylation of eIF2α, and expression of downstream transcription factor ATF4 within cells, which may lead to increased production of mtROS, cytoplasmic leakage of damaged mtDNA, and accumulation of oxidized DNA, leading to IFI16 recognition and activation of type I IFN signaling pathway.

[0054] In one specific example, the antiviral composition may additionally include an antiviral agent.

[0055] The above antiviral drugs include Tenofovir, Entecavir, Adefovir, Sofosbuvir, Cidofovir, Acyclovir, Famciclovir, Valacyclovir, Ganciclovir, Amprenavir, Abacavir, Ansamycin, Darunavir, Delaviridine, Efavirenz, Etravirine, Hypericin, Indinavir, Lamivudine, Lobucavir, Nelfinavir, It may be at least one selected from the group consisting of Nevirapine, Novapren, Ritonavir, Saquinavir, Stavudine, Tipranavir, Virazole, Ribavirin, Zalcitabine, Zidovudine, Maraviroc, Raltegravir, Elvitegravir, Didanosine, Emtricitabine, Lopinavir, Atazanavir, Enfuvirtide, and Clevudine.

[0056]

[0057] Another aspect provides a pharmaceutical composition for preventing or treating viral infections, comprising Spiperone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0058] Another aspect provides a method for preventing or treating a viral infection comprising administering spiperone or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0059] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of a viral infection.

[0060] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the prevention or treatment of viral infections.

[0061] Another aspect provides a method for preventing or treating a viral infection comprising administering spiperone to a subject in need thereof.

[0062] A pharmaceutical composition for preventing or treating a viral infection according to one specific example; Or in a method for preventing or treating a viral infection, the virus is Hepatitis B virus, Adenovirus, Herpes virus, Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Varicella-Zoster Virus (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), Kaposi's Sarcoma-associated Herpesvirus (KSHV / HHV-8), smallpox Variola virus, Monkeypox virus, Human Papillomavirus (HPV), JC virus, BK virus, Merkel Cell Polyomavirus (MCV), Human Parvovirus B19, Bocavirus, Hepatitis D virus, Coronavirus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Hepatitis A virus, Hepatitis C virus,It may be one or more selected from the group consisting of COVID-19 virus (or Severe Acute Respiratory Syndrome Coronavirus-2: SARS-CoV-2), Human Immunodeficiency Virus (HIV), Severe Fever with Thrombocytopenia Syndrome, polio virus, dengue virus, measles virus, virus, influenza virus, rotavirus, herpes virus, flavivirus, toga virus, rubivirus, pestivirus, Marburg virus, encephalitis virus, Japanese encephalitis virus, Ebola virus, Nipah virus, and rhinovirus.

[0063] A pharmaceutical composition for preventing or treating a viral infection according to one specific example; or a method for preventing or treating a viral infection, wherein the virus may be at least one selected from the group consisting of a DNA virus, an RNA virus, and a retrovirus. Specifically, the virus may be a DNA virus.

[0064] A pharmaceutical composition for preventing or treating a viral infection according to one specific example; or a method for preventing or treating a viral infection, wherein the virus may be a virus that produces cccDNA within the nucleus of a host cell.

[0065] A pharmaceutical composition for preventing or treating a viral infection according to one specific example; or a method for preventing or treating a viral infection, wherein the pharmaceutical composition or the treatment method may have cccDNA inhibitory activity and / or removal activity.

[0066] CccDNA (covalently closed circular DNA) is a circular, closed DNA molecule produced by some viruses during their replication within the host cell nucleus. It plays a crucial role in viral replication and maintaining persistent infection. Therefore, suppressing cccDNA is a key strategy for preventing or treating viral infections.

[0067] A pharmaceutical composition for preventing or treating a viral infection according to one specific example; or a method for preventing or treating a viral infection, wherein the pharmaceutical composition or the treatment method may have an activity for preventing or treating a viral infection through endoplasmic reticulum stress (ER stress) and interferon induction mechanisms. Specifically, the pharmaceutical composition or the treatment method may have an activity for preventing or treating a viral infection by activating the PERK-eIF2α-ATF4 signaling pathway, which is one of the ER stress pathways, and inducing the secretion of type 1 interferon.

[0068] Specifically, the composition containing spiperone may exhibit preventive or therapeutic activity against viral infection by inducing an ER stress pathway centered on the PERK-eIF2α-ATF4 axis, thereby activating the STING-TBK1-IRF3 / 7 signaling pathway through the leakage of viral DNA into the cytoplasm and activation of IFI16, a DNA sensor, thereby inducing expression of type I interferon (type I IFN) and interferon-stimulated genes (ISGs).

[0069] More specifically, spiperone significantly activates ER stress response by inducing phosphorylation of PERK, phosphorylation of eIF2α, and expression of downstream transcription factor ATF4 within cells, which may lead to increased production of mtROS, cytoplasmic leakage of damaged mtDNA, and accumulation of oxidized DNA, leading to IFI16 recognition and activation of type I IFN signaling pathway.

[0070] The term "viral infection" in this specification refers to a disease caused by a virus, encompassing a variety of symptoms and diseases caused by viral infection. "Viral infection" includes "viral infectious disease."

[0071] In one specific example, the pharmaceutical composition for preventing or treating a viral infection may additionally include an antiviral agent.

[0072] In one specific example, the method for preventing or treating the viral infection may further comprise a step of administering an antiviral agent.

[0073] The above antiviral drugs include Tenofovir, Entecavir, Adefovir, Sofosbuvir, Cidofovir, Acyclovir, Famciclovir, Valacyclovir, Ganciclovir, Amprenavir, Abacavir, Ansamycin, Darunavir, Delaviridine, Efavirenz, Etravirine, Hypericin, Indinavir, Lamivudine, Lobucavir, Nelfinavir, It may be at least one selected from the group consisting of Nevirapine, Novapren, Ritonavir, Saquinavir, Stavudine, Tipranavir, Virazole, Ribavirin, Zalcitabine, Zidovudine, Maraviroc, Raltegravir, Elvitegravir, Didanosine, Emtricitabine, Lopinavir, Atazanavir, Enfuvirtide, and Clevudine.

[0074]

[0075] Another aspect provides a pharmaceutical composition for the prevention or treatment of hepatitis B, comprising Spiperone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0076] Another aspect provides a method for preventing or treating hepatitis B, comprising administering to a subject in need thereof spiperone or a pharmaceutically acceptable salt thereof.

[0077] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of hepatitis B.

[0078] Another aspect provides the use of spiperone or a pharmaceutically acceptable salt thereof for the prevention or treatment of hepatitis B.

[0079] Another aspect provides a method for preventing or treating hepatitis B, comprising administering spiperone to a subject in need thereof.

[0080] A pharmaceutical composition for preventing or treating hepatitis B according to one specific example; or a method for preventing or treating hepatitis B, wherein the pharmaceutical composition or the treatment method may have cccDNA inhibitory activity and / or removal activity.

[0081] Covalently closed circular DNA (cccDNA) is a circular, closed DNA molecule that occurs during the replication of some viruses within the nucleus of a host cell. It plays a crucial role in viral replication and maintaining persistent infection. In particular, cccDNA is a major factor in causing chronic hepatitis B virus (HBV) infection. Currently, no effective drugs have been developed to inhibit cccDNA in HBV, and inhibition of cccDNA is a key strategy for the prevention or treatment of hepatitis B.

[0082] A pharmaceutical composition for preventing or treating hepatitis B according to one specific example; or a method for preventing or treating hepatitis B, wherein the pharmaceutical composition or the treatment method may have an activity for preventing or treating viral infection through endoplasmic reticulum stress (ER stress) and interferon induction mechanisms. Specifically, the pharmaceutical composition or the treatment method may have an activity for preventing or treating hepatitis B by activating the PERK-eIF2α-ATF4 signaling pathway, which is one of the ER stress pathways, and inducing the secretion of type 1 interferon. Specifically, the composition containing spiperone may exhibit preventive or therapeutic activity against hepatitis B by inducing an ER stress pathway centered on the PERK-eIF2α-ATF4 axis, thereby activating the STING-TBK1-IRF3 / 7 signaling pathway through the leakage of HBV DNA into the cytoplasm and the activation of IFI16, a DNA sensor, thereby inducing the expression of type I interferon (type I IFN) and interferon-stimulated genes (ISGs).

[0083] More specifically, spiperone significantly activates ER stress response by inducing phosphorylation of PERK, phosphorylation of eIF2α, and expression of downstream transcription factor ATF4 within cells, which may lead to increased production of mtROS, cytoplasmic leakage of damaged mtDNA, and accumulation of oxidized DNA, leading to IFI16 recognition and activation of type I IFN signaling pathway.

[0084] In one specific example, the pharmaceutical composition for preventing or treating hepatitis B may additionally include an antiviral agent.

[0085] In one specific example, the method for preventing or treating hepatitis B may further comprise a step of administering an antiviral agent.

[0086] The above antiviral drugs include Tenofovir, Entecavir, Adefovir, Sofosbuvir, Cidofovir, Acyclovir, Famciclovir, Valacyclovir, Ganciclovir, Amprenavir, Abacavir, Ansamycin, Darunavir, Delaviridine, Efavirenz, Etravirine, Hypericin, Indinavir, Lamivudine, Lobucavir, Nelfinavir, It may be at least one selected from the group consisting of Nevirapine, Novapren, Ritonavir, Saquinavir, Stavudine, Tipranavir, Virazole, Ribavirin, Zalcitabine, Zidovudine, Maraviroc, Raltegravir, Elvitegravir, Didanosine, Emtricitabine, Lopinavir, Atazanavir, Enfuvirtide, and Clevudine.

[0087]

[0088] The term "comprising an active ingredient" as used herein means that the pharmaceutical composition contains an effective amount capable of exhibiting a preventive or therapeutic effect against a viral infection or hepatitis B.

[0089] As used herein, the term “pharmaceutically acceptable” means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reactions, within the scope of pharmaceutical judgment.

[0090] As used herein, the term "pharmaceutically acceptable salt" means a salt according to one aspect of the present invention which is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Salts of the parent compound can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base, for example, sodium, calcium, magnesium or potassium, or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. These reactions are typically carried out in water or in an organic solvent or in a mixture of the two. Generally, when practical, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile can be used. The pharmaceutically acceptable salts include both addition salts of acids or bases and stereochemically isomeric forms thereof, and may be, for example, addition salts of organic or inorganic acids. The above salt includes any salt that maintains the activity of the parent compound in the subject of administration and does not cause undesirable effects, and is not particularly limited thereto.

[0091] These salts include inorganic and organic salts, for example, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idet, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, esilinic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucic acid, It can be naphthylic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilinic acid, methanesulfonic acid. In addition, the salt form includes salts of alkali and alkaline earth metals such as ammonium salt, lithium salt, sodium salt, potassium salt, magnesium salt, and calcium salt, salts with organic bases such as benzathine, N-methyl-D-glucamine, and hydrabamine salts, and salts with amino acids such as arginine and lysine. In addition, the salt form can be converted into a free form by treating with a suitable base or acid.

[0092] In this specification, the term “prevention” means any action that inhibits or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0093] In one specific example, the pharmaceutical composition for preventing or treating a viral infection or the pharmaceutical composition for preventing or treating hepatitis B may be administered in combination with an antiviral agent. The antiviral agent is as described above.

[0094] The term “combination administration” as used herein refers to administering the individual components of a treatment regimen simultaneously, sequentially, in reverse order, or individually. It refers to obtaining a combined therapeutic effect by administering two or more drugs simultaneously, sequentially, or in reverse order, or by administering them alternately at regular or indefinite intervals. Combination therapy is not limited thereto, but can be defined as providing a synergistic effect while providing efficacy that is therapeutically superior to the efficacy that can be obtained by administering one or the other of the components of the combination therapy at the usual dose, as measured by, for example, the degree of response, the rate of response, the time until disease progression, or the duration of survival. Combination administration includes simultaneous or sequential administration in any order.

[0095] As used herein, the combination therapy of Spiperone and an antiviral agent according to the present invention can provide a "synergistic effect," i.e., the effect achieved when the active substances are used together is greater than the sum of the effects achieved when the active substances are used individually. The synergistic effect can be achieved when the active substances are (1) co-formulated and combined in a unit dosage formulation and administered or delivered simultaneously; (2) delivered sequentially, alternately, or in parallel as separate formulations; or (3) by some other route of administration. When delivered in alternating therapy, the synergistic effect can be achieved, for example, by sequential administration or delivery of the active substances by each injection using a separate syringe. A "synergistic combination" produces an effect that is superior to the sum of the effects of the individual active substances of the combination.

[0096] Combination therapy can provide an "additive" effect, meaning that the effect achieved when the active substances are used together is equal to the sum of the effects achieved when the active substances are used separately.

[0097] The terms "subject" and "patient" are used interchangeably herein. The subject may be an animal. In some embodiments, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0098] As used herein, the term "therapeutically effective amount" refers to an amount of a drug, such as Spiperone, effective to achieve a desired therapeutic or preventive result. In some cases, the desired result is treatment of a disease or disorder in a subject. The therapeutically effective amount level can be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route and excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field. The compositions of the present disclosure can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. That is, the total effective amount of the compositions of the present disclosure can be administered to a patient as a single dose, or can be administered in a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into account, it is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0099] As used herein, terms such as "treating," "treatment," "to treat," "palliating," or "to palliate" refer to therapeutic measures aimed at curing, slowing, alleviating symptoms, and / or arresting the progression of a diagnosed pathological condition or disorder. Therefore, those requiring treatment include those who have already been diagnosed with or are suspected of having a disorder.

[0100] Meanwhile, the pharmaceutical composition for preventing or treating a viral infection or the pharmaceutical composition for preventing or treating hepatitis B of the present specification may additionally include a pharmaceutically acceptable carrier and may be formulated together with the carrier.

[0101] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0102] The pharmaceutical composition according to one embodiment of the present disclosure and the composition comprising a pharmaceutically acceptable carrier can be applied to any dosage form containing the pharmaceutical composition as an active ingredient, and can be prepared as an oral or parenteral dosage form, and can be formulated in a unit dosage form for ease of administration and uniformity of dosage. The pharmaceutical dosage form of the present disclosure includes a form suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or a form suitable for administration by inhalation or insufflation.

[0103] Oral administration dosage forms containing the composition of the present specification as an active ingredient may be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

[0104] The composition of the present disclosure may be formulated as a parenteral dosage form containing the active ingredient, such as a subcutaneous injection, intravenous injection, or intramuscular injection; a suppository injection; or a spray formulation such as an aerosol that can be inhaled through the respiratory tract. To formulate the composition of the present disclosure as an injectable dosage form, the composition of the present disclosure may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which may then be formulated into a unit dosage form in an ampoule or vial.

[0105] The dosage of the pharmaceutical composition of this specification varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage is preferably 0.01 μg to 100 mg per kg of body weight per day when administered parenterally, more preferably 1 μg to 50 mg. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, sex, body weight, age, etc., the above dosage does not limit the scope of this specification in any way.

[0106]

[0107] Another aspect provides an antiviral health functional food containing Spiperone.

[0108] Another aspect provides the use of spiperone for use in the manufacture of antiviral health functional foods.

[0109] In the antiviral health functional food of the present invention according to one specific example, “virus,” “antivirus,” and “antiviral activity” are as described in relation to the antiviral composition in this specification.

[0110] Another aspect provides a health functional food for preventing or improving viral infections, including spiperone.

[0111] Another aspect provides the use of spiperone for use in the manufacture of a health functional food for the prevention or amelioration of viral infections.

[0112] Another aspect provides a health functional food for preventing or improving hepatitis B, including spiperone.

[0113] Another aspect provides the use of spiperone for use in the manufacture of a health functional food for the prevention or improvement of hepatitis B.

[0114] In the health functional food for preventing or improving viral infection or the health functional food for preventing or improving hepatitis B according to one specific example of the present invention, “virus,” “viral infection,” “prevention,” “improvement,” “preventive or therapeutic activity for viral infection,” and “preventive or therapeutic activity for hepatitis B” are as described herein with respect to the antiviral composition.

[0115] In one specific example, the health functional food may additionally include an antiviral agent, and the antiviral agent is as described above.

[0116] In one specific example, the health functional food may additionally include a food-wise acceptable salt (e.g., a food-wise acceptable salt of spiperone).

[0117] As used herein, the term "food-acceptable salt" refers to a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. For example, the food-acceptable salt can be obtained by reacting the compound with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, and the like. In addition, it can be obtained by reacting the compound with a base to form salts such as ammonium salts, alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts of amino acids such as arginine and lysine, but is not limited thereto.

[0118] The health functional food herein may be formulated into any one form selected from the group consisting of powders, tablets, capsules, pills, granules, and liquids, using conventional methods known in the art, but is not limited thereto. Various forms may be manufactured using methods known in the art.

[0119] Additionally, it can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have antiviral activity.

[0120] In addition, the health functional food of the present invention may contain conventional food additives, and the suitability as the "food additive" is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0121] In addition to the above, the health functional food of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination.

[0122]

[0123] Another aspect provides an antiviral feed composition comprising Spiperone.

[0124] Another aspect provides the use of spiperone for the preparation of an antiviral feed composition.

[0125] The antiviral feed composition of the present invention according to one specific example has antiviral activity, and in the antiviral feed composition of the present invention, “virus,” “antivirus,” and “antiviral activity” are as described in relation to the antiviral composition in this specification.

[0126] Another aspect provides a feed composition for preventing or improving viral infection comprising spiperone.

[0127] Another aspect provides the use of spiperone for the preparation of a feed composition for the prevention or amelioration of viral infections.

[0128] Another aspect provides a feed composition for preventing or improving hepatitis B, comprising spiperone.

[0129] Another aspect provides the use of spiperone for the preparation of a feed composition for the prevention or amelioration of hepatitis B.

[0130] In the feed composition for preventing or improving a viral infection or the feed composition for preventing or improving hepatitis B according to one specific example of the present invention, “virus,” “viral infection,” “prevention,” “improvement,” “preventive or therapeutic activity for viral infection,” and “preventive or therapeutic activity for hepatitis B” are as described herein with respect to the antiviral composition.

[0131] In one specific example, the feed composition may further comprise an antiviral agent, the antiviral agent being as described above.

[0132] In this specification, “feed composition” may mean any natural or artificial diet, meal, etc. or a component of said meal, especially for or suitable for eating, ingesting and digesting by an animal.

[0133] The above feed composition may include, but is not particularly limited to, nutrients such as energy, protein, lipids, vitamins, and minerals required by the individual consuming the feed. The individual refers to the subject of breeding, and includes, without limitation, any living organism capable of consuming the feed of the present invention, including companion animals and livestock.

[0134] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant-based feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal-based feed such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more types.

[0135] The feed composition according to the present invention can be manufactured by adding Spiperone of the present invention in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

[0136] The feed composition according to the present invention can be applied to any subject for which the antiviral effect of the present invention is desired, without limitation. For example, it can be applied to any subject, including non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, and fish.

[0137] The antiviral composition comprising spiperone of the present invention exhibits antiviral activity against a wide range of viruses, based on its cccDNA inhibitory efficacy and mechanism of action. Furthermore, the pharmaceutical composition comprising spiperone exhibits excellent cccDNA inhibitory efficacy, antiviral efficacy, and therapeutic efficacy against viruses, making it useful for the prevention or treatment of viral infections such as hepatitis B. Furthermore, spiperone exhibits a synergistic effect with existing antiviral agents, making it effective in the treatment of viral infections.

[0138] Figure 1 is a schematic diagram of the antiviral activity evaluation of spiperone (in vitro).

[0139] Figure 2 is a graph evaluating the degree of cytotoxicity of spiperone.

[0140] Figure 3 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA according to spiperone treatment in an in vitro system.

[0141] Figure 4 shows the results of Western blot analysis measuring HBcAg and Capsid levels according to spiperone treatment in an in vitro system.

[0142] Figure 5 shows the results of confocal analysis measuring HBcAg and Capsid levels according to spiperone treatment in an in vitro system.

[0143] Figure 6 shows the results of agarose gel electrophoresis analysis measuring the cccDNA level according to spiperone treatment in an in vitro system.

[0144] Figure 7 shows the results of RT-qPCR analysis measuring cccDNA levels according to spiperone treatment in an in vitro system.

[0145] Figures 8 and 9 show the results of evaluating the synergistic effect of spiperone and tenofovir.

[0146] Figure 10 is a schematic diagram of the antiviral activity evaluation of spiperone (in vivo).

[0147] Figure 11 shows the schedule of in vivo experiments to evaluate the antiviral activity and therapeutic efficacy against viral infections following intraperitoneal injection of spiperone.

[0148] Figure 12 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA following treatment with spiperone (intraperitoneal injection) in an in vivo system.

[0149] Figure 13 shows the results of immunohistochemical staining (IHC) analysis of HBcAg in liver tissue following spiperone treatment (intraperitoneal injection) in an in vivo system.

[0150] Figure 14 shows the results of fluorescence immunostaining analysis of nuclei and HBsAg in liver tissue following spiperone treatment (intraperitoneal injection) in an in vivo system.

[0151] Figure 15 shows the schedule of in vivo experiments to evaluate the antiviral activity and therapeutic efficacy against viral infections following oral administration of spiperone.

[0152] Figure 16 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA following spiperone treatment (oral administration) in an in vivo system.

[0153] Figure 17 shows the results of immunohistochemical staining (IHC) analysis of HBcAg in liver tissue following spiperone treatment (oral administration) in an in vivo system.

[0154] Figure 18 shows the results of fluorescence immunostaining analysis of nuclei and HBsAg in liver tissue following spiperone treatment (oral administration) in an in vivo system.

[0155] Figures 19 to 21 are Volcano plots showing the results of RNA-seq-based gene expression change analysis according to spiperone treatment.

[0156] Figures 22 and 23 are a heat map showing the results of analysis of changes in expression of ER stress-related genes based on RNA-seq according to spiperone treatment, and a graph showing the results of comparison of mRNA levels.

[0157] Figures 24 and 25 are a heat map showing the results of an analysis of changes in the expression of Type I IFN-related genes based on RNA-seq according to spiperone treatment, and a graph showing the results of a comparison of mRNA levels.

[0158] Figure 26 shows the results of Western blot analysis evaluating the ability of spiperone to induce ER stress by activating the PERK-eIF2α-ATF4 axis.

[0159] Figure 27 shows the results of a fluorescent immunostaining analysis evaluating the ability of spiperone to induce ER stress by activating the PERK-eIF2α-ATF4 axis.

[0160] Figure 28 shows the results of immunohistochemical staining analysis evaluating the ability of spiperone to induce ER stress by activating the PERK-eIF2α-ATF4 axis.

[0161] Figures 29 to 31 are graphs and fluorescent immunostaining photographs showing the results of analyzing whether spiperone treatment increases mtROS, leaks mtDNA into the cytoplasm, and induces oxidative DNA damage.

[0162] Figure 32 shows the results of Western blot analysis evaluating the expression levels of proteins involved in the type I IFN activation pathway when treated with spiperone.

[0163] Figure 33 is a graph showing the results of analyzing the dependence of eIF2α and IFI16 on the type I IFN induction mechanism by spiperone.

[0164] Figure 34 shows the in vivo experimental schedule to evaluate the type I IFN dependence of the anti-HBV effect of spiperone.

[0165] Figure 35 is a graph evaluating HBsAg, HBeAg, and HBV DNA levels to evaluate the type I IFN pathway dependency of spiperone using IFNAR deficient mice.

[0166] Figure 36 shows the results of a fluorescent immunostaining analysis to evaluate the type I IFN pathway dependency of spiperone using IFNAR deficient mice.

[0167] Figure 37 shows the results of immunohistochemical staining analysis to evaluate the type I IFN pathway dependency of spiperone using IFNAR deficient mice.

[0168] Figure 38 shows the results of a ChIP assay evaluating the epigenetic inhibition mechanism by which spiperone acts on HBV cccDNA.

[0169] Figure 39 is a schematic diagram of the evaluation of the HBV cccDNA reduction ability of spiperone in an AAV-HBV mouse model (in vivo).

[0170] Figure 40 shows the in vivo experimental schedule for evaluating the cccDNA inhibition ability of spiperone.

[0171] Figure 41 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA to evaluate the anti-HBV activity of spiperone in an in vivocccDNA mouse model.

[0172] Figure 42 shows the results of a fluorescence immunostaining analysis to evaluate the anti-HBV activity of spiperone in an in vivocccDNA mouse model.

[0173] Figure 43 shows the results of immunohistochemical staining analysis to evaluate the anti-HBV activity of spiperone in an in vivocccDNA mouse model.

[0174] Figure 44 shows the results of RT-qPCR quantitative analysis to evaluate the cccDNA inhibitory effect of spiperone using an in vivo cccDNA mouse model.

[0175] Figure 45 shows the results of cccDNA-selective PCR electrophoresis to evaluate the cccDNA inhibitory effect of spiperone using an in vivo cccDNA mouse model.

[0176] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0177] Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0178] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0179] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0180]

[0181] Research for this patented invention was conducted using a compound library provided by the Korea Chemical Bank.

[0182] The research results of this patented invention are the result of research (Project ID: 2710079365) conducted with support from the National Research Foundation of Korea with funding from the government (Ministry of Science and ICT).

[0183] The research results of this patented invention are the result of research (Project ID: 2460002949) conducted with support from the Health and Medical Technology Research and Development Project of the Korea Health Industry Development Institute with funding from the Ministry of Health and Welfare.

[0184]

[0185] Example 1. Evaluation of the antiviral activity and safety of Spiperone (in vitro)

[0186] The antiviral activity of Spiperone was evaluated by assessing various indicators of the HBV virus, such as HBV viral DNA levels, HBsAg and HBeAg levels, pgRNA levels, cccDNA levels, and Capsid protein levels. The stability of Spiperone was evaluated by assessing cytotoxicity.

[0187] Figure 1 is a schematic diagram of the antiviral activity evaluation of spiperone (in vitro).

[0188]

[0189] 1.1 Safety evaluation of spiperone

[0190] Spiperone (SPP) was treated on HepG2.2.15, a subtype of HepG2 (human hepatocellular carcinoma) that stably expresses HBV, and the degree of cytotoxicity was analyzed using the LDH assay (Lactate Dehydrogenase assay) (OD 492 nm).

[0191] Figure 2 is a graph evaluating the degree of cytotoxicity of spiperone.

[0192] As shown in Figure 2, spiperone exhibited a similar level of cytotoxicity to the control group (PBS) and a similar level of cytotoxicity to the existing antiviral agents, entecavir (ETV) and tenofovir (TDF).

[0193]

[0194] 1.2 Measurement of HBsAg, HBeAg, HBV DNA, and HBV pgRNA levels following spiperone treatment in HBV stable cell lines and HBV injection systems

[0195] To evaluate the antiviral effect of spiperone, in vitro experiments were performed using HepG2.2.15 and HBV virion-infected HepG2-NTCP-C4 cell lines.

[0196] HepG2.2.15 is a human hepatoma cell line HepG2, in which the HBV genome is stably integrated, enabling sustained HBV replication and viral antigen expression. After treatment with spiperone, the cell line was compared for its antiviral activity against the commercially available antiviral drug tenofovir (TDF).

[0197] In addition, HepG2-NTCP-C4 is a cell line that enables HBV virion infection through overexpression of the NTCP receptor on the cell membrane of HepG2, and is most similar to human liver cells, so it is widely used in liver disease research. Accordingly, after treating the HepG2-NTCP-C4 cell line infected with HBV virion with spiperone, the virus inhibition ability was compared with that of the existing commercial antiviral agent tenofovir at 5 to 7 days.

[0198] Specifically, the amounts of HBsAg and HBeAg in cell cultures treated with spiperone were measured using HBsAg ELISA kit (KA0289) and HBeAg ELISA kit (NBP2-60029-1), and the OD values ​​were read using TECAN equipment.

[0199] For quantification of HBV DNA, HBV DNA was extracted from cells using a Qiagen blood extraction kit, and DNA levels were measured using RT-qPCR analysis.

[0200] And to measure the pgRNA level during sphepiron treatment, cell pellets were obtained and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). The extracted RNA sample was incubated at 37℃ for 60 minutes using RQ1 DNase (Promega, United Kingdom), and then mixed with 1 μl of stop solution and left at 65℃ for 10 minutes to inactivate DNAse. RNA samples were stored at -80℃, and 1 μg of RNA was reverse-transcribed using Reverse Transcription System (Promega, United Kingdom) for measurement of viral pgRNA. 2 μl of the generated cDNA was used for RT-qPCR analysis, and GAPDH was used as an internal calibration gene for quantification.

[0201] Figure 3 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA according to spiperone treatment in an in vitro system.

[0202] As shown in Fig. 3, in vitro experiments were performed using HepG2.2.15 and HBV virion-infected HepG2-NTCP-C4 cell lines to evaluate the antiviral effect of spiperone. As a result, compared to the existing antiviral drug tenofovir (TDF), the expression of HBsAg and HBeAg was significantly reduced in the spiperone-treated group, and the HBV DNA level was also confirmed to be reduced. In addition, pgRNA, a key indicator reflecting the transcription and replication activity of HBV, was also significantly reduced in the spiperone-treated group compared to the control group.

[0203]

[0204] 1.3 Determination of HBV structural proteins, HBcAg, and Capsid levels following spiperone treatment

[0205] After treating HepG2.2.15 cells with spiperone, the cells were cultured for 24 to 48 hours, and the amount of Capsid protein in the culture medium was confirmed by Western blot and confocal analysis using anti-Hepatitis B Virus Core Antigen antibody (Abcam, #ab8638). The results are shown in Figs. 4 and 5.

[0206] Figure 4 shows the results of Western blot analysis measuring HBcAg and Capsid levels according to spiperone treatment in an in vitro system.

[0207] Figure 5 shows the results of confocal analysis measuring HBcAg and Capsid levels according to spiperone treatment in an in vitro system.

[0208] As shown in Figure 4, the Western blot results showed that the expression of Capsid and HBcAg decreased in a concentration-dependent manner in the spiperone-treated group, and this decrease in expression was also confirmed when compared to the tenofovir (TDF)-treated group.

[0209] As shown in Fig. 5, confocal analysis also showed that the intracellular fluorescence signal of HBcAg was significantly reduced when spiperone was treated compared to the PBS and tenofovir (TDF) treatment groups, confirming that spiperone effectively inhibits HBV capsid protein expression and core particle formation.

[0210]

[0211] 1.4 Confirmation of the level of cccDNA, the template for HBV replication and the core of infection

[0212] To evaluate the inhibitory effect of spiperone on HBV cccDNA, cccDNA was purified from HepG2.2.15 and HepG2-NTCP-C4, an HBV-infected cell line, and selectively amplified, followed by agarose gel electrophoresis and RT-qPCR analysis.

[0213] Specifically, to measure cccDNA, linearized 1.2X genotype C2 HBV plasmid was transfected into HepG2.2.15 and HepG2-NTCP-C4 cell lines, and lysis buffer A (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 50 mM NaCl, 1% NP-40) was added to the obtained cells and incubated at 4°C for 10 minutes. The lysate was then centrifuged at 14,000 rpm for 10 minutes to obtain a nuclear pellet, which was resuspended in lysis buffer B (10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 0.5% SDS). Next, sonication (3 to 4 pulses, 5 seconds) was performed to disrupt the nuclei, and then 0.5 mg / ml of proteinase K was added and incubated overnight at 37°C.

[0214] The next day, the lysate was treated with phenol-chloroform (1:1) and ethanol precipitation to extract DNA, and 1 μg of the extracted DNA was treated with 10 U of PSAD (Plasmid Safe DNase I, Epicenter, PA, United States) at 37°C for 45 min, and then the enzyme action was stopped at 70°C to remove linear DNA, thereby purifying only cccDNA. The cccDNA thus obtained was quantified by RT-qPCR using cccDNA primers [forward: 5'- CCG TGT GCA CTT CGC TTC A - 3' (SEQ ID NO: 1); reverse: 5'- GCA CAG CTT GGA GGC TTG A - 3' (SEQ ID NO: 2)], and mtDNA was used as an internal calibration.

[0215] HBV cccDNA was amplified using cccDNA-selective primers [forward: 5′ GCG CAC CTC TCT TTA-3′1523-1540) (SEQ ID NO: 3); reverse: 5′ CAC AGC TTG GAG GC-3′ (position 1886-1870) (SEQ ID NO: 4)] and cccDNA-nonselective primers [forward: 5′ TCT ATG GAA GGC GGG TA-3′ (position 2755-2774) (SEQ ID NO: 5); reverse: 5′ TCC AGC TCC TAC CTT GT-3′ (position 3002-2983) (SEQ ID NO: 6)], and the amplified products were detected by 1% agarose gel electrophoresis.

[0216] The results are shown in Figures 6 and 7.

[0217] Figure 6 shows the results of agarose gel electrophoresis analysis measuring the cccDNA level according to spiperone treatment in an in vitro system.

[0218] Figure 7 shows the results of RT-qPCR analysis measuring cccDNA levels according to spiperone treatment in an in vitro system.

[0219] As shown in Fig. 6, the electrophoresis analysis results showed that the cccDNA band intensity was significantly reduced in the spiperone-treated group compared to the PBS and tenofovir (TDF)-treated groups.

[0220] As shown in Figure 7, the RT-qPCR results also confirmed a significant decrease in cccDNA only in the spiperone-treated group.

[0221]

[0222] Example 2. Evaluation of the synergistic effect of spiperone and antiviral agents.

[0223] Synergy Finder was used to evaluate the synergistic effect of spiperone and antiviral drugs currently used in clinical practice.

[0224] Specifically, HepG2.2.15 cells were treated with spiperone and tenofovir (TDF) at various concentrations, and the amount of HBsAg in the culture medium was measured 48 hours later using ELISA. Synergy scores were calculated using the Synergy Finder program. A synergistic effect was considered to exist if the synergy score was 5 or higher using the ZIP calculation method.

[0225] Figures 8 and 9 show the results of evaluating the synergistic effect of spiperone and tenofovir.

[0226] As shown in Figures 8 and 9, when HepG2.2.15 cells were treated with spiperone and tenofovir together, a high score of 9 or more was confirmed, confirming a high synergistic effect.

[0227] Through this synergistic effect of spiperone and antiviral agents, it can be judged that the combined use of spiperone can be very advantageously applied in clinical practice in the administration of antiviral agents or viral infection treatment agents, such as applying spiperone as an additional drug for HBV patients taking existing nucleotide analog antiviral drugs (e.g., tenofovir).

[0228]

[0229] Example 3. Evaluation of the antiviral activity of spiperone (in vivo)

[0230] After injecting spiperon and standard drugs into HBV transgenic mice (C57B1 / 6N mice expressing pHY92-1.1x-HBV-full genome plasmid), serum was isolated and obtained through orbital blood collection, and the levels of HBV DNA, HBeAg, and HBsAg in the serum were measured using RT-PCR or ELISA. In addition, the level of viral pgRNA in mouse liver tissue was analyzed to evaluate the antiviral activity of spiperon against HBV in the mouse model.

[0231] Figure 10 is a schematic diagram of the antiviral activity evaluation of spiperone (in vivo).

[0232]

[0233] 3.1 Confirmation of antiviral activity following intraperitoneal injection of spiperone

[0234] To evaluate the antiviral activity of spiperone in HBV-transgenic (C57BL / 6) mice, PBS, tenofovir (TDF) (500 μg / mouse), and spiperone (1.5 mg / kg) were administered intraperitoneally daily for 8 weeks.

[0235] Figure 11 shows the schedule of in vivo experiments to evaluate the antiviral activity and therapeutic efficacy against viral infections following intraperitoneal injection of spiperone.

[0236] The experimental results are shown in Figures 12 to 14.

[0237] Figure 12 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA following treatment with spiperone (intraperitoneal injection) in an in vivo system.

[0238] Figure 13 shows the results of immunohistochemical staining (IHC) analysis of HBcAg in liver tissue following spiperone treatment (intraperitoneal injection) in an in vivo system.

[0239] Figure 14 shows the results of fluorescence immunostaining analysis of nuclei and HBsAg in liver tissue following spiperone treatment (intraperitoneal injection) in an in vivo system.

[0240] As shown in Figures 12 to 14, the serum analysis results showed that the levels of HBsAg, HBeAg, and HBV DNA in the spiperone-treated group were significantly reduced compared to the control group (PBS) and tenofovir (TDF)-treated groups, and the pgRNA expression level in the liver was also significantly suppressed. HBcAg immunohistochemical staining (IHC) of liver tissue also confirmed that HBV-infected cells were significantly reduced in the spiperone-treated group, and the HBsAg fluorescent immunostaining results also visually confirmed that HBV antigen expression in the liver tissue was significantly reduced in the spiperone-treated group.

[0241]

[0242] 3.2 Confirmation of antiviral activity following oral administration of spiperone (oral gavage)

[0243] To evaluate the antiviral activity of spiperone when administered orally to HBV-transgenic (C57BL / 6) mice, PBS, tenofovir (TDF) (500 μg / mouse), and spiperone (1.5 mg / kg) were administered orally daily for 8 weeks.

[0244] Figure 15 shows the schedule of in vivo experiments to evaluate the antiviral activity and therapeutic efficacy against viral infections following oral administration of spiperone.

[0245] The experimental results are shown in Figures 16 to 18.

[0246] Figure 16 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA following spiperone treatment (oral administration) in an in vivo system.

[0247] Figure 17 shows the results of immunohistochemical staining (IHC) analysis of HBcAg in liver tissue following spiperone treatment (oral administration) in an in vivo system.

[0248] Figure 18 shows the results of fluorescence immunostaining analysis of nuclei and HBsAg in liver tissue following spiperone treatment (oral administration) in an in vivo system.

[0249] As shown in Figures 16 to 18, the serum analysis results showed that the levels of HBsAg and HBeAg were significantly reduced in the spiperone-treated group compared to the control group (PBS) and tenofovir (TDF)-treated group, and the levels of HBV DNA and pgRNA expression in the liver were also significantly suppressed. In HBcAg immunohistochemical staining (IHC) of liver tissue, it was confirmed that the number of HBcAg was significantly reduced in the spiperone-treated group, indicating a significant decrease in HBV-infected cells. In addition, the results of HBsAg fluorescent immunostaining also visually confirmed that the fluorescence intensity was significantly reduced in the spiperone-treated group, indicating a significant decrease in HBV antigen expression in the liver tissue.

[0250]

[0251] Example 4. Analysis of the antiviral and cccDNA inhibitory mechanisms of spiperone.

[0252] 4.1 RNA-seq and mRNA level analysis

[0253] RNA sequencing analysis was performed using HepG2-NTCP-C4 cells infected with HBV virions 12 hours prior to infection. The experimental results are shown in Figures 19 to 25.

[0254] Figures 19 to 21 are Volcano plots showing the results of RNA-seq-based gene expression change analysis according to spiperone treatment.

[0255] Figures 22 and 23 are a heat map showing the results of analysis of changes in expression of ER stress-related genes based on RNA-seq according to spiperone treatment, and a graph showing the results of comparison of mRNA levels.

[0256] Figures 24 and 25 are a heat map showing the results of an analysis of changes in the expression of Type I IFN-related genes based on RNA-seq according to spiperone treatment, and a graph showing the results of a comparison of mRNA levels.

[0257] As shown in Figures 19 to 25, RNA-seq-based pathway enrichment analysis results showed that ER stress response, NF-κB signaling, and Type I IFN-related signaling pathways were significantly activated, and in particular, the expression of ER stress-related genes was extensively increased (Figures 19 to 21). In addition, not only in Volcano plot analysis but also in heatmaps and graphs, the main ER stress pathways, including the PERK-eIF2α-ATF4 axis, were strongly induced (Figures 22 and 23), and the expression of Type I IFN and NF-κB-related genes (MX1, ISG15, IFNB1, OAS1, etc.) was also significantly increased (Figures 24 and 25).

[0258] These results suggest that ER stress induced by spiperone activates the STING-TBK1-IRF3 / 7 pathway via cytoplasmic HBV DNA leakage and IFI16 recognition, which in turn induces type I IFN secretion and interferon-stimulated gene (ISG) induction, leading to HBV suppression. Furthermore, this immune activation response can further amplify ER stress, suggesting that spiperone may have a novel mechanism of action that effectively inhibits HBV replication based on the link between ER stress and the immune response.

[0259]

[0260] 4.2 Proof of ER stress induction ability of spiperone

[0261] To determine whether spiperone induces endoplasmic reticulum stress (ER stress), the expression of proteins belonging to the PERK-eIF2α-ATF4 pathway was analyzed using HepG2.2.15 cells. The results are shown in Figure 26.

[0262] Figure 26 shows the results of Western blot analysis evaluating the ability of spiperone to induce ER stress by activating the PERK-eIF2α-ATF4 axis.

[0263] As shown in Figure 26, Western blot analysis results showed that the expression of p-PERK and p-eIF2α in the spiperone-treated group significantly increased compared to the control group (PBS-treated group) and the comparison group (TDF-treated group), and the expression of the downstream transcription factor ATF4 also significantly increased. These results demonstrate that spiperone strongly induces the ER stress signaling pathway centered on the PERK axis.

[0264]

[0265] In addition, to confirm the ER stress-inducing effect of spiperone at the cellular and tissue levels, the expression of ATF4 and p-PERK was analyzed using immunofluorescence staining and immunohistochemistry (IHC) staining, respectively, in HepG2.2.15 cells and HBV transgenic mice (HBV TG mice). The results are shown in Figures 27 and 28.

[0266] Figure 27 shows the results of a fluorescent immunostaining analysis evaluating the ability of spiperone to induce ER stress by activating the PERK-eIF2α-ATF4 axis.

[0267] Figure 28 shows the results of immunohistochemical staining analysis evaluating the ability of spiperone to induce ER stress by activating the PERK-eIF2α-ATF4 axis.

[0268] As shown in Figures 27 and 28, in HepG2.2.15 cells, the fluorescence signal of ATF4 (Alexa Fluor 488) in the nucleus was significantly increased when spiperone was treated compared to the PBS and TDF treatment groups, suggesting that spiperone strongly induces ATF4 expression within the cells. In addition, in mouse liver tissues, the immunoreactivity of p-PERK and ATF4 was significantly increased in the spiperone treatment group, and strong positive signals were observed throughout the liver. These results demonstrate that spiperone induces an ER stress response centered on the PERK-eIF2α-ATF4 axis even at the tissue level.

[0269]

[0270] 4.3 Analysis of ER stress-induced mitochondrial damage and mtDNA leakage by spiperone

[0271] To determine whether endoplasmic reticulum (ER) stress induced by spiperone treatment leads to mitochondrial dysfunction and oxidative stress, MitoSOX-based flow cytometry, quantitative analysis of cytosolic mitochondrial DNA (mtDNA), and immunofluorescence staining analysis of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidized DNA damage, were performed. The results are shown in Figures 29 to 31.

[0272] Figures 29 to 31 are graphs and fluorescent immunostaining photographs showing the results of analyzing whether spiperone treatment increases mtROS, leaks mtDNA into the cytoplasm, and induces oxidative DNA damage.

[0273] As shown in Figures 29 to 31, the MitoSOX fluorescence signal in the spiperone-treated group significantly increased compared to the control group (PBS) and the comparison group (TDF), suggesting a significant increase in mitochondrial ROS production. In particular, the fluorescence signal was at a similar level to that in the positive control group, Rotenone-treated group, confirming that spiperone induces strong mitochondrial oxidative stress.

[0274] In addition, qPCR-based cytoplasmic mtDNA quantitative analysis showed that the ratios of ND1 / 18S and ND2 / 18S were significantly increased in the spiperone-treated group, indicating that damaged mtDNA was leaked into the cytoplasm.

[0275] And according to the results of immunofluorescence staining analysis, the 8-OHdG signal increased in a concentration-dependent manner as the concentration of spiperone increased, which means that DNA oxidized by spiperone treatment accumulated in the cells.

[0276]

[0277] 4.4 Confirmation of activation of type I interferon (IFN) signaling pathway by spiperone

[0278] To confirm whether spiperone activates the type I IFN immune pathway, the expression and phosphorylation levels of proteins involved in the IFI16-STING-TBK1-IRF3 axis, a pathway that detects damaged DNA within cells and transmits immune signals, were examined using Western blot analysis. The results are shown in Figure 32.

[0279] Figure 32 shows the results of Western blot analysis evaluating the expression levels of proteins involved in the type I IFN activation pathway when treated with spiperone.

[0280] As shown in Figure 32, in the spiperone-treated group, the phosphorylation levels of proteins included in the signal transduction pathway were significantly increased compared to the control group (PBS) and the comparison group (TDF), demonstrating that spiperone can activate the type I IFN-induced pathway that detects damaged DNA within cells at the molecular level.

[0281]

[0282] In addition, to confirm whether the antiviral effect of spiperone is related to type I IFN signaling induced by the eIF2α and IFI16 pathways, HBV DNA quantitative analysis was performed using HepG2.2.15 cells into which siRNA for the respective genes was introduced, and IFN-β promoter activity analysis was performed using hMH55-ISRE-IFNβreporter cells. The results are shown in Fig. 33.

[0283] Figure 33 is a graph showing the results of analyzing the dependence of eIF2α and IFI16 on the type I IFN induction mechanism by spiperone.

[0284] As shown in Figure 33, in the eIF2α-siRNA treatment group, the HBV DNA inhibitory effect of spiperone was significantly reduced compared to the scramble-siRNA treatment group, and the level of luciferase expression linked to the IFNβ promoter was also reduced. In the IFI16-siRNA treatment condition, both the HBV DNA inhibitory effect and luciferase expression were similarly reduced. These results demonstrate at the molecular level that spiperone exhibits antiviral activity through the ER stress and DNA sensing-based type I IFN pathway.

[0285]

[0286] Example 5. Type I IFN-dependent evaluation of the anti-HBV effect of spiperone using IFNAR-deficient mice (in vivo)

[0287] To determine whether the antiviral effect of spiperone is dependent on the type I IFN signaling pathway, C57BL / 6 wild-type mice (WT) and IFNAR knockout mice (IFNAR knockout; IFNAR KO) were infected with HBV via hydrodynamic injection (HDI) using pAAV-HBV1.2x plasmid. After 2 weeks, spiperone (1.5 mg / kg) or PBS was administered intraperitoneally daily.

[0288] Figure 34 shows the in vivo experimental schedule to evaluate the type I IFN dependence of the anti-HBV effect of spiperone.

[0289] The experimental results are shown in Figs. 35 to 37.

[0290] Figure 35 is a graph evaluating HBsAg, HBeAg, and HBV DNA levels to evaluate the type I IFN pathway dependency of spiperone using IFNAR deficient mice.

[0291] Figure 36 shows the results of a fluorescent immunostaining analysis to evaluate the type I IFN pathway dependency of spiperone using IFNAR deficient mice.

[0292] Figure 37 shows the results of immunohistochemical staining analysis to evaluate the type I IFN pathway dependency of spiperone using IFNAR deficient mice.

[0293] As shown in Figures 35 to 37, in wild-type (WT) mice, the levels of HBsAg, HBeAg, HBV DNA, and pgRNA were all significantly reduced when spiperone was treated, but most of these inhibitory effects were lost in IFNAR KO mice. In addition, in immunofluorescence staining analysis of liver tissues, the fluorescence signal of HBcAg was significantly reduced in WT mice when spiperone was treated, whereas there was no significant difference in IFNAR KO mice compared to the PBS treatment group. The results of immunohistochemical staining (IHC) for HBsAg also showed the same trend.

[0294] These results demonstrate in vivo that the antiviral effect of spiperone is dependent on the type I IFN signaling pathway.

[0295]

[0296] Example 6. Confirmation of the ability of spiperone to control and eliminate HBV cccDNA.

[0297] 6.1 Confirmation of the epigenetic cccDNA regulation ability of spiperone through ChIP analysis

[0298] To determine the effect of spiperone on the regulation of HBV cccDNA transcriptional activity, a ChIP (Chromatin Immunoprecipitation) assay was performed using HBV-infected HepG2-NTCP-C4 cells. After HBV virion infection, spiperone was treated. Histone proteins bound to cccDNA were immunoprecipitated using antibodies against histone acetylation markers (H3K5ac, H3K27ac, AcH3, and AcH4). The binding levels of the corresponding histone markers were then quantified by qPCR analysis using cccDNA-specific primers. The results are shown in Figure 38.

[0299] Figure 38 shows the results of a ChIP assay evaluating the epigenetic inhibition mechanism by which spiperone acts on HBV cccDNA.

[0300] As shown in Figure 38, the levels of histone acetylation markers (H3K5ac, H3K27ac, AcH3, AcH4) bound to HBV cccDNA were generally confirmed to decrease in the spiperone-treated group compared to the virus-only group. These results suggest that spiperone can induce an antiviral effect through an epigenetic regulatory mechanism that suppresses the transcriptional activity of HBV genes by inhibiting histone acetylation of HBV cccDNA.

[0301]

[0302] 6.2 Confirmation of the cccDNA inhibition ability of spiperone using an AAV-HBV-induced cccDNA mouse model (in vivo)

[0303] Mice transduced with AAV-HBV1.04x (cccDNA mouse model) are an experimental animal model for evaluating HBV replication and cccDNA persistence. Unlike transgenic mice, the HBV genome does not integrate into the host DNA, and the formation and regulation of HBV cccDNA within hepatocytes are possible. Using this mouse model, the antiviral efficacy and cccDNA inhibition ability of the drug can be evaluated by analyzing changes in various viral markers and cccDNA levels in liver tissue after injection of spiperone.

[0304] Figure 39 is a schematic diagram of the evaluation of the HBV cccDNA reduction ability of spiperone in an AAV-HBV mouse model (in vivo).

[0305] Specifically, to evaluate the in vivocccDNA inhibition ability of spiperone, AAV-HBV1.04x (8×10 10A mouse model of HBV infection was established by intravenous administration of (vg / mouse) to C57BL / 6 male mice. After 7 days of infection, spiperone (1.5 mg / kg) or PBS was administered intraperitoneally (ip) daily for 4 weeks. At the end of the experiment, serum and liver tissues were collected, and the levels of HBsAg, HBeAg, HBV DNA, pgRNA, and cccDNA were quantitatively analyzed using ELISA, RT-qPCR, and cccDNA-selective PCR, respectively. In addition, the expression of HBV antigens in liver tissues was evaluated through immunofluorescence staining for HBsAg and immunohistochemical staining for HBcAg.

[0306] Figure 40 shows the in vivo experimental schedule for evaluating the cccDNA inhibition ability of spiperone.

[0307] The experimental results are shown in Figs. 41 to 45.

[0308] Figure 41 is a graph evaluating the levels of HBsAg, HBeAg, HBV DNA, and pgRNA to evaluate the anti-HBV activity of spiperone in an in vivocccDNA mouse model.

[0309] Figure 42 shows the results of a fluorescence immunostaining analysis to evaluate the anti-HBV activity of spiperone in an in vivocccDNA mouse model.

[0310] Figure 43 shows the results of immunohistochemical staining analysis to evaluate the anti-HBV activity of spiperone in an in vivocccDNA mouse model.

[0311] Figure 44 shows the results of RT-qPCR quantitative analysis to evaluate the cccDNA inhibitory effect of spiperone using an in vivo cccDNA mouse model.

[0312] Figure 45 shows the results of cccDNA-selective PCR electrophoresis to evaluate the cccDNA inhibitory effect of spiperone using an in vivo cccDNA mouse model.

[0313] As shown in Figures 41 to 43, in AAV-HBV-infected mice administered spiperone, the levels of HBsAg, HBeAg, and HBV DNA in the serum were significantly reduced, and the pgRNA level in the liver tissue was also significantly suppressed as determined by RT-qPCR analysis. The results of immunofluorescence staining (HBsAg) and immunohistochemical staining (HBcAg) analyses of liver tissue also showed that the expression of HBV antigens was significantly reduced in the spiperone-treated group.

[0314] In particular, as shown in Figure 44, when cccDNA in liver tissue was quantified using RT-qPCR, it was confirmed that the amount of cccDNA was significantly reduced in the spiperone-treated group, suggesting that spiperone can quantitatively suppress the amount of HBV cccDNA itself in vivo.

[0315] Furthermore, as shown in Figure 45, PCR electrophoresis analysis using cccDNA-selective primers and non-selective primers performed separately revealed that a strong cccDNA band was detected only under virus-only conditions, whereas the intensity of the band was significantly weakened in the spiperone-treated group. This serves as qualitative evidence that spiperone substantially reduces cccDNA levels in liver tissue.

[0316]

[0317] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the experimental examples and embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. An antiviral composition containing Spiperone.

2. An antiviral composition according to claim 1, wherein the virus is at least one selected from the group consisting of DNA viruses, RNA viruses, and retroviruses.

3. An antiviral composition according to claim 1, wherein the virus is a virus that produces cccDNA in the nucleus of a host cell.

4. In the first paragraph, the antiviral composition has an antiviral activity by activating the PERK-eIF2α-ATF4 signal transduction pathway, which is one of the endoplasmic reticulum stress pathways, and inducing the secretion of type 1 interferon.

5. An antiviral composition according to claim 1, wherein the antiviral composition further comprises an antiviral agent.

6. In paragraph 5, the antiviral agent is at least one selected from the group consisting of tenofovir, entecavir, adefovir, sofovir, cidofovir, acyclovir, famciclovir, valacyclovir, ganciclovir, amprenavir, abacavir, ansamycin, darunavir, delaviridine, efavirenz, etravirine, hypericin, indinavir, lamivudine, robucavir, nelfinavir, nevirapine, novaprene, ritonavir, saquinavir, stavudine, tipranavir, virazole, ribavirin, zalcitabine, zidovudine, maraviroc, raltegravir, elvitegravir, didanosine, emtricitabine, lopinavir, atazanavir, enfuvertide, and clevudine. An antiviral composition.

7. A pharmaceutical composition for the prevention or treatment of viral infection, comprising spironolactone or a pharmaceutically acceptable salt thereof as an active ingredient.

8. A pharmaceutical composition according to claim 7, wherein the virus is at least one selected from the group consisting of DNA viruses, RNA viruses, and retroviruses.

9. A pharmaceutical composition according to claim 7, wherein the virus is a virus that produces cccDNA in the nucleus of a host cell.

10. A pharmaceutical composition according to claim 7, wherein the pharmaceutical composition further comprises an antiviral agent.

11. In the 10th paragraph, the antiviral agent is at least one selected from the group consisting of tenofovir, entecavir, adefovir, sofovir, cidofovir, acyclovir, famciclovir, valacyclovir, ganciclovir, amprenavir, abacavir, ansamycin, darunavir, delaviridine, efavirenz, etravirine, hypericin, indinavir, lamivudine, robucavir, nelfinavir, nevirapine, novaprene, ritonavir, saquinavir, stavudine, tipranavir, virazol, ribavirin, zalcitabine, zidovudine, maraviroc, raltegravir, elvitegravir, didanosine, emtricitabine, lopinavir, atazanavir, enfuvertide, and clevudine. A pharmaceutical composition.

12. A pharmaceutical composition for the prevention or treatment of hepatitis B, comprising spironolactone or a pharmaceutically acceptable salt thereof as an active ingredient.

13. A pharmaceutical composition according to claim 12, wherein the pharmaceutical composition further comprises an antiviral agent.

14. In paragraph 13, the antiviral agent is at least one selected from the group consisting of tenofovir, entecavir, adefovir, sofovir, cidofovir, acyclovir, famciclovir, valacyclovir, ganciclovir, amprenavir, abacavir, ansamycin, darunavir, delaviridine, efavirenz, etravirine, hypericin, indinavir, lamivudine, robucavir, nelfinavir, nevirapine, novaprene, ritonavir, saquinavir, stavudine, tipranavir, virazole, ribavirin, zalcitabine, zidovudine, maraviroc, raltegravir, elvitegravir, didanosine, emtricitabine, lopinavir, atazanavir, enfuvertide, and clevudine. A pharmaceutical composition.

15. Antiviral health functional food containing spironolactone.

16. Antiviral feed composition containing spirferon.

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