Antiviral compositions comprising nucleoside analogs derived from nucleic acids or pharmaceutically acceptable salts thereof

Abstract

The present application relates to an antiviral composition, an immunity enhancing composition, a feed or a feed additive comprising a nucleoside analog derived from a nucleotide.

Description

Technical Field

[0001] The present application relates to an antiviral composition, an immunomodulatory composition and a feed composition, which contain nucleoside analogs derived from nucleic acids and pharmaceutically acceptable salts thereof. Background Art

[0002] Viruses are infectious pathogens that infect not only animals and plants but also microorganisms, causing a variety of diseases, disrupting the host's life cycle, or even leading to death. In recent years, various animal viruses, such as SARS-CoV-2, MERS, and influenza viruses, have been widespread infecting humans worldwide and causing significant harm, making their presence a significant concern for contemporary humanity. As a solution to this situation, there may be a way to overcome the fear of cross-species infectious viruses derived from animals by controlling viral diseases in animals.

[0003] Until recently, scientists have been conducting research with boundless enthusiasm and effort to control animal viruses through various methods, but a clear method for perfect control has yet to be achieved. For example, methods such as bait vaccines for wildlife and commercial vaccinations for commercial and companion animals have been used. However, due to the frequent mutation of viruses and the slow pace of vaccine development, it is difficult to expect these methods to achieve a truly perfect preventive effect. Furthermore, it is known that the combined use of vaccines and therapeutic agents, in full compliance with quarantine regulations, can at least achieve the desired level of effectiveness.

[0004] To support this assertion, we already know from experience that it is difficult to completely control local or global animal viral diseases through the various vaccination programs and large-scale national quarantines currently underway. Many scientists agree that effective viral disease control will become possible when virotherapeutics are appropriately used as the final piece in the puzzle to create effective virus-related solutions in the current situation.

[0005] Furthermore, since various viruses constantly emerge due to natural laws and cannot be prevented in advance, a therapeutic agent or inhibitor that allows for post-event viral control is likely to be needed, rather than a vaccine, which is difficult to prevent at a rapid level. However, currently, humans do not have access to effective and highly effective therapeutic agents against animal viruses, and as a result, it can be said that humans still suffer from enormous economic and human damage caused by various viruses transmitted from animals.

[0006] Thus, in the case where a virus therapeutic agent is urgently needed, firstly, this patent will describe a substance that inhibits the viral proliferation of the following economic animals and companion animals that are closely related to human life,

[0007] African swine fever virus (ASFV), Classical Swine Fever Virus (CSFV), Low Pathogenic Avian Influenza Virus (LPAIV), Canine Coronavirus (CCoV), Canine Adenovirus (CAV), Canine Distemper Virus (CDV), Feline Parvovirus (FPV), Feline Calicivirus (FCV), Feline Infectious Peritonitis Virus (FIPV, also known as Feline Coronavirus, FCoV) and Foot-and-Mouth Disease Virus (FMDV). Summary of the Invention

[0008]

Technical Issues

[0009] One object of the present application is to provide an antiviral composition, an immunomodulatory composition or a feed composition, which comprises a nucleoside analogue derived from a nucleic acid and a pharmaceutically acceptable salt thereof.

[0010]

Technical solution

[0011] One aspect of the present application may be an antiviral composition comprising nucleosides in the following forms: the dialdehyde form of inosine, the dialdehyde form of xanthosine, the dialdehyde form of guanosine, or the acyclic diol form of inosine, the acyclic diol form of xanthosine, the acyclic diol form of guanosine.

[0012] One aspect of the present application may be an immunomodulatory composition comprising a nucleoside in the form of a dialdehyde form of inosine, a dialdehyde form of xanthosine, a dialdehyde form of guanosine, or a non-cyclic diol form of inosine, a non-cyclic diol form of xanthosine, a non-cyclic diol form of guanosine.

[0013] One aspect of the present application may be a medicament, feed or feed additive comprising the antiviral composition.

[0014] Specifically, the composition can be a composition that exhibits an antiviral effect against at least one virus, the virus being selected from the group consisting of: African swine fever virus (ASFV), classical swine fever virus (CSFV), low pathogenic avian influenza virus (LPAIV), canine coronavirus (CCoV), canine adenovirus (CAV), canine distemper virus (CDV), feline parvovirus (FPV), feline calicivirus (FCV), feline infectious peritonitis virus (FIPV, also known as feline coronavirus, FCoV) and foot-and-mouth disease virus (FMDV).

[0015] One aspect of the present application may be a method for preventing, ameliorating or treating viruses, comprising administering to a subject a nucleoside in the following forms: a dialdehyde form of inosine, a dialdehyde form of xanthine riboside, a dialdehyde form of guanosine, or a non-cyclic diol form of inosine, a non-cyclic diol form of xanthine riboside, or a non-cyclic diol form of guanosine.

[0016] One aspect of the present application may be a method of modulating immunity comprising administering to a subject a nucleoside in the form of a dialdehyde form of inosine, a dialdehyde form of xanthine riboside, a dialdehyde form of guanosine, or a non-cyclic diol form of inosine, a non-cyclic diol form of xanthine riboside, a non-cyclic diol form of guanosine nucleoside.

[0017] One aspect of the present application may be the use of the following forms of nucleosides for the prevention or treatment of viruses: the dialdehyde form of inosine, the dialdehyde form of xanthine riboside, the dialdehyde form of guanosine, or the non-cyclic diol form of inosine, the non-cyclic diol form of xanthine riboside, the non-cyclic diol form of guanosine.

[0018] One aspect of the present application may be the use of the following forms of nucleosides for regulating immunity: the dialdehyde form of inosine, the dialdehyde form of xanthine riboside, the dialdehyde form of guanosine, or the non-cyclic diol form of inosine, the non-cyclic diol form of xanthine riboside, the non-cyclic diol form of guanosine.

[0019] Beneficial effects

[0020] According to the present application, nucleoside analogs derived from nucleic acids are useful as antiviral agents.

[0021] Best Practice

[0022] Hereinafter, the present application will be described in detail.

[0023] The antiviral or immunomodulatory composition of the present application is a nucleoside analog in dialdehyde form or acyclic diol form, and as a specific aspect, is characterized in that it contains inosine, xanthine nucleoside, guanosine and pharmaceutically acceptable salts thereof in dialdehyde form or acyclic diol form as active ingredients, and the nucleoside analog is derived from a nucleic acid represented by the following chemical formulas 1 to 6.

[0024] [Chemical Formula 1]

[0025]

[0026] [Chemical Formula 2]

[0027]

[0028] [Chemical Formula 3]

[0029]

[0030] [Chemical Formula 4]

[0031]

[0032] [Chemical Formula 5]

[0033] [Chemical Formula 6]

[0034]

[0035] This active ingredient, inosine, xanthosine or guanosine in dialdehyde form or acyclic diol form can be produced by an optimized method. As a specific embodiment, it can be prepared by the method described in the examples of the present invention, but is not limited thereto.

[0036] Then, based on 100 parts by weight of the antiviral composition, it may contain 0.0001 to 20 parts by weight, 0.0001 to 15 parts by weight, 0.0001 to 10 parts by weight, 0.0001 to 5 parts by weight, 0.0001 to 1 part by weight, 0.0001 to 0.5 parts by weight, 0.0001 to 0.1 parts by weight, 0.0001 to 0.05 parts by weight, 0.0001 to 0.01 parts by weight, 0.0001 to 0.005 parts by weight, 0.0001 to 0. 0.001 to 0.0005 parts by weight, 0.001 to 20 parts by weight, 0.001 to 15 parts by weight, 0.001 to 10 parts by weight, 0.001 to 5 parts by weight, 0.001 to 1 part by weight, 0.001 to 0.5 parts by weight, 0.001 to 0.1 parts by weight, 0.001 to 0.05 parts by weight, 0.001 to 0.01 parts by weight, 0.001 to 0.005 parts by weight, 0.01 to 20 parts by weight, 0.01 parts by weight 0.1 to 15 parts by weight, 0.01 to 10 parts by weight, 0.01 to 5 parts by weight, 0.01 to 1 part by weight, 0.01 to 0.5 parts by weight, 0.01 to 0.1 parts by weight, 0.01 to 0.05 parts by weight, 0.1 to 20 parts by weight, 0.1 to 15 parts by weight, 0.1 to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 to 1 part by weight, 0.1 to 0.5 parts by weight, 1 to 20 parts by weight, 1 to 15 parts by weight, 1 to 10 parts by weight, 1 to 5 Parts by weight, 5 to 20 parts by weight, 5 to 15 parts by weight, 5 to 10 parts by weight, 10 to 20 parts by weight, 10 to 15 parts by weight or 15 to 20 parts by weight of the compound represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, therefore, when the compound or a pharmaceutically acceptable salt thereof is included in an amount of less than 0.0001 parts by weight, the antiviral effect of the compound is not properly manifested, and when its content exceeds 20 parts by weight, the increase in the antiviral effect is insufficient compared to the increase in the compound content, and thus is not preferred.

[0037] The virus is not limited as long as it is a virus species, but specifically, it can be a virus from a companion animal or an industrial animal. Examples of such companion animals or industrial animals can be even-toed ungulates such as cattle, pigs, goats, sheep, deer, etc., fish, arthropods, dogs, cats or birds.

[0038] Examples of viruses derived from artiodactyls such as cattle, pigs, goats, sheep, deer, etc., fish, arthropods, dogs, cats or birds, although not limited thereto, may be at least one selected from the group consisting of African swine fever virus (ASFV), classical swine fever virus (CSFV), low pathogenic avian influenza virus (LPAIV), canine coronavirus (CCoV), canine adenovirus (CAV), canine distemper virus (CDV), feline parvovirus (FPV), feline calicivirus (FCV), feline infectious peritonitis virus (FIPV, also known as feline coronavirus, FCoV) and foot-and-mouth disease virus (FMDV).

[0039] Representative viruses among those for which antiviral efficacy was verified in this application are described below.

[0040] African swine fever, caused by the African swine fever virus (ASFV), is a lethal viral hemorrhagic disease that causes significant economic losses to the pig industry due to its 100% mortality rate. ASFV has been present in Africa since the 1920s and is endemic throughout much of sub-Saharan Africa. It also appeared in Europe and South America, where it was largely eradicated, though complete eradication took 30 years or more in Spain and Portugal. Since its introduction into Europe in 2007 via Georgia, the virus has spread widely to domestic and wild pigs in the region, and is currently endemic to many Eastern European countries and parts of the Russian Federation.

[0041] ASFV cannot infect humans or other animals; it is only susceptible to members of the Suidae family. Domestic pigs and wild boars are natural reservoirs. Wild boars, warthogs, and giant forest pigs in Africa, in particular, do not show clinical symptoms even when infected, thus acting as vectors for the virus. Besides pigs, the only other species of ticks, belonging to the genus Ornithodoros, are the soft ticks that carry the virus and bite pigs or wild boars, spreading the disease. Currently, there is no vaccine or treatment available globally, so quarantine measures are recommended for each country to prevent its introduction.

[0042] Classical swine fever is an acute systemic infectious disease caused by classical swine fever virus (CSFV). It is a type 1 livestock infectious disease that, upon infection, manifests as a severe febrile reaction and characteristic external symptoms such as spots on the skin, and, upon autopsy, hemorrhagic spots in the bladder and kidneys, button-shaped ulcers in the ileocecal colon, and hemorrhagic infarction of the spleen. However, these specific symptoms do not appear in most cases in partially immunized pigs or on farms where the disease persists.

[0043] Prevention involves vaccination with a live attenuated vaccine, and when administered according to appropriate vaccination protocols and stored well under refrigeration, the disease is effectively prevented. While the disease has been eradicated in many countries, such as the United States, neighboring Japan has established and is currently promoting an eradication program. Furthermore, in South Korea, a highly effective vaccine has been developed and commercialized, and while outbreaks can be controlled through proper vaccination, the disease continues to occur because pig farmers avoid vaccination for economic reasons.

[0044] Avian influenza (AI) is a viral infectious disease caused by pathogenic influenza viruses that infect wild birds and domestic poultry. It is categorized into non-pathogenic, low-pathogenic, and highly pathogenic avian influenza, with clinical symptoms and pathogenicity varying depending on the pathogenicity of the virus. Highly pathogenic avian influenza (HPAI) is classified as a Type 1 livestock infectious disease in South Korea and a List A disease by the Office International des Epizooties (OIE). It is subject to thorough surveillance and inspection in most countries, and when an outbreak occurs, quarantine measures aimed at eradication are implemented, such as establishing emergency quarantine lines, blocking movement, and culling. Meanwhile, in South Korea, low-pathogenic avian influenza is managed as a Type 2 livestock infectious disease, and quarantine measures are implemented to suppress its occurrence, minimize damage, and reduce its spread through vaccination.

[0045] All low-pathogenic avian influenza viruses that have appeared in South Korea are H9N2. The first outbreak was reported in 1996, and while it has a low mortality rate, it can cause problems in laying hen farms, such as reduced egg production, eggshell discoloration, deteriorating egg quality, and reduced feed intake (in broilers), resulting in significant economic losses. Avian influenza viruses can be transmitted through droplets, air, and water, primarily through direct contact with feces. Specifically, they are transmitted directly to other chickens through feces on the boots or clothing of handlers, feed carts, utensils, equipment, and eggs. One gram of feces secreted by an infected chicken contains enough virus to infect approximately one million chickens. Therefore, thorough disinfection is crucial to prevent transmission. For prevention, an inactivated H9N2 vaccine is commercially available, which helps minimize economic losses for poultry farms by reducing the symptoms of infection. However, there are no commercially available treatments or preventive drugs, so the reality is that simple quarantine is the only option.

[0046] Canine coronavirus (CCoV) belongs to the Coronaviridae family and is a virus with a single strand of RNA as its genome, and causes canine coronavirus infection. In 1971, a collective outbreak of canine coronavirus infection occurred in the United States, and a pathogenic virus was isolated at this time. Many outbreaks of this disease have also been reported in Australia, Japan and South Korea. Currently, it is considered to be one of the pathogens of canine viral diarrhea because it co-infects with canine parvovirus and further worsens the symptoms. It causes an acute viral gastrointestinal infectious disease with the main symptoms of vomiting, diarrhea and dehydration. Both puppies and adult dogs are infected, but it is particularly common in puppies and the symptoms are obvious. Regardless of the breed, age, etc. of the dog, it is susceptible, and it shows rapid spread and morbidity. In particular, it develops in group-domesticated dogs in a short period of time.

[0047] Commercially available vaccinations are available as preventative measures against canine coronavirus infection, but safety precautions are required. For example, when live attenuated canine distemper vaccine and canine parvovirus vaccine are administered together, there is a concern about encephalitis as a side effect. Currently, there are no effective treatments, and symptomatic treatment based on early diagnosis is likely to be the most effective. To keep the dog stable and warm and to minimize stress, fluid therapy and antibiotics are administered to prevent secondary bacterial infections as part of symptomatic treatment.

[0048] Canine adenovirus (CAV) is divided into CAV-1, which causes canine infectious hepatitis, and CAV-2, which causes canine cough (canine bronchitis or cold). The natural reservoir for infection is canines, such as dogs, foxes, and coyotes. The virus is transmitted globally, affecting all breeds, sexes, and ages. Infection is typically oral, and after recovery, some virus remains in the kidneys, with the virus excreted in the urine for approximately 6 to 9 months. Therefore, canines infected with this virus act as reservoirs for a long period of time. After the initial infection, after an incubation period of 3 to 8 days, the virus becomes weak, with a runny nose, lethargy, and an abnormal fever. Gastrointestinal symptoms such as vomiting may also occur. Hepatic edema, in particular, causes severe abdominal pain. Jaundice develops in the late stages of hepatitis, and dogs recovering from the infection exhibit a blue eye, known as "hepatitis blue eye."

[0049] Currently, there is no clear treatment method developed, and the main treatment method is symptomatic treatment (antibiotic treatment to eliminate secondary infection, fluid therapy for electrolyte balance in the body, and treatment of dehydration), which allows infected subjects to acquire immunity and be cured. As a preventive method, there is a commercially available vaccine, and a CAV-2 virus vaccine that shows protective activity against CAV-1 and CAV-2 types is used.

[0050] Canine distemper, caused by the canine distemper virus (CDV), is a typical acute, febrile viral disease in dogs. After an incubation period of approximately 3 to 6 days, symptoms include rhinitis, fever, severe respiratory symptoms, indigestion, sclerosis of the soles of the feet, and neurological symptoms. Due to its high infectiousness and mortality rate, the disease is almost 100% fatal when neurological symptoms appear. The disease primarily affects puppies under one year old, but occasionally occurs in older dogs. The disease is known to be transmitted through feces, urine, and nasal secretions excreted by infected dogs, and the virus is excreted 60 to 90 days after infection.

[0051] The only preventive measure is vaccination, with the first vaccination given at 2 months of age, followed by booster vaccinations every 3 to 4 weeks, and additional vaccinations required annually. Current treatment options include symptomatic treatments, such as sulfonamides or antibiotics to suppress secondary bacterial infections, glucose or electrolyte supplements for dehydration, and adjuvants (immunostimulants) for rapid recovery.

[0052] Feline panleukopenia (panleukopenia, feline infectious enteritis, panleukocytosis), caused by feline parvovirus (FPV), is fatal to all cat species due to its high infectiousness and mortality rate. The name "panleukopenia" stems from the marked decrease in white blood cells (leukocytes) observed in infected animals. Transmission is primarily through contact with the body fluids and excreta of infected animals, but even without direct contact with the vector, infection can occur through fleas, bedbugs, and other insects that have come into contact with the vector. Transmission can occur not only through bedding and food that have come into contact with the vector, but also through worn clothing and shoes. Clinical symptoms generally appear within 4 to 6 days of exposure, but can also occur within 2 to 14 days. The virus is not contagious to humans. Given the right environment, the virus can survive for up to a year because the pathogenic FPV is structurally stable. Furthermore, even animals that have recovered from the disease may still contain the virus in their secretions for up to 6 weeks after recovery.

[0053] Vaccines are available for prevention, but they are primarily used as a combination vaccine for various other diseases. Feline panleukopenia is a serious disease in cats, so preventive vaccines are recommended for all cats. To date, there are no commercially available treatments, but recombinant interferon omega has been studied in vitro to inhibit the proliferation of FPB. As symptomatic treatment, whole blood transfusions are administered to increase white blood cell counts, and intravenous fluids containing antibiotics and vitamins A, B, and C are administered to prevent sepsis due to dehydration.

[0054] Feline calicivirus (FCV) causes severe acute and chronic respiratory disease in cats, and cats infected with this virus may develop acute clinical signs or develop symptoms slowly. Rarely, no clinical signs may appear, but symptoms may develop during times of stress or when the immune system is compromised. Common clinical signs include rhinitis, conjunctivitis, stomatitis, gingivitis, and glossitis, with pneumonia, fever, abortion, and cystitis occasionally observed. Even after treatment of the infection, cats may shed the virus for months to years.

[0055] To date, there is no specific treatment for FCV infection. To treat and prevent secondary infections, antibiotics and immunomodulators are used. Stomatitis is difficult to treat, and upper respiratory tract infection symptoms may worsen when steroids are prescribed, necessitating close observation. Feline calicivirus vaccines have been widely used as a preventative measure for this disease over the past 20 years. However, vaccination does not provide 100% protection against the disease, necessitating the development of therapeutic agents. Nevertheless, their use is recommended as they can alleviate symptoms of FCV infection.

[0056] Feline infectious peritonitis is caused by mutations in the feline coronavirus (FCoV) that infect cats. It is known that viral mutations, triggered by various factors, are responsible for approximately 10% of cats infected with FCoV, and that FCoV proliferates in macrophages, causing systemic disease with immune-mediated vasculitis and pyogranulomatous lesions.

[0057] The clinical symptoms caused by feline infectious peritonitis include weight loss, loss of appetite, high fever, etc., and various clinical symptoms are manifested depending on the affected organs. According to the type of lesion, it can be divided into exudative type (wet type) and non-exudative type (dry type). The exudative type of feline infectious peritonitis is characterized by fibrinous peritonitis and pleurisy, which is triggered by a humoral immune response and produces effusions in the abdominal cavity, chest cavity or pericardium, and can develop into a systemic disease. It is known that the non-exudative type mainly involves humoral immunity, but cell-mediated immunity is also partially involved, and granulomatous lesions occurring in the affected organs trigger clinical symptoms. In particular, in the case of the non-exudative type, the manifestation of neurological symptoms is more common than in the exudative type.

[0058] Vaccines are available as a preventative measure, but each veterinarian has different opinions on their effectiveness. In other words, even if the vaccine is administered, peritonitis may occur, and there are also safety issues (vaccination is pathogenic), so vaccination is generally not recommended. Unfortunately, this is an incurable disease, and it is impossible to completely treat or cure it. Only symptomatic treatment can be given based on the symptoms. Symptomatic treatment also focuses on alleviating symptoms through immune regulation. In Japan, some veterinarians believe that omega interferon can be used for treatment, but it is not an official treatment method, and even if it is used, there have been many cases of death, and the cost is high, so it is not widely used.

[0059] Foot-and-mouth disease virus (FMDV) is a small RNA virus classified into seven serotypes: A, O, C, Asia1, SAT1, SAT2, and SAT3. These major serotypes are further divided into approximately 80 or more subtypes. This virus infects animals with bifurcated hooves, such as cattle, pigs, goats, sheep, and deer (ungulate and even-toed ungulate mammals). While the mortality rate is not very high, it significantly reduces the value of livestock products due to the formation of blisters on the lips, tongue, nose, and between the hooves, as well as decreased appetite, increased body temperature, decreased growth, decreased locomotion, and, in the case of dairy cows, decreased milk production. Furthermore, its high contagiousness has led it to be classified as an Office International des Epizooties (OIE) List A disease (diseases with the potential for rapid spread in international trade and significant economic losses). Even in South Korea, it is designated a Type 1 livestock infectious disease.

[0060] Currently, vaccines are the only available countermeasure and preventive method. However, the inconvenience is that vaccines offer very weak cross-protection between viruses, so different vaccines must be used depending on the serotype and subtype. Furthermore, the vaccine's preventive effect lasts only about six months and doesn't last long, so frequent vaccination is necessary. Furthermore, vaccines produce antibodies identical to those actually produced by the disease, making it difficult to distinguish between infected and vaccinated livestock through blood tests. Consequently, when vaccination is underway, it becomes impossible to obtain approval as a country free of foot-and-mouth disease, causing problems in the export of related livestock products and potentially leading to significant economic problems.

[0061] As pharmaceutically acceptable salt, the acid addition salt formed by pharmaceutically acceptable free acid is useful.As free acid, inorganic acid and organic acid can be used, as inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid etc. can be used, as organic acid, citric acid, maleic acid, fumaric acid, dextran, methanesulfonic acid, acetic acid, glycolate, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, pamoic acid (embonic acid), glutamic acid, aspartic acid etc. can be used.In addition, the pharmaceutical composition containing the nucleoside analogs represented by Chemical Formula 1 to 6 of the present application can not only include pharmaceutically acceptable salt, but also can include all salts, hydrates and solvates that can be prepared by conventional methods.

[0062] The addition salts described herein can be prepared by conventional methods, for example, by dissolving a compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and adding an excess of an organic acid or an acidic aqueous solution of an inorganic acid, followed by soaking or crystallization. Subsequently, the addition salt can be prepared by evaporating the solvent or excess acid in the mixture and drying to obtain the addition salt or filtering the precipitated salt.

[0063] In addition, the composition can be selected from a pharmaceutical composition or a health food composition. In a specific embodiment, when the antiviral composition is a pharmaceutical composition, it can further comprise at least one additive selected from the group consisting of carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, glidants, flavoring agents, antioxidants, buffers, antibacterial agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the preparation of pharmaceutical compositions. Particularly, as carrier, excipient and diluent, lactose, glucose (dextrose), sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talcum, magnesium stearate and mineral oil can be used, and the solid preparation for oral administration comprises tablet, pill, powder, granule, capsule etc., and these solid preparations can be by by at least one excipient, for example starch, calcium carbonate, sucrose or lactose, gelatin etc. are mixed in composition and prepare.In addition, except simple excipient, lubricant can also be used, as magnesium stearate and talcum.Oral liquid preparation comprises suspension, oral liquid, emulsion, syrup etc., except commonly used simple diluent water and liquid paraffin, can also comprise various excipients, for example wetting agent, sweetener, flavoring, preservative etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents or suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used.

[0064] As other embodiments, the pharmaceutical composition can be formulated into granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, suspensions, emulsions, drops or liquids. According to an example of the present application, the pharmaceutical composition can be administered to a subject by methods well known in the art, for example, by conventional methods of oral, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrasternal, cutaneous, intranasal, inhalation, topical, rectal, intraocular or intradermal routes, but is not limited thereto.

[0065] The specific dosage of the compounds represented by Chemical Formulas 1 to 6 or pharmaceutically acceptable salts thereof may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the administration route and cycle, and may be appropriately selected by those skilled in the art.

[0066] In other embodiments of the present application, the pharmaceutical composition may contain 0.01 to 90 parts by weight, 0.01 to 50 parts by weight, 0.01 to 10 parts by weight, 0.01 to 5 parts by weight, 0.01 to 1 part by weight, 0.01 to 0.1 parts by weight, 0.1 to 90 parts by weight, 0.1 to 50 parts by weight, 0.1 to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 to 1 part by weight, 1 to 90 parts by weight, 1 to 50 parts by weight, 1 to 10 parts by weight, 1 to 5 parts by weight, 10 to 90 parts by weight, 10 to 70 parts by weight, 10 to 50 parts by weight, 10 to 30 parts by weight or 10 to 20 parts by weight of a compound selected from the group consisting of compounds of Chemical Formulas 1 to 6 and pharmaceutically acceptable salts thereof, but is not limited thereto.

[0067] According to one example of the present application, although not limited thereto, as clearly indicated below, when an appropriate amount of each livestock is orally administered to animals, it is absorbed through intestinal epithelial cells and introduced into the bloodstream, diffuses to each organ tissue, and in particular, it is delivered / absorbed into cells in tissues where viruses proliferate, and inhibits viral proliferation in infected cells through viral attack, and finally, it can be used as a therapeutic agent for virus-infected animals.

[0068] In the present application, the subject can be a mammal, a bird, a fish, or an arthropod, but is not limited thereto. The mammalian subject can be a pig, a cow, a dog, a cat, or a chicken. The fish subject can be a flounder, a salmon, a sea bream, an eel, a grouper, or a trout. The arthropod subject can be a shrimp or a lobster.

[0069] In other embodiments of the present application, the health food may contain 0.01 to 90 parts by weight, 0.01 to 50 parts by weight, 0.01 to 10 parts by weight, 0.01 to 5 parts by weight, 0.01 to 1 part by weight, 0.01 to 0.1 parts by weight, 0.1 to 90 parts by weight, 0.1 to 50 parts by weight, 0.1 to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 to 1 part by weight, 1 to 90 parts by weight, 1 to 50 parts by weight, 1 to 10 parts by weight, 1 to 5 parts by weight, 10 to 90 parts by weight, 10 to 70 parts by weight, 10 to 50 parts by weight, 10 to 30 parts by weight or 10 to 20 parts by weight of a compound selected from the group consisting of compounds of Chemical Formulas 1 to 6 and pharmaceutically acceptable salts thereof, but is not limited thereto. In other embodiments of the present application, the health food may further include at least one additive selected from the group consisting of: organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar, and sorbitol. The organic acid, although not limited thereto, may be citric acid, malic acid, adipic acid, or lactose; the phosphate, although not limited thereto, may be sodium phosphate, potassium phosphate, acid pyrophosphate, or polyphosphate (polymeric phosphate); and the antioxidant, although not limited thereto, may be a natural antioxidant such as polyphenols, catechins, α-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid, or phytic acid. In other embodiments of the present application, the health food may contain various nutritional supplements, probiotics, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, colorants and fillers (cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerol, alcohol, carbonating agents for carbonated beverages, etc. According to an embodiment of the present application, the preparation of the health food, although not limited thereto, can be in the form of solid, powder, granule, tablet, capsule, liquid or beverage. In addition, the health food, although not limited thereto, can be used to prepare foods such as candy, sugar, ice cream products, dairy products, meat products, fish products, tofu or jelly, edible oils and fats, noodles, tea, beverages, special nutritional foods, health supplements, seasoning foods, ice, ginseng products, pickled foods, dried fish and shellfish, fruits, vegetables, dried and cut products of fruits or vegetables, fruit juices, vegetable juices, mixed juices of the two, potato chips, noodles, processed animal husbandry foods, processed seafood, processed dairy products, fermented oil foods, bean foods, cereal foods, fermented microbial foods, candy and bread, spices, processed meat, acidic beverages, licorice, herbs, etc.

[0070] In addition, the present application provides a feed additive containing a compound selected from the group consisting of compounds of Chemical Formulas 1 to 6 and pharmaceutically acceptable salts thereof as an active ingredient.

[0071] In one example, the feed additive of the present application can be applied in various forms similar to the antiviral composition. Examples of formulation forms, although not limited thereto, include liquids, suspensions, powders, granules, tablets, capsules, pills, and the like. In addition, in order to prepare a dosage form, at least one additive, excipient, such as a diluent, a glidant, a binder, a disintegrant, a sweetener, a stabilizer, and a preservative can be selected and used. In addition to the active ingredient, they can be included in ordinary feed additives, and flavorings, adjuvants, and the like for providing additional functions can be mixed and used. Specifically, the diluent can be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, the glidant can be magnesium stearate or talc, and the binder can be polyvinyl pyrrolidone or hydroxypropyl cellulose. In addition, the disintegrant may be carboxymethylcellulose calcium, sodium starch glycolate, polacrilin potassium or cross-linked polyvinylpyrrolidone, and the sweetener may be white sugar, fructose, sorbitol, aspartame or nucleic acid (IMP, GMP), and the stabilizer may be sodium carboxymethylcellulose, β-cyclodextrin, white beeswax or xanthan gum, and the preservative may be methylparaben, propylparaben or potassium sorbate.

[0072] The feed additive can be selected from the group consisting of: mammals, fish, birds or arthropods, specifically, pigs, cattle, chickens, goats, sheep, horses, fish, shrimps, insects, dogs and cats. Specifically, it can be pigs, cattle, chickens, dogs or cats, but is not limited to this. The feed additive can inhibit the activity of viruses selected from the group consisting of: African swine fever virus (ASFV), classical swine fever virus (CSFV), low pathogenic avian influenza virus (LPAIV), canine coronavirus (CCoV), canine adenovirus (CAV), canine distemper virus (CDV), feline parvovirus (FPV), feline calicivirus (FCV), feline infectious peritonitis virus (FIPV, also known as feline coronavirus, FCoV) and foot-and-mouth disease virus (FMDV).

[0073] The feed additive can be fed to a subject, such as a mammal, fish, bird, or arthropod, specifically, a pig, cattle, chicken, goat, sheep, horse, fish, shrimp, insect, dog, or cat, in the same system and dosage as the antiviral composition described herein. Furthermore, the feeding method can be a feeding method known in the art, for example, oral feeding by mixing with feed, but is not limited thereto.

[0074] In one example, according to the above system and dosage, the feed additive of the present application can be added to 1 kg of feed on a dry weight basis in various proportions, such as 0.01 to 300 g, 1 g to 200 g or 10 g to 100 g (i.e., 0.001 wt % to 30 wt %, 0.1 wt % to 20 wt % or 1 wt % to 10 wt % based on the total feed dry weight), but is not limited thereto.

[0075] The antiviral substance selected from the compounds of Chemical Formulas 1 to 6 and their pharmaceutically acceptable salts contained in the antiviral composition, the pharmaceutical composition or feed additive according to the present application, shows a mechanism for effectively inhibiting viral infection and proliferation by reducing infectivity, wherein the method for reducing infectivity is: inhibiting the function of inosine monophosphate dehydrogenase (IMPDH) to inhibit smooth viral gene replication by reducing the source of viral gene replication, wherein the inosine monophosphate dehydrogenase produces the required GMP when the virus invades the cell and performs gene replication; or by inserting into the viral gene replication process as a guanosine analog during the replication process, causing abnormalities in the viral functional protein produced as the final product.

[0076] Several known mechanisms of action of nucleoside analogs, such as well-known efficacy mechanisms such as immunomodulatory effects, increased expression of interferon-stimulating factors, and inhibition of viral RNA polymerase, may also be the mechanisms of the compounds selected from Chemical Formulas 1 to 6 and their pharmaceutically acceptable salts contained in the antiviral composition, pharmaceutical composition, or feed additive according to the present application. DETAILED DESCRIPTION

[0077] Invention Mode

[0078] Hereinafter, in order to help understand the present application, the present application will be described in detail by way of examples. However, the following examples only illustrate the content of the present application, but the scope of the present application is not limited by the following examples. The examples of the present application are provided in order to more completely describe the present application to those skilled in the art.

[0079] [Example]

[0080] Preparation Example 1: Preparation of IMP, Xanthine Nucleoside Monophosphate (XMP) and Guanosine Monophosphate (GMP) Method for preparing nucleoside analogs

[0081] 1-1. Preparation of Dialdehyde Nucleosides

[0082] For oxidative cleavage, IMP, XMP, or GMP (prepared and supplied by CJ CheilJedang) dissolved in water was prepared using periodate (NaIO4) as a catalyst and an organic solvent, followed by filtration using filter paper, and the permeate was purified using an anion exchange resin (WA30) and then freeze-dried to obtain the final product (Maria Meurillon et al., 2014 Eur. J. Med. Chem. 77: 18-37).

[0083] 1-2. Preparation of Nucleosides in the Form of Acyclic Diols

[0084] For oxidative cleavage, IMP, XMP, or GMP (prepared and supplied by CJ CheilJedang) dissolved in water was prepared using periodate (NaIO4) as a catalyst and an organic solvent, followed by filtration using filter paper, and the permeate was reduced by adding sodium borohydride (NaBH4) and then freeze-dried to obtain the final product (Maria Meurillon et al., 2014 Eur. J. Med. Chem. 77: 18-37).

[0085] Example 1: African swine fever virus (ASFV)

[0086] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms against African swine fever virus (ASFV, China / 2018 / Anhui XCGQ), porcine alveolar macrophages (PAM cell line) were cultured at 0.5×10 5 Cells / well were inoculated in 48-well plates and then cultured for one day. The next day, 1 MOI of ASFV and inosine, xanthine nucleoside, and guanosine in dialdehyde and acyclic diol forms were simultaneously introduced into the cells. Two days later, DNA was extracted from the infected cells using a viral gene (DNA / RNA) extraction kit (iNtRON, 101410754). For the extracted DNA, ASFV-specific primers (F-AGTTCGGATGTCACAACGCT, R-ACTGGTTCCCTCCACCGATA) and real-time RT-qPCR experimental methods (95°C, 5 minutes for 1 cycle, 95°C, 10 seconds, 65°C, 30 seconds, 56°C, 60 seconds, 40 cycles) were used to verify the antiviral efficacy of the material.

[0087] As shown in Table 1, when inosine, xanthosine, and guanosine in their dialdehyde and acyclic diol forms were administered into cells, ASFV infection and proliferation were inhibited. Guanosine in its dialdehyde form exhibited the highest efficacy (IC50: 79.0 μM), with the antiviral effects ranking after inosine and xanthosine. Similarly, among the acyclic diol forms, the antiviral efficacy against ASFV was ranked in the order of guanosine, inosine, and xanthosine.

[0088]

Table 1

[0089] ASFV infection inhibitory concentrations, cytotoxicity, and selectivity indices of inosine, xanthosine, and guanosine in their dialdehyde and acyclic diol forms

[0090] Classification <![CDATA[IC 50 ]]> <![CDATA[CC 50 ]]> SI dialdehyde inosine <96.6uM(±1.26) >10000uM >103.5 dialdehyde xanthine riboside <110.0uM(±1.1) >10000uM >90.9 dialdehyde guanosine <79.0uM(±1.0) >6000uM >75.9 Acyclic diol inosine <353.0uM(±1.1) >10000uM >28.3 Acyclic diol xanthine riboside <1165uM(±1.1) >10000uM >8.6 Acyclic diol guanosine <270.0uM(±1.6) >10000uM >37.0

[0091] Example 2: Classical Swine Fever Virus (CSFV)

[0092] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms against classical swine fever virus (CSFV), porcine kidney cells (PK15 cell line) were cultured at 1×10 4 Cells / well were seeded in 96-well plates and cultured for one day. Then, 100 TCID 50 CSFV and dialdehyde and acyclic diol forms of inosine, xanthine nucleoside and guanosine were simultaneously treated into cells. After 2 days, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TM vRDⅡ) was used to isolate RNA from infected cells. The antiviral efficacy of the material was verified using CSFV-specific primers (F-CTCTGGTCAGGGTGCTCAAG, R-GAGGGACTGTGCAACCATCA) and real-time RT-qPCR (95°C, 15 minutes, 1 cycle, 95°C, 20 seconds, 58°C, 40 seconds, 40 cycles).

[0093] As can be seen from [Table 2], the inhibitory effects of inosine, xanthosine and guanosine in the dialdehyde form and the acyclic diol form on CSFV infection and proliferation were confirmed. Xanthosine in the dialdehyde form showed the best efficacy (IC 50 :156.7μM), the antiviral effect was ranked by guanosine and inosine. In addition, in the non-cyclic diol form, IC 50 The values ​​are shown in the order of inosine, xanthosine, and guanosine, and show relatively higher IC values ​​compared to the dialdehyde form. 50 value.

[0094]

Table 2

[0095] CSFV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0096]

[0097] Example 3: Low Pathogenic Avian Influenza Virus (LPAIV)

[0098] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms against low pathogenic avian influenza virus (LPAIV, H9N2), canine kidney cells (MDCK cell line) were cultured at 1.4×10 4 Cells / well were seeded in a 96-well plate and cultured for one day. The next day, 20 TCID 50 100 μg / well of LPAIV and dialdehyde and acyclic diol forms of inosine, xanthine nucleoside and guanosine were simultaneously treated into the cells. After 2 days, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TM The antiviral efficacy of the material was verified by real-time RT-qPCR (95°C, 15 minutes, 1 cycle, 95°C, 20 seconds, 58°C, 40 seconds, 40 cycles) using LPAIV-specific primers (F-GCTAGGCAGATGGTACAGGC, R-TGCACTCCCATCCGTTTCTG) and the vRDⅡ (vRDⅡ)-infected cells.

[0099] As can be seen from [Table 3], the dialdehyde form of guanosine showed the best efficacy (IC 50 : 102.2 μM), and confirmed that the inhibitory effect of virus infection and proliferation was in the order of xanthosine and inosine. In addition, when the material was in the form of acyclic diol, the antiviral effect on LPAIV was shown to be in the order of guanosine, inosine and xanthosine.

[0100]

Table 3

[0101] LPAIV infection inhibitory concentration, cytotoxicity and selectivity index of dialdehyde and acyclic diol forms of inosine, xanthosine and guanosine

[0102]

[0103]

[0104] Example 4: Canine Coronavirus (CCoV)

[0105] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine, and guanosine in their dialdehyde and acyclic diol forms against canine coronavirus (CCoV), canine fibroblasts (A-72 cell line) were cultured at 1.4 × 10 4 Cells / well were seeded in a 96-well plate and cultured for one day. The next day, 50 TCID 50 CCoV and dialdehyde and non-cyclic diol forms of inosine, xanthine nucleoside and guanosine were simultaneously treated into cells. After 2 days, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TM vRDⅡ) were used to extract RNA from infected cells. The antiviral efficacy of the material was verified using CCoV-specific primers (F-TGAAGGTGTGCCAACTGGTGT, R-GCCCATCCTGTCGCACTACT) and real-time RT-qPCR experimental methods (95°C, 15 minutes, 1 cycle, 95°C, 20 seconds, 58°C, 40 seconds, 40 cycles).

[0106] As can be seen from [Table 4], the dialdehyde forms of inosine and xanthine riboside showed excellent efficacy (IC 50 : 45.1 μM and 43.0 μM), guanosine showed an IC of 109.7 μM 50 Furthermore, in the case of materials in the form of acyclic diols, the antiviral potency against CCoV was observed to be in the order of inosine, guanosine, and xanthine nucleosides.

[0107]

Table 4

[0108] CCoV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0109]

[0110]

[0111] Example 5: Canine Adenovirus (CAV)

[0112] To evaluate the viral infection and inhibitory effects of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms on canine adenovirus (CAV), monkey kidney cells (Vero cell line) were cultured at 2×10 4 Cells / well were seeded in a 96-well plate and cultured for one day. The next day, 100 TCID 50 / well of CAV and dialdehyde form and acyclic diol form of inosine, xanthine nucleoside and guanosine were simultaneously treated into the cells. After 2 days, DNA was extracted from the infected cells using a commercial viral gene (DNA / RNA) extraction kit (iNtRON, 101410754). For the extracted DNA, CAV-specific primers (F-CGCTGAACATTACTACCTTGTC, R-GCAGAGTCTAGAACAAATGGC) and real-time RT-qPCR experimental methods (95°C, 5 minutes 1 cycle, 95°C, 15 seconds, 60°C, 30 seconds, 40 cycles) were used to verify the antiviral efficacy of the material.

[0113] As can be seen from [Table 5], the inhibitory effects of inosine, xanthosine, and guanosine in the dialdehyde and acyclic diol forms on CAV infection and proliferation were confirmed. Guanosine in the dialdehyde form showed the best efficacy (IC 50 :85.9μM), and the antiviral effect was ranked by xanthosine and inosine. In addition, in the acyclic diol form, the antiviral efficacy was ranked by xanthosine, inosine, and guanosine, and showed a relatively high IC compared to the dialdehyde form. 50 value.

[0114]

Table 5

[0115] CAV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0116]

[0117]

[0118] Example 6: Canine Distemper Virus (CDV)

[0119] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms against canine distemper virus (CDV), monkey kidney cells (Vero cell line) were cultured at 2×10 4 Cells / well were seeded in a 96-well plate and cultured for one day. The next day, 100 TCID 50 CDV and dialdehyde and non-cyclic diol forms of inosine, xanthine nucleoside and guanosine were simultaneously treated into the cells. After 3 days, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TMvRDII) were used to extract RNA from infected cells. The antiviral efficacy of the material was verified using CDV-specific primers (F-GCTTACTTCAGACTCGGGCAAGAAATGGTTA, R-CAGTAGCTCGAATTGTCCGGTCCTCTGTTGT) and real-time RT-qPCR (95°C, 10 minutes, 1 cycle, 95°C, 15 seconds, 60°C, 30 seconds, 72°C, 30 seconds, 40 cycles).

[0120] As can be seen from [Table 6], the dialdehyde form of xanthine riboside showed the best efficacy (IC 50 :79.9μM), and confirmed that the inhibitory potency of viral infection and proliferation was ranked by inosine and guanosine. Compared with the dialdehyde form of the material, the non-cyclic diol form of the material was significantly inhibited at IC 50 The values ​​showed the antiviral potency, and the potency ranking was confirmed to be xanthosine, guanosine, and inosine.

[0121]

Table 6

[0122] CDV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0123]

[0124] Example 7: Feline Parvovirus (FPV)

[0125] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms against feline parvovirus (FPV), feline kidney cells (CRFK cell line) were cultured at 3×10 4 Cells / well were seeded in a 48-well plate and cultured for one day. The next day, 100 TCID 50 / well FPV and dialdehyde form and acyclic diol form of inosine, xanthine nucleoside and guanosine were simultaneously treated into the cells. After 2 days, DNA was extracted from the infected cells using a commercial viral gene (DNA / RNA) extraction kit (iNtRON, 101410754). For the extracted DNA, FPV-specific primers (F-AGAGCATTGGGCTTACCACC, R-CCCCATTTGAGTTACACCACG) and real-time RT-qPCR experimental methods (95°C, 15 minutes 1 cycle, 95°C, 20 seconds, 58°C, 30 seconds, 72°C, 30 seconds, 40 cycles) were used to verify the antiviral efficacy of the material.

[0126] As can be seen from [Table 7], when inosine, xanthosine and guanosine in the dialdehyde form and the acyclic diol form were used to treat cells, it was confirmed that the infection and proliferation of FPV were inhibited. Xanthosine in the dialdehyde form showed the best efficacy (IC 50 :31.0μM), and confirmed the excellent effect in the order of guanosine and inosine. In addition, in the acyclic diol form, IC 50 The values ​​can be determined in the order of guanosine, inosine and xanthosine.

[0127]

Table 7

[0128] FPV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0129]

[0130] Example 8: Verification of the inhibitory effect on feline calicivirus (FCV) infection

[0131] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms against feline calicivirus (FCV), feline kidney cells (CRFK cell line) were cultured at 3×10 4 Cells / well were seeded in a 48-well plate and cultured for one day. 50 FCV and dialdehyde and non-cyclic diol forms of inosine, xanthine nucleoside and guanosine were simultaneously treated into the cells. After 24 hours, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TM vRDⅡ) were used to extract RNA from infected cells. The antiviral efficacy of the material was verified using FCV-specific primers (F-GCAAAGATCCGGCTTGCCTC, R-CGCTGTTGACCAAGTGCAGC) and real-time RT-qPCR (95°C, 15 minutes for 1 cycle, 95°C, 20 seconds, 58°C, 30 seconds, 72°C, 30 seconds, 40 cycles).

[0132] As can be seen from [Table 8], when cells were treated with inosine, xanthosine and guanosine in the dialdehyde form and the acyclic diol form, it was confirmed that FCV infection and proliferation were inhibited. Guanosine in the dialdehyde form showed the most excellent efficacy (IC 50 :18.2μM), and confirmed excellent effects in the order of xanthosine and inosine. In addition, the non-cyclic diol form of the material also showed efficacy in the order of guanosine, xanthosine and inosine.

[0133]

Table 8

[0134] FCV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0135]

[0136] Example 9: Feline Infectious Peritonitis Virus (FIPV)

[0137] To evaluate the viral infection and inhibitory efficacy of inosine, xanthosine and guanosine in their dialdehyde and acyclic diol forms on FIPV, feline embryonic cells (FCWF-4 cell line) were cultured at 2×10 4 Cells / well were seeded in a 96-well plate and cultured for one day. The next day, 100 TCID 50 FIPV and dialdehyde and acyclic diol forms of inosine, xanthine nucleoside and guanosine were simultaneously treated into the cells. After 2 days, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TM vRDⅡ) were used to extract RNA from infected cells. The antiviral efficacy of the material was verified using FIPV-specific primers (F-TGGCATCTTGCTAACTGGAACT, R-TGCCATAAACGAGCCAGCTA) and real-time RT-qPCR (95°C, 15 minutes, 1 cycle, 95°C, 30 seconds, 58°C, 40 seconds, 40 cycles).

[0138] As can be seen from [Table 9], the dialdehyde form of inosine showed excellent inhibitory efficacy on the infection and proliferation of FIPV (IC 50 :45.5μM), and it can be confirmed that the order of excellent effect is guanosine and xanthine nucleoside. However, the IC 50 The differences between the values ​​were not large. In addition, in the case of the acyclic diol form, the antiviral potency against FIPV was shown to be in the order of inosine, xanthosine, and guanosine.

[0139]

Table 9

[0140] FIPV infection inhibitory concentrations, cytotoxicity, and selectivity indices of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine

[0141]

[0142] Example 10: Foot-and-mouth disease virus (FMDV)

[0143] To evaluate the inhibitory effects of the dialdehyde forms of inosine and guanosine and the acyclic diol forms of inosine, xanthosine and guanosine on the viral infection of foot-and-mouth disease virus (FMDV), hamster kidney cells (BHK-21 cell line) were cultured at 5×10 4Cells / well were seeded in 48-well plates and cultured for one day. The next day, 0.01 MOI of FMDV and dialdehyde forms of inosine and guanosine, as well as non-cyclic diol forms of inosine, xanthine nucleosides, and guanosine were simultaneously treated into the cells. After 36 hours, viral RNA was extracted using a commercial viral RNA extraction kit (Ribospin TM vRDⅡ) were used to extract RNA from infected cells. The antiviral efficacy of the material was verified using FMDV-specific primers (F-CCGACCCCTCATTCAGCAGACCTC, R-GAGGGTTCTTTTCCGCGTCGCC) and real-time RT-qPCR experimental methods (95°C, 5 minutes for 1 cycle, 95°C, 10 seconds, 60°C, 30 seconds, 40 cycles, 95°C, 15 seconds, 60°C, 60 seconds, 95°C, 15 seconds, 1 cycle melting curve).

[0144] As can be seen from [Table 10], the dialdehyde form of guanosine showed very good inhibitory efficacy on the infection and proliferation of FIPV (IC 50 :114.8μM). The acyclic diol form of the material showed antiviral efficacy against FMDV in the order of inosine, xanthosine, and guanosine, but showed a slightly higher IC compared to the dialdehyde form. 50 value.

[0145]

Table 10

[0146] FMDV infection inhibitory concentration, cytotoxicity and selectivity index of dialdehyde and acyclic diol forms of inosine, xanthosine and guanosine

[0147]

[0148] Example 11: Evaluation of the Immunopotentiating Potency of Nucleoside Analogs Derived from Nucleic Acid Analogs

[0149] To confirm the effectiveness of enhancing interferon-stimulated gene expression, which is one of the representative antiviral mechanisms of nucleoside analogs (Johnson YN Lau, et al. 2002. Mechanism of action of Ribavirin in the combination treatment of chronic HCV infection. Hepatology. 35(5): 1002-9. Doi: 10.1053 / jhep.2002.32672, Paeshuyse J., et al., 2011. Ribavirin for the treatmentof chronic hepatitis C virus infection: a review of the proposed mechanism ofaction.Curr.Opin.Virol.1:590-598.Doi:10.1016 / j.coviro.2011.10.030), the dialdehyde forms of inosine, xanthosine, and guanosine, as well as the acyclic diol forms of inosine, xanthosine, and guanosine were used to evaluate the effects of increasing the expression of ISG15, Mx1, and RNaseL, which are representative interferon-stimulated genes that are very important in inducing antiviral immunity.

[0150] Porcine alveolar macrophages (PAM cell line) were cultured at 1×10 5Cells were seeded per well in a 48-well plate and cultured for one day. The following day, cells were treated with the dialdehyde forms of inosine, xanthosine, and guanosine, as well as the acyclic diol forms of inosine, xanthosine, and guanosine. RNA was extracted from infected cells 12 hours later using a commercial easy-spin [DNA-free] total RNA extraction kit (iNtRON). The same gene amount was quantified using porcine β-actin (F-GACCACCTTCAACTCGATCA, R-GTGTTGGCGTAGAGGTCCTT) and then real-time RT-qPCR experiments were performed using ISG15, Mx1, and RNaseL specific primers (ISG15: F-GGTGCAAAAGCTTCAGAGACC, R-GTCAGCCAGACCTCATAGGC / Mx1: F-AGCGCAGTGACACCAGCGAC, R-GCCCGGTTCAGCCTGGGAAC / RNaseL: F-GCCAGACCTAGTGGCTTCTG, R-AGAGGCCCAGAGAGTTGTGA) to verify the expression rate of the immunomodulatory factors of the test materials.

[0151] As shown in [Table 11], it was confirmed that dialdehyde and non-cyclic diol forms of inosine, xanthosine and guanosine increased the expression of interferon-stimulated genes (ISG15, Mx1, RNaseL). Observed that, by dialdehyde forms of inosine, xanthosine and guanosine, the expression of ISG15 gene increased by 4 to 4.7 times, the expression of Mx1 gene increased by 3.5 times, and the expression of RNaseL gene increased by 2.5 to 3.4 times. Due to non-cyclic diol forms of inosine, xanthosine and guanosine, the expression of ISG15 gene increased by 4.2-6.3 times, the expression of Mx1 gene increased by 2.8-4.0 times, and the expression of RNaseL gene increased by 1.7-3.0 times. The increase in interferon-stimulated genes indicates that dialdehyde and non-cyclic diol forms of materials have an immunopotentiating effect that induces antiviral efficacy.

[0152]

Table 11

[0153] Interferon-stimulated gene expression values ​​of dialdehyde and acyclic diol forms of inosine, xanthosine and guanosine (2 -ΔΔCt value).

[0154]

[0155]

Claims

1. An antiviral composition comprising at least one nucleoside analog selected from the group consisting of nucleoside analogs of the following chemical formulas 1-6 and pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 2. The composition of claim 1, wherein the virus is at least one virus selected from the group consisting of African swine fever virus (ASFV), classical swine fever virus (CSFV), low pathogenic avian influenza virus (LPAIV), canine coronavirus (CCoV), canine adenovirus (CAV), canine distemper virus (CDV), feline parvovirus (FPV), feline calicivirus (FCV), feline infectious peritonitis virus (FIPV, also known as feline coronavirus, FCoV) and foot-and-mouth disease virus (FMDV).

3. An immunomodulatory composition comprising at least one nucleoside analog selected from the group consisting of nucleoside analogs of the following chemical formulas 1-6 and pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] The composition according to claim 1 or 2, which is a pharmaceutical composition or a food composition.

5. A feed additive comprising at least one nucleoside analogue selected from the group consisting of nucleoside analogues of the following Chemical Formulas 1-6 and pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] The feed additive according to claim 5 , which is used for mammals, birds, fish or arthropods.

7. A feed comprising at least one nucleoside analogue selected from the group consisting of nucleoside analogues of the following Chemical Formulas 1-6 and pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 8. The feed according to claim 7, which is for mammals, birds, fish or arthropods.

9. A method for preventing, ameliorating or treating a virus, comprising administering to a subject at least one nucleoside analogue selected from the group consisting of the nucleoside analogues of the following Chemical Formulas 1-6 and pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 10. A method for regulating immunity, comprising administering to a subject at least one nucleoside analog selected from the group consisting of nucleoside analogs of the following Chemical Formulas 1-6 and pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6]

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