Prophylactic administration method against respiratory virus, comprising administering interferon-beta to potential respiratory virus-infected subject

Abstract

To provide a pharmaceutical composition for restraining self-replication infection of respiratory viruses in a subject exposed to potential respiratory virus infection, the composition comprising interferon-beta as an active ingredient.SOLUTION: Provided is a pharmaceutical composition for pre-exposure prophylaxis or post-exposure prophylaxis of infection by a coronavirus, an influenza virus, or an RS virus in a subject, the composition comprising mutant interferon-beta as an active ingredient, the mutant interferon-beta being administered by inhalation and the mutant interferon-beta comprising a substitution of threonine at arginine 27 of wild-type interferon-beta.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority based on Korean Patent Application No. 2021-0004780, filed on January 13, 2021, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a method for prophylactic administration against respiratory viruses, which comprises administering interferon beta to an individual with a potential respiratory virus infection. More specifically, the present invention relates to a method for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection, which comprises administering interferon beta as an active ingredient to respiratory cells by inhalation, by administering the interferon beta directly to cells infected or potentially infected with the respiratory virus. [Background technology]

[0003] Respiratory diseases caused by viral infections of the respiratory tract are the most common illnesses, accounting for approximately half of all infectious diseases. Respiratory virus infections occur primarily in immunocompromised patients such as children and the elderly, and the most well-known representative respiratory virus infections include adenovirus, parainfluenza virus (PIV), respiratory syncytial virus (RSV), rhinovirus, and coronavirus.

[0004] Coronaviruses, a type of respiratory virus, are divided into four genera: the alpha and beta genera infect both humans and animals, while the gamma and delta genera only infect animals. There are six known species of coronaviruses that can infect humans. Of these, four (229E, OC43, NL63, and HKU1) are known to cause the common cold, while the remaining two, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), can cause severe pneumonia in humans.

[0005] In particular, a new respiratory virus that was first identified in pneumonia patients in Wuhan City, Hubei Province, China in December 2019 spread widely and severely impacted the medical system. The novel coronavirus that emerged in 2019 is a different lineage from the previous MERS (MERS-CoV) and SARS (SARS-CoV) viruses and is the seventh coronavirus species to infect humans. This virus was named "SARS-CoV-2" and is the cause of the disease known as "Coronavirus Disease 2019 (COVID-19)."

[0006] The initial symptoms of respiratory viral infections are similar regardless of the virus, making it difficult to distinguish the cause based on clinical symptoms alone. Because respiratory viral infections are highly contagious, they can cause major epidemics in a short period of time. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Registration No. 10-2018201 [Patent Document 2] Korean Patent Registration No. 10-1800366 [Patent Document 3] Korean Patent Publication No. 10-2019-0063512 [Non-patent literature]

[0008] [Non-Patent Document 1] Zhu, Na, et al. "A novel coronavirus from patients with pneumonia in China, 2019." New England Journal of Medicine (2020). Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, while researching the use of interferon beta to suppress respiratory viral infections, the inventors discovered that when interferon beta is appropriately administered to the respiratory area where respiratory viral infections mainly occur, it can suppress self-replicating infection of respiratory viruses in individuals who are at risk of being exposed to respiratory viral infections, i.e., individuals who have been exposed to potential respiratory viral infections, and thus completed the present invention.

[0010] It is therefore an object of the present invention to provide a pharmaceutical composition for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory viral infection, comprising interferon beta as an active ingredient.

[0011] It is also an object of the present invention to provide a pharmaceutical composition comprising interferon beta for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection.

[0012] It is also an object of the present invention to provide a pharmaceutical composition for suppressing self-replicating respiratory viral infection in an individual exposed to a latent respiratory viral infection, consisting essentially of interferon beta.

[0013] Another object of the present invention is to provide a method for post-exposure prophylaxis (PEP) to protect an individual from self-replicating infection by a respiratory virus after exposure to a potential respiratory viral infection, characterized by administering a prophylactically effective amount of interferon beta to an individual.

[0014] It is yet another object of the present invention to provide a method for protecting an individual from self-replicating infection by a respiratory virus, comprising administering a prophylactically effective amount of interferon beta to the individual after exposure to a potential respiratory viral infection.

[0015] Another object of the present invention is to provide the use of interferon beta for the manufacture of a medicament for the suppression of self-replicating respiratory viral infection in an individual exposed to a potential respiratory viral infection.

[0016] Another object of the present invention is to provide a method for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory viral infection, comprising administering to the individual in need thereof an effective amount of a composition comprising interferon beta as an active ingredient. [Means for solving the problem]

[0017] To achieve the above object, the present invention provides a pharmaceutical composition for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection, comprising interferon beta as an active ingredient.

[0018] The present invention also provides a pharmaceutical composition comprising interferon beta for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection.

[0019] The present invention also provides a pharmaceutical composition for suppressing self-replicating respiratory viral infection in an individual exposed to a latent respiratory viral infection, the composition consisting essentially of interferon beta.

[0020] To achieve another object of the present invention, the present invention provides a method for post-exposure prophylaxis (PEP) to protect an individual from self-replicating infection by a respiratory virus after exposure to a potential respiratory viral infection, characterized by administering a prophylactically effective amount of interferon beta to an individual.

[0021] In order to achieve another object of the present invention, the present invention provides a method for protecting an individual from self-replicating infection by a respiratory virus, comprising administering a prophylactically effective amount of interferon beta to the individual after exposure to a potential respiratory virus infection.

[0022] In order to achieve another object of the present invention, the present invention provides the use of interferon beta for producing a preparation for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection.

[0023] In order to achieve another object of the present invention, the present invention provides a method for suppressing self-replicating infection of a respiratory virus in an individual who has been exposed to a potential respiratory virus infection, which method comprises administering to the individual in need thereof an effective amount of a composition containing interferon beta as an active ingredient.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide one of skill with general definitions of some terms used in the specification of the present invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

[0025] The present invention will be described in detail below.

[0026] The present invention provides a pharmaceutical composition for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection, comprising interferon beta as an active ingredient.

[0027] Interferon beta is a globular protein with five alpha helices and is 22 kDa in size. It has been reported to be effective in treating cancer, autoimmune diseases, viral infections, HIV-related diseases, hepatitis C, and rheumatoid arthritis due to its various immunological activities, such as antiviral activity, cell growth inhibition or antiproliferation activity, lymphocyte toxicity enhancement activity, immunomodulatory activity, target cell differentiation induction or inhibition activity, macrophage activation activity, cytokine production enhancement activity, cytotoxic T cell effectiveness enhancement activity, and natural killer cell enhancement activity.

[0028] The interferon beta of the present invention may be natural (wild-type) or mutant interferon beta. Preferably, the interferon beta (interferon beta polypeptide or interferon beta protein) of the present invention is a polypeptide in which arginine at position 27 (R27) of wild-type interferon beta has been mutated to threonine (R27T), and which consists of the amino acid sequence represented by SEQ ID NO: 1. The interferon beta of the present invention can be bound to two sugar chains at amino acids 25 and 80 of SEQ ID NO: 1 and should be understood as a polypeptide having interferon beta activity. Preferably, the interferon beta of the present invention is human interferon beta.

[0029] In the present invention, interferon beta is administered by directly reaching cells infected or latently infected with a coronavirus. More specifically, interferon beta is administered by inhalation, contacting cells infected or latently infected with a respiratory virus, thereby reaching the cells infected or latently infected with a respiratory virus and functioning by binding to the interferon beta receptor of the cells.

[0030] Administration by inhalation is via the oral or nasal cavity due to the structure of the respiratory tract, and can be administered, for example, by spraying or injecting interferon beta or a carrier containing interferon beta in a liquid, aerosol, or gas phase so that it contacts respiratory cells via the oral or nasal cavity.

[0031] In the present invention, the respiratory system generally refers to the trachea through which breathing takes place, and refers to each trachea that connects the nose and mouth to the airways and lungs. Preferably, in the present invention, the respiratory system includes the nasal mucosa, nasopharynx, oropharynx, laryngopharynx, larynx, trachea, bronchi, bronchioles, and lungs.

[0032] In the present invention, the respiratory virus may be selected from the group consisting of adenovirus, avian influenza virus, bocavirus, coronavirus, cytomegalovirus, hantavirus, herpes simplex virus, influenza virus, measles virus, metapneumovirus, parainfluenza virus, respiratory syncytial virus, rhinovirus, and varicella-zoster virus. Preferably, the respiratory virus of the present invention is a coronavirus.

[0033] The individual in the present invention may be uninfected with coronavirus or in the early stage of coronavirus infection. The individual may be an individual with clinical or regulatory concerns about exposure to coronavirus (e.g., an individual who has been in close contact with a coronavirus-infected individual, or an individual who has been in contact with a close contact, an individual who has been advised or forced to quarantine, or medical staff involved in coronavirus treatment), an individual who is an asymptomatic coronavirus transmitter, or an individual with early coronavirus infection. Coronavirus-infected individuals can be easily identified by those skilled in the art using known coronavirus test kits. Preferably, in the present invention, the individual may be uninfected with coronavirus or in the early stage of coronavirus infection. Therefore, the present invention can suppress or reduce the asymptomatic spread of the virus to those around them by individuals infected with coronavirus.

[0034] Such pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) can be applied in a similar manner to methods for preventing HIV (human immunodeficiency virus).

[0035] In the present invention, an individual in the early stage of coronavirus infection may be an individual who does not exhibit any of the symptoms of coronavirus infection selected from the group consisting of fever, dry cough, fatigue, feeling unwell, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, and discoloration of the fingers or toes.

[0036] In the present invention, the coronavirus is defined as i) an alphacoronavirus, such as 229E or NL63 that infect humans, or porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), canine coronavirus (CCoV), feline coronavirus (FCoV), Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Rhinolophus bat coronavirus HKU2, or Asian yellow house bat (Scotophilus bat coronavirus 512; ii) an alphacoronavirus, such as OC43, HKU1, SARS-CoV, MERS-CoV, or SARS-CoV-2 that infect humans, or porcine hemagglutinating encephalomyelitis virus that does not infect humans. encephalomyelitis virus (PHEV), bovine coronavirus (BCoV), equine coronavirus (EqCoV), murine coronavirus (MuCoV), Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Rousettus bat coronavirus HKU9 are betacoronaviruses; iii) avian coronaviruses that do not infect humans, such as beluga whale coronavirus SW1, and gammacoronaviruses;iv) It may be one of the deltacoronaviruses, which are Bulbul coronavirus HKU11, Thrush coronavirus HKU12, and Munia coronavirus HKU13, which do not infect humans;

[0037] In the present invention, the self-replicating infection refers to the fact that the coronavirus replicates in the respiratory cells of the individual and infects surrounding cells. The life cycle of coronaviruses is well known.

[0038] Meanwhile, the present invention provides a post-exposure prophylaxis (PEP) method for protecting an individual from self-replicating infection by a respiratory virus after exposure to a potential respiratory viral infection, the method comprising administering a prophylactically effective amount of interferon beta to the individual.

[0039] The present invention also provides a method of protecting an individual from self-replicating infection by a respiratory virus, comprising administering a prophylactically effective amount of interferon beta to the individual after exposure to a potential respiratory viral infection.

[0040] Also provided is a method for protecting an individual from self-replicating infection by coronavirus, which additionally comprises administering in combination or sequentially a currently clinically active or known coronavirus therapeutic agent.

[0041] In the above, the administration of the coronavirus therapeutic agent can be oral, inhaled, intraperitoneal, or intravenous administration.

[0042] The pharmaceutical composition according to the present invention may contain the coronavirus therapeutic agent of the present invention alone, or may additionally contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0043] The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Furthermore, carriers for parenteral administration may include water, a suitable oil, physiological saline, aqueous glucose, glycol, etc., and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be found in the art.

[0044] The composition of the present invention preferably contains the active ingredient and the pharmaceutically acceptable carrier in a weight ratio of 0.1 to 99.9:99.9 to 0.1, but is not limited to this.

[0045] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method, for example, orally or parenterally, and can be formulated into a preparation for oral administration or parenteral administration depending on the administration route.

[0046] For oral administration, the compositions of the present invention can be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and the like, using methods known in the art. For example, oral formulations can be prepared by blending the active ingredient with a solid excipient, pulverizing the mixture, and adding appropriate additives to form a granular mixture to obtain tablets or sugar-coated tablets. Examples of suitable excipients include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or potassium alginate can be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may additionally contain an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

[0047] For parenteral administration, the formulations may be formulated in the form of injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art, and these formulations are described in formulas generally known in all medicinal chemistry fields.

[0048] The total effective amount of the composition of the present invention can be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The content of the active ingredient in the pharmaceutical composition of the present invention can be varied depending on the severity of the disease. Preferably, the total dose of the pharmaceutical composition of the present invention is approximately 0.01 to 10,000 mg per patient body weight per day, most preferably 0.1 to 500 mg. However, the effective dose of the pharmaceutical composition for a patient is determined by taking into account various factors, such as the formulation method, administration route, number of treatments, the patient's age, body weight, health condition, sex, severity of the disease, diet, and excretion rate. Taking these factors into consideration, a person skilled in the art can determine the appropriate effective dose of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its formulation, administration route, or administration method, as long as it exhibits the effects of the present invention.

[0049] The present invention provides the use of interferon beta for the manufacture of a formulation for suppressing self-replicating respiratory viral infection in an individual exposed to a potential respiratory viral infection.

[0050] The present invention provides a method for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory viral infection, comprising administering to the individual in need thereof an effective amount of a composition comprising interferon beta as an active ingredient.

[0051] The "effective amount" of the present invention refers to an amount that, when administered to an individual, exhibits an effect of improving, treating, preventing, detecting, diagnosing a respiratory virus infection, or suppressing or reducing the disease, and the "individual" may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect.

[0052] As used herein, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components or method steps not specifically mentioned in a composition or method according to the present invention. The term "consisting of" means excluding additional elements, steps, or ingredients not otherwise described. The term "essentially consisting of" means that a composition or method may include, in addition to the materials or steps described, materials or steps that do not substantially affect the basic characteristics of the composition or method. [Effects of the Invention]

[0053] As described above, in the present invention, interferon beta can be administered by inhalation to the respiratory tract, including the nasal mucosa, nasopharynx, oropharynx, laryngopharynx, larynx, trachea, bronchi, bronchioles, and lungs, to effectively suppress self-replicating coronavirus infection in individuals exposed to a latent coronavirus infection. [Brief explanation of the drawings]

[0054] [Figure 1a] Figures 1a and 1b show the results of treating cells with interferon beta before (Figure 1a) or after (Figure 1b) infection with SARS-CoV-2 to confirm the effect of interferon in suppressing coronavirus infection. [Figure 1b] Figures 1a and 1b show the results of treating cells with interferon beta before (Figure 1a) or after (Figure 1b) infection with SARS-CoV-2 to confirm the effect of interferon in suppressing coronavirus infection. [Figure 2a] 2a and 2b show the results of quantitative analysis of the results of FIGS. 1a and 1b. [Figure 2b]2a and 2b show the results of quantitative analysis of the results of FIGS. 1a and 1b. [Figure 3] Figure 3 shows the effects of remdesivir and interferon beta on suppressing coronavirus infection, confirmed by plaque assay (left) and real-time quantitative RT-qPCR (right). [Figure 4] Figure 4 shows the results of confirming the effect of interferon beta in suppressing lung damage caused by coronavirus infection in a hamster infection model. [Figure 5] Figure 5 shows a schematic experimental sequence for analyzing the efficacy of interferon beta in pre-exposure prophylaxis against the Wuhan or Delta strains of coronavirus. [Figure 6] Figure 6 shows the results of plaque assay confirming the pre-exposure prophylactic effect of interferon beta against the Wuhan or Delta strains of coronavirus. [Figure 7] Figure 7 shows a schematic experimental sequence for analyzing the preventive or therapeutic effect of interferon beta after exposure to the Wuhan or Delta strains of coronavirus. [Figure 8] Figure 8 shows the results of plaque assay confirming the preventive or therapeutic effect of interferon beta after exposure to the Wuhan or Delta strains of coronavirus. [Figure 9] FIG. 9 shows a schematic experimental sequence for analyzing the preventive effect of interferon beta against influenza (H1N1). [Figure 10] FIG. 10 shows the results of plaque assay confirming the preventive effect of interferon beta against influenza (H1N1). DETAILED DESCRIPTION OF THE INVENTION

[0055] [Best Mode for Carrying Out the Invention] Preferred examples are presented below to facilitate understanding of the present invention. However, the following examples are provided to facilitate understanding of the present invention and are not intended to limit the scope of the present invention. [Example]

[0056] Example 1: Interferon beta suppresses coronavirus infection To test the effect of interferon beta on coronavirus infection, Vero E6 cells were infected and treated with interferon beta. RNA was extracted and subjected to RT-qPCR. The treatment conditions were as follows:

[0057] Vero cells were grown in DMEM medium containing 2% heat-inactivated FBS and 2 mM L-glutamine, and 5 × 10 Vero cells were plated in 6 wells. 5 Cells were seeded at 1000 x 1000 cells / well and cultured in a 37°C, 5% CO2 incubator to maintain approximately 80% confluence. For pre-infection experiments the day after seeding, SARS-CoV-2 stock was initially injected at approximately 2 x 10 6 After infection with plaque-forming units (PFU / mL), the cells were treated with ABN101 material, and for post-infection, the cells were first treated with ABN101 material at various concentrations and then infected with SARS-CoV-2 stock.

[0058] In preinfection experiments, Vero E6 cells were infected with SARS-CoV-2 for 1 hour and 30 minutes. After the infected virus was completely removed, 3 ml of culture medium was added and treated with interferon beta (ABN101, R27T interferon beta) diluted to 5,000 IU / mL, 1,000 IU / mL, 2,000 IU / mL, and 5,000 IU / mL. After incubation in an incubator at 37°C and 5% CO2 for 3 days, the culture supernatants were collected. Each supernatant was filtered to remove cells, and total RNA was isolated. Using the isolated RNA as a template, quantitative real-time RT-qPCR was performed using primers specifically detecting the SARS-CoV-2 E gene to determine whether SARS-CoV-2 virus was reduced by interferon beta treatment. The primer sequences and probes used in this experiment to detect the E gene of SARS-CoV-2 are as follows: Probe sequence: 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 2) Primer 1 sequence: 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 3) Primer 2 sequence: 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 4)

[0059] For post-infection experiments, interferon beta (ABN101, R27T interferon beta) was diluted to 5000 IU / mL, 1000 IU / mL, 2000 IU / mL, and 5000 IU / mL concentrations and treated for 24 hours. The culture medium was then removed, and Vero E6 cells were infected with SARS-CoV-2 for 1 hour and 30 minutes. The infected virus was then completely removed, and 3 mL of culture medium was added. The cells were then cultured in a 37°C, 5% CO2 incubator. On day 2, the culture supernatant was collected, filtered, the cells removed, and total RNA was isolated. RT-qPCR experiments were performed in the same manner as for the pre-infection experiments.

[0060] As a result, as shown in Figures 1 and 2, when only virus was treated (virus only) at both pre-infection and post-infection, the Ct value was 14.896, while when interferon beta was treated at 500 IU, 1,000 IU, 2,000 IU, and 5,000 IU (ABN101 (treatment amount)), the Ct value increased as the amount of interferon beta treated increased, indicating that the amount of virus decreased in a concentration-dependent manner.

[0061] Quantitative analysis of this is shown in Figure 2a and Figure 2b, and the EC50 of ABN101 against SARS-CoV-2 was confirmed to be 121.5 pM (pre-infection) and 72.3 pM (post-infection), respectively.

[0062] Example 2: Comparison of coronavirus infection suppression effects with remdesivir To compare the efficacy of interferon beta in suppressing coronavirus infection with remdesivir, the results were compared using plaque assays and real-time quantitative RT-qPCR.

[0063] In the plaque assay, cells were overlaid with agarose, stained with crystal violet, and analyzed for viral plaques to determine the extent to which the cells were infected with the virus.

[0064] Vero cells were grown in DMEM medium containing 2% heat-inactivated FBS and 2 mM L-glutamine, and 5 × 10 Vero cells were plated in 6 wells. 5 Cells were seeded per well and cultured in a 37°C, 5% CO2 incubator to maintain approximately 80% confluence. The next day after seeding, SARS-CoV-2 stock was added at approximately 2 x 10 6 Plaque-forming units (PFU / mL) were treated.

[0065] Vero E6 cells were infected with SARS-CoV-2 for 1 hour and 30 minutes, after which the infected virus was completely removed. 3 mL of culture medium was added, and the cells were treated with various concentrations of remdesivir and cultured in an incubator at 37°C and 5% CO2 for 3 days, after which the culture supernatants were collected.

[0066] Remdesivir was prepared by dissolving it in DMSO, and since the measurement standard was to control substances containing DMSO, Mock-1 and Mock-2 were prepared.

[0067] Real-time quantitative RT-qPCR was performed in the same manner as in Example 1, using samples treated with remdesivir at concentrations of 0.01 μM (10 nM), 0.05 μM (50 nM), 0.1 μM (100 nM), 0.2 μM (200 nM), 0.5 μM, 1 μM, 2 μM, and 3 μM.

[0068] The treatment materials for Mock-1 and Mock-2 are as follows. Mock-1: After virus infection, culture medium only Mock-2: Culture medium + DMSO after virus infection

[0069] The left side of Figure 3 shows the results of a plaque assay, which showed that when interferon beta was treated pre-infection and post-infection (bottom left), viral infection was not significantly reduced when treated with remdesivir (top left), compared to when few infected cells were confirmed. The EC50 of remdesivir is known to be 74 nM, but it was confirmed that interferon beta showed good efficacy at a concentration approximately 600 times lower than that.

[0070] The right side of Figure 3 shows the results of real-time quantitative RT-qPCR, which was conducted using the same experimental method as Figure 1. As a result, while there was almost no difference in Ct values ​​between remdesivir and virus when treated with virus only (virus only), when 500 IU of interferon beta was treated (ABN101 (treatment amount)), the difference in Ct values ​​between interferon beta and virus only treatment increased, confirming that the efficacy of interferon beta was superior to the EC50 of remdesivir.

[0071] Example 3: Effect of interferon beta on suppressing lung damage caused by coronavirus infection in a hamster infection model Since it is well known that SARS-CoV-2 infection causes lung damage, we investigated whether interferon beta protects against lung damage caused by coronavirus infection.

[0072] To assess the severity of lung damage caused by SARS-CoV-2 infection, 6-week-old male Syrian hamsters were infected intranasally with the virus at 100 PFU / head. They were treated with interferon beta (ABN101) at 0.15 MIU / head and 1 MIU / head immediately after infection (0 day post-injection (dpi)), 2 days later (2 dpi), and 4 days later (4 dpi), respectively, and the severity of lung tissue damage was assessed.

[0073] The control group received only the virus (control, virus only), while the experimental group received 0.15 MIU (1 million IU) and 1 MIU of interferon beta administered intranasally into the respiratory tract, respectively. The degree of lung damage was assessed by a third-party veterinarian. After sacrificing the hamsters, the entire lungs were collected and blinded to the lungs of the virus-only and interferon beta groups. The lungs were numbered and assessed for damage in a blinded manner. The lung damage was scored on a 0-10 scale, with 10 representing total lung area damage and 0 representing normal levels.

[0074] As a result, as shown in Figure 4 and Table 1, when only the virus was administered, the lung injury score was 3.8 ± 0.98, whereas when interferon beta was administered, the lung injury score decreased in a dose-dependent manner.

[0075] [Table 1]

[0076] Example 4: Effect of interferon beta on preventing the Wuhan strain of coronavirus or its variant, the Delta strain To test the efficacy of interferon beta in preventing coronavirus infection (pre-exposure prophylaxis), Vero E6 cells were treated with interferon beta, removed, and then infected with the Wuhan or Delta strain of coronavirus and analyzed by plaque assay.

[0077] In the plaque assay, cells were overlaid with agarose, stained with crystal violet, and analyzed for viral plaques to determine how many cells were infected with the virus.

[0078] Vero E6 cells were grown in complete DMEM medium (10% FBS) and seeded in 12-well plates. They were cultured at 37°C in a 5% CO2 incubator to maintain approximately 80% confluence. SARS-CoV-2 stock (Wuhan or Delta strain) was administered at approximately 100 plaque-forming units (PFU / mL) to measure the prophylactic effect.

[0079] To confirm prophylactic activity, ABN101 was administered at concentrations of 25 IU / mL, 50 IU / mL, 100 IU / mL, 250 IU / mL, 500 IU / mL, 1,000 IU / mL, 2,500 IU / mL, 5,000 IU / mL, 10,000 IU / mL, and 25,000 IU / mL. After 24 hours, ABN101 was removed. Vero E6 cells were then infected with the Wuhan or Delta strain of SARS-CoV-2 for 1 hour. After complete virus removal, the cells were cultured in fresh culture medium for 72 hours and then subjected to plaque assays.

[0080] The experimental procedure is shown in Figure 5.

[0081] As a result, as shown in Table 2 and Figure 6, 250 IU / mL showed a preventive effect of more than 90% against both coronavirus strains, and 500 IU / mL or more showed a preventive effect of more than 95% against both coronavirus strains. The EC50 activity of ABN101 against the Wuhan strain was 28.125 IU / mL, and the EC50 activity against the Delta strain was 184.38 IU / mL.

[0082] [Table 2]

[0083] Example 5: Preventive or therapeutic effects of interferon beta after exposure to coronavirus Wuhan or Delta strains To test the efficacy of interferon beta in treating coronavirus infection, Vero E6 cells were infected with either the Wuhan or Delta strains of the novel coronavirus, then treated with different concentrations of interferon beta and analyzed by plaque assay.

[0084] In the plaque assay, cells were overlaid with agarose, stained with crystal violet, and analyzed for viral plaques to determine how many cells were infected with the virus.

[0085] Vero E6 cells were grown in complete DMEM medium (10% FBS) and seeded in 12-well plates. They were cultured at 37°C in a 5% CO2 incubator to maintain approximately 80% confluence. SARS-CoV-2 stock (Wuhan strain or Delta strain) was administered at approximately 100 plaque-forming units (PFU / mL) to assess therapeutic efficacy.

[0086] To confirm efficacy, Vero E6 cells were infected with the Wuhan or Delta strain of SARS-CoV-2 for one hour, and the infected virus was then completely removed. After that, the cells were treated with ABN101 at concentrations of 25 IU / mL, 50 IU / mL, 100 IU / mL, 250 IU / mL, 500 IU / mL, 1,000 IU / mL, 2,500 IU / mL, 5,000 IU / mL, 10,000 IU / mL, and 25,000 IU / mL. The medium was then collected and virus titration was measured to confirm the infection-suppressing effect.

[0087] The flow chart of this experiment is shown in Figure 7.

[0088] As a result, as shown in Table 3 and Figure 8, ABN101 demonstrated an infection-inhibiting efficacy of over 95% at concentrations of 500 IU / mL or higher against both coronavirus strains. The infection-inhibiting effect of ABN101 was as follows: the EC50 activity against the Wuhan strain was 59.11 IU / mL, and the EC50 activity against the Delta strain was 25 IU / mL. [Table 3]

[0089] Example 6: Preventive effect of interferon beta on influenza (H1N1) To test the preventive effect of interferon beta on influenza infection, Vero E6 cells were treated with interferon beta, removed, and then infected with influenza virus (H1N1 / A / PR8) and analyzed by plaque assay.

[0090] In the plaque assay, cells were overlaid with agarose, stained with crystal violet, and analyzed for viral plaques to determine how many cells were infected with the virus.

[0091] MDCK cells were grown in complete DMEM medium (10% FBS) and seeded in 12-well plates. MDCK cells were maintained at approximately 80% confluence in a 37°C, 5% CO2 incubator. Influenza virus stock (H1N1 / A / PR8) was administered at approximately 100 plaque-forming units (PFU / mL) to measure the prophylactic effect.

[0092] To confirm prophylactic activity, ABN101 was administered at concentrations of 25 IU / mL, 50 IU / mL, 100 IU / mL, 250 IU / mL, 500 IU / mL, 1,000 IU / mL, 2,500 IU / mL, 5,000 IU / mL, 10,000 IU / mL, and 25,000 IU / mL. ABN101 was removed 24 hours later. MDCK cells were then infected with influenza virus (H1N1 / A / PR8) for 1 hour. After complete removal of the infected virus, the cells were cultured in fresh culture medium for 72 hours and then subjected to plaque assay.

[0093] The procedure for this experiment is shown in Figure 9.

[0094] As a result, as shown in FIG. 10, the ABN101-treated group showed a 20% or greater influenza infection prevention effect compared to the untreated group. [Industrial Applicability]

[0095] As described above, in the present invention, interferon beta can be administered by inhalation to the respiratory tract, such as the nasal mucosa, nasopharynx, oropharynx, laryngopharynx, larynx, trachea, bronchi, bronchioles, or lungs, to effectively suppress self-replicating coronavirus infection in individuals exposed to a latent coronavirus infection.

Claims

1. A pharmaceutical composition for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection, comprising interferon beta as an active ingredient.

2. 2. The pharmaceutical composition of claim 1, wherein the interferon beta has the amino acid sequence of SEQ ID NO:

1.

3. 2. The pharmaceutical composition of claim 1, wherein the interferon beta is administered by inhalation.

4. The pharmaceutical composition according to claim 3, wherein the administration by inhalation is administered by contact with respiratory cells by inhalation.

5. 5. The pharmaceutical composition of claim 4, wherein the respiratory tract is the nasal mucosa, nasopharynx, oropharynx, laryngopharynx, larynx, trachea, bronchi, bronchioles, or lungs.

6. 2. The pharmaceutical composition of claim 1, wherein the respiratory virus is a coronavirus and the individual is not infected with a coronavirus or is in the early stages of coronavirus infection.

7. 7. The pharmaceutical composition of claim 6, wherein the individual in the early stage of coronavirus infection does not exhibit symptoms of coronavirus infection selected from the group consisting of fever, dry cough, fatigue, feeling unwell, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, and discoloration of the fingers or toes.

8. 2. The pharmaceutical composition of claim 1, wherein the respiratory virus is selected from the group consisting of adenovirus, avian influenza virus, bocavirus, coronavirus, cytomegalovirus, hantavirus, herpes simplex virus, influenza virus, measles virus, metapneumovirus, parainfluenza virus, respiratory syncytial virus, rhinovirus, and varicella-zoster virus.

9. The coronaviruses include 229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), canine coronavirus (CCoV), feline coronavirus (FCoV), Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Rhinolophus bat coronavirus HKU2, Asian yellow house bat (Scotophilus bat) coronavirus 512, porcine hemagglutinating encephalomyelitis virus (PHEV), bovine coronavirus (BCoV), equine coronavirus (EqCoV), murine coronavirus (murine 9. The pharmaceutical composition according to claim 8, wherein the coronavirus is any one selected from the group consisting of MuCoV, Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Avian coronavirus, Beluga whale coronavirus SW1, Bulbul coronavirus HKU11, Thrush coronavirus HKU12, and Munia coronavirus HKU13.

10. 2. The pharmaceutical composition of claim 1, wherein the self-replicating infection is characterized in that the coronavirus replicates in the respiratory cells of the individual and infects surrounding cells.

11. A post-exposure prophylaxis (PEP) method for protecting an individual from self-replicating infection by a respiratory virus after exposure to a potential respiratory viral infection, characterized in that the method comprises administering to the individual a prophylactically effective amount of interferon beta.

12. A method of protecting an individual from self-replicating infection by a respiratory virus comprising administering to the individual a prophylactically effective amount of interferon beta after exposure to a potential respiratory virus infection.

13. Use of interferon beta for producing a preparation for suppressing self-replicating infection of a respiratory virus in a subject exposed to a potential respiratory virus infection.

14. A method for suppressing self-replicating infection of a respiratory virus in an individual exposed to a potential respiratory virus infection, comprising administering to the individual in need thereof an effective amount of a composition comprising interferon beta as an active ingredient.

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