Antiviral compositions containing nucleic acid derived nucleoside analogs and pharma-ceutically acceptable salts thereof

Abstract

The present application relates to an antiviral composition, an immune enhancing composition, a feed or a feed additive comprising a nucleotide-derived nucleoside analogue.

Description

[Technical field]

[0001] The present application relates to antiviral, immunomodulatory and feed compositions comprising nucleic acid-derived nucleoside analogues and their pharma- ceutically acceptable salts. [Background technology]

[0002] The damage to humans and animals caused by viruses is gradually increasing worldwide, and the scale of the damage is also showing a tendency to become enormous. The significant impact on humanity of zoonotic viruses such as SARS-CoV-2, MERS, and influenza that have emerged or are continuing in recent years, as well as the social and economic damage caused by animal viruses such as avian influenza and African swine fever, are urging the development of more effective measures to control viral diseases.

[0003] In particular, there is no doubt that complete control of viral diseases is impossible in the current situation where only a few vaccines are the only virus countermeasures. Efforts to develop animal virus therapeutics as a powerful solution to solve this problem have been ongoing for the past few decades. Research is ongoing into the development of virotherapy agents (agents that suppress virus infection and proliferation) to control many animal viruses, such as influenza viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), swine influenza virus (SIV) or avian influenza virus (AIV), porcine rotavirus (PRV), porcine circovirus (PCV), infectious bronchitis virus (IBV), avian Newcastle disease virus (NDV), bovine viral diarrhea (BVD), bovine rotavirus (BRV), and canine parvovirus (CPV) (Kim et al., 2013 Virus Res. 171(1):44-53, Paul C Jordan et al., 2018 Antivir Chem Chemother. 26:1-19, Andrea J Pruijssers et al., 2019 Current Opinion in Virology 35:57-62), there are currently no highly effective antiviral drugs on the market, and with viral diseases now posing a major threat, there is a great need for the development and commercialization of antiviral drugs. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kim et al., 2013 Virus Res. 171(1):44-53 [Non-Patent Document 2] Paul C Jordan et al., 2018 Antivir Chem Chemother. 26:1-19 [Non-Patent Document 3] Andrea J Pruijssers et al., 2019 Current Opinion in Virology 35:57-62 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present application is to provide antiviral, immunomodulatory or feed compositions comprising nucleic acid-derived nucleoside analogues and pharma- ceutically acceptable salts thereof. [Means for solving the problem]

[0006] One embodiment of the present application may be an antiviral composition comprising a nucleoside of a dialdehyde form of inosine, a dialdehyde form of xanthosine, a dialdehyde form of guanosine, or an acyclic diol form of inosine, an acyclic diol form of xanthosine, or an acyclic diol form of guanosine.

[0007] Another aspect of the present application may be an immunomodulatory composition comprising a nucleoside of 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, or the acyclic diol form of guanosine.

[0008] Another aspect of the present application may be a medicine, a feed, or a feed additive comprising the antiviral composition.

[0009] Specifically, the composition may be an influenza virus, such as Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Porcine Epidemic Diarrhea virus (PEDV), Swine Influenza virus (SIV) or Avian Influenza virus (AIV), Porcine Rotavirus (PRV), Porcine Circovirus (PCV), Infectious Bronchitis virus (IBV), Chicken Newcastle Disease virus (NDV), Bovine Viral Diarrhea virus (BVDV), Bovine Rotavirus (BRV), Canine Parvovirus (CPV), or any of the following viruses: The composition may exhibit an antiviral effect against one or more viruses selected from the group consisting of: Effect of the Invention

[0010] The nucleic acid-derived nucleoside analogues according to the present application can be useful as antiviral agents. [Brief description of the drawings]

[0011] [Figure 1a] 1 is a diagram showing the conversion of IMP, XMP and GMP into dialdehyde forms to obtain the structures of dialdehyde-form nucleosides of Formulas 1 to 3 according to the present application. [Figure 1b] The chemical structures of nucleosides of the present invention, represented by Chemical Formulas 4 to 6, are shown in which IMP, XMP and GMP are converted into acyclic diol forms, respectively. [Diagram 2]The anti-PRRSV efficacy of dialdehyde-type inosine confirmed through immunofluorescence staining (top) and a graph showing the PRRSV infection rate using this (bottom). [Diagram 3] The anti-PRRSV efficacy of dialdehyde-type xanthosine confirmed through immunofluorescence staining (top) and a graph showing the PRRSV infection rate using this (bottom). [Figure 4] The anti-PRRSV efficacy of dialdehyde-type guanosine confirmed through immunofluorescence staining (top) and a graph showing the PRRSV infection rate using this (bottom). [Diagram 5] The anti-PRRSV efficacy of acyclic diol inosine confirmed through immunofluorescence staining (top) and a graph showing the PRRSV infection rate using this (bottom). [Figure 6] The anti-PRRSV efficacy of acyclic diol xanthosine confirmed through immunofluorescence staining (top) and a graph showing the PRRSV infection rate using this (bottom). [Figure 7] The anti-PRRSV efficacy of acyclic diol guanosine confirmed through immunofluorescence staining (top) and a graph showing the PRRSV infection rate using this (bottom). [Figure 8] This shows the concentration of the substance in the blood at different times after administering the dialdehyde form of xanthosine via oral (intragastric) and IP (intraperitoneal) injection. [Figure 9] The results show the concentration of the substance in the blood at different times after the dialdehyde form of guanosine was administered by oral (intragastric) and IP (intraperitoneal) injection. [Figure 10] The schedule for the influenza virus challenge test using mice is shown below. [Figure 11] The graph shows changes in mouse body weight in each group during a 7-day monitoring period after SIV infection. [Figure 12] The graph shows the change in survival rate of mice in each group during a 7-day monitoring period after SIV infection. [Figure 13] The results show the lesions in lung tissues removed through autopsy 3, 5, and 7 days after SIV infection in each group. [Figure 14] The residual virus titer in lung tissues excised through autopsy on days 3, 5, and 7 after SIV infection was measured by PCR. The results are shown for each group. [Figure 15] These are representative photographs of lung tissue lesions for each group at final necropsy on day 7 after SIV infection. [Figure 16] The schedule for the PRRS virus challenge test using piglets is shown below. [Figure 17] The changes in piglet weight in each group during the 14-day monitoring period after PRRSV infection are shown. [Figure 18] The amount of virus present in lung tissue collected from piglets in each group two weeks after PRRSV infection was compared with that in the positive control group. [Figure 19] These are photographs of lungs removed from representative piglets from each group two weeks after PRRSV infection, and interstitial pneumonia lesions were observed with the naked eye. [Figure 20] The photographs show the results of lung extraction from a representative piglet from each group two weeks after PRRSV infection, and the presence of PRRS virus particles in the tissue was observed by immunohistochemistry (IHC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present application will now be described in detail.

[0013] The antiviral or immunomodulatory composition of the present application is a nucleoside analog in the form of a dialdehyde or acyclic diol, and in one specific embodiment, is characterized by comprising, as an active ingredient, one or more selected from the group consisting of nucleic acid-derived dialdehyde forms, acyclic diol forms of inosine, xanthosine, and guanosine, as represented by the following chemical formulas 1 to 6, and pharma- ceutical acceptable salts thereof. [ka] JPEG2024545703000003.jpg111168

[0014] The active ingredients, inosine, xanthosine, or guanosine in the form of dialdehyde or acyclic diol, can be produced through an optimized process. As a specific example, they can be produced by the method described in the examples of the present invention, but are not limited thereto.

[0015] In this case, the compound represented by Chemical Formulas 1 to 6 or a pharma- ceutically acceptable salt thereof may be used in an amount of 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, 0.0001~0.001 parts by weight, 0.0001~0.0005 parts by weight, 0.001 parts by weight~20 parts by weight, 0.001 parts by weight~15 parts by weight, 0.001 parts by weight~10 parts by weight, 0.001~5 parts by weight, 0.001 Part by weight ~ 1 part by weight, 0.001 ~ 0.5 part by weight, 0.001 ~ 0.1 part by weight, 0.001 ~ 0.05 part by weight, 0.001 ~ 0.01 part by weight, 0.001 ~ 0.005 part by weight, 0.01 part by weight ~ 20 part by weight, 0. 01 parts by weight ~ 15 parts by weight, 0.01 parts by weight ~ 10 parts by weight, 0.01 ~ 5 parts by weight, 0.01 parts by weight ~ 1 part by weight, 0.01 ~ 0.5 parts by weight, 0.01 ~ 0.1 parts by weight, 0.01 ~ 0.05 parts by weight, 0.1 parts by weight ~ 2 0 parts by weight, 0.1 parts to 15 parts by weight, 0.1 parts to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 parts 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, The compound or a pharmaceutically acceptable salt thereof may be contained in an amount of 5 to 20 parts by weight, 5 to 15 parts by weight, 5 to 10 parts by weight, 10 to 15 parts by weight, 10 to 20 parts by weight, or 15 to 20 parts by weight. When the compound or a pharmaceutically acceptable salt thereof is contained in an amount of less than 0.0001 part by weight, the antiviral effect of the compound is not exhibited satisfactorily, whereas when the compound or a pharmaceutically acceptable salt thereof is contained in an amount of more than 20 parts by weight, the increase in the antiviral effect is insignificant compared to the increase in the compound content, which is not preferable.

[0016] The virus is not limited to the above as long as it is a type of virus, and may be an influenza virus such as Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Porcine Epidemic Diarrhea virus (PEDV), Swine Influenza virus (SIV) or Avian Influenza virus (AIV), Porcine Rotavirus (PRV), Porcine Circovirus (PCV), Infectious Bronchitis virus (IBV), Chicken Newcastle Disease virus (NDV), Bovine Viral Diarrhea virus (BVDV), Bovine Rotavirus (BRV), and Canine Parvovirus (CPV). The virus may be one or more selected from the group consisting of:

[0017] Representative viruses among those for which antiviral efficacy has been verified in this application are described below.

[0018] Porcine reproductive and repiratory syndrome virus (PRRSV) is a typical chronic wasting disease in pigs. It belongs to the Ateriviridae family, the Nidovirales order, and is distantly related to the Coronaviridae family. This virus is broadly classified into European (EU strain) and North American (NA strain) types based on antigenic and genetic characteristics, and has an envelope protein and a 15kb positive single-stranded RNA gene. This gene has at least 10 or more open reading frames (ORFs), and ORF1a and ORF1b at the 5' end, which code for nonstructural proteins (NSPs), account for two-thirds of the entire gene. This virus mainly infects porcine alveolar macrophages (PAMs) that exist in the lungs, suppressing the immune function of macrophages, making them vulnerable to secondary infections by other viruses and bacteria, and is known to be a very serious virus. Infected pregnant sows can suffer miscarriages and stillbirths (mummies), while piglets can suffer from severe respiratory symptoms and die. The damage caused by the Porcine Circovirus and Postweaning Systemic Wasting Syndrome has been a major problem in the pig farming industry for many years.

[0019] Porcine epidemic diarrhea virus (PEDV), which belongs to the Coronavirus family, infects the small intestine of pigs, as its name suggests, causing the loss of villi and severe dehydration and diarrhea, resulting in serious economic losses for pig farmers. It is a viral disease that causes particularly large economic losses in young pigs, as it shows a high mortality rate (50-100%). PEDV has an envelope and contains a positive single-stranded RNA of about 28 kb, with seven ORFs. It is broadly divided into two genetic forms (G1 and G2) based on the Spike gene, and each form is further divided into a and b. Recently, the G2c type has also been identified. Additionally, there are S INDEL (G1b type) that contains many deletions and insertions in the S1 subunit of the spike protein, and non-S INDEL (G2b type) that does not, and the G2b type generally shows high viral virulence. Because cross-immunity is not well formed between the G1 and G2 types, vaccines must also be developed for each type. There is still a lack of reliable countermeasures against PEDV, as there is no treatment and vaccines do not form cross-immunity by type.

[0020] Porcine circovirus, first identified in 1982, is the causative agent of postweaning multisystemic wasting syndrome (PMWS), which has been occurring in North America, the UK, and many other European countries, and in Korea, where its occurrence began in 1999, causing viral diseases in pig farms. Porcine circovirus, the smallest form of animal virus, is classified into serotypes 1 and 2, with type 1 being non-pathogenic and type 2 being known to be a pathogenic virus. In addition, mixed infections with porcine reproductive and respiratory syndrome (PRRS) virus or porcine parvovirus have been reported in many cases on farms, making it a troublesome virus that causes serious economic problems.

[0021] IBV, which belongs to the coronavirus family, has been occurring worldwide, with its first outbreak in Korea officially confirmed in 1986. While some viruses, such as the Massachusetts type, are isolated worldwide, it has been reported that there are also viruses that show regional characteristics that are prevalent in Europe, the United States, Southeast Asia, and other regions. In Korea, a virus that causes decreased egg production and respiratory symptoms was first isolated in 1986, and since 1990, there has been a sustained high incidence of viral infections with renal-type symptoms as the main symptom. Recently, the prevalent isolates are roughly divided into the Korean I group, which is a respiratory type, and the Korean II group, which is a renal type, and it has been analyzed that the renal type is similar to isolates from Japan and China.

[0022] BVDV induces bovine viral diarrhea, a Class 2 legal infectious disease in cattle, causing ulcers in the digestive tract mucosa, diarrhea, and respiratory lesions, and in severe cases, can even lead to the death of cattle. It was first reported by Olafson et al. in the United States in 1946. BVDV is an RNA virus classified in the genus Pestivirus of the family Flaviviridae, and is classified into cytopathic strains (NADL, Singer, T20 strains, etc.) and non-cytopathic strains (New York strain, etc.) depending on whether they cause cytopathic effects in cell lines. The main routes of infection for BVDV are oral infection through feed contaminated with feces and placental infection, but it can also be transmitted through the respiratory and reproductive tracts. Reproductive infection is known to be possible through semen and fertilized eggs.

[0023] CPV infections were reported worldwide before 1978, and in Korea, it was first confirmed in Gyeonggi Province in the 1980s and has since spread nationwide. Clinically or pathologically, it can be broadly divided into two types: cardiac type, which is seen mainly in young puppies aged 3 to 8 weeks, and enteritis type, which is seen in weaned dogs. Enteritis type symptoms include severe vomiting, dehydration due to bloody diarrhea, and leukopenia, which can lead to death. There is no cure for CPV, and intravenous drip injections are used to alleviate symptoms, and antibiotics are administered to suppress complex bacterial infections. The only preventive measures are attenuated and inactivated vaccines.

[0024] Porcine rotavirus infection, along with transmissible gastroenteritis virus (TGE) and porcine epidemic diarrhea (PED), is one of the major viral diseases that cause diarrhea in young pigs and impair growth performance. The mortality rate is around 10%, but it increases in cases of mixed infection. It is prevented by vaccination, but it is not easy to completely control it through vaccination because it is an RNA virus that frequently mutates and there are a total of eight serotype groups from A to H due to the VP6 capsid protein (of which type A causes the greatest damage). Furthermore, since recombinant rotaviruses can easily be created that cause not only homogeneous rotavirus infection but also heterogeneous infection, it is necessary to quickly prepare measures other than vaccines against rotavirus infection that could cause great damage due to new recombinant rotaviruses in the event of an outbreak.

[0025] In pigs and calves, especially young calves aged 2-4 weeks, infection with rotavirus (Bovine Rotavirus (BRV)) induces severe diarrhea, which causes serious economic damage. BRV is known as a disease that causes diarrhea in young calves, along with bovine coronavirus (BCV) and bovine viral diarrhea virus (BVDV), which cause calf diarrhea worldwide, and among these, rotavirus infection is the most serious in Korea. As with pigs, vaccines are expected to have a preventive effect, but since there is no treatment, it is difficult to completely control the disease. Bovine rotavirus is structurally classified into 11 P type viruses classified by the VP4 protein that constitutes the outermost layer of the rotavirus, and 12 G type viruses classified by the VP7 protein.

[0026] Newcastle disease is a disease caused by the Newcastle disease virus in birds. There are 12 genetic types (Paramyxovirus (PMV)-1 to PMV12), and PMV-1 is the most important disease in birds, especially chickens and turkeys. In chickens, it is classified into lentogenic strains with low pathogenicity, mesogenic strains with intermediate pathogenicity, and velogenic strains with high pathogenicity, and lentogenic strains are used as live-attenuated virus vaccines. It is a legal type 1 livestock infectious disease (in Korea) that causes 100% death within 2 to 14 days in unvaccinated chickens. Typical symptoms of Newcastle disease include difficulty in breathing, coughing, green or bloody stools, and neurological symptoms such as tilted head (a symptom where the neck is twisted). High mortality rates, along with reduced growth performance and egg production, can cause severe damage to farmers, and although prevention is effective, due to the nature of RNA viruses, mutant strains can easily occur. Therefore, complete disease control will only be possible if effective treatment methods or substances are developed.

[0027] The swine influenza virus infects pigs and causes acute respiratory disease with high transmissibility. Symptoms include upper respiratory symptoms such as fever, coughing, tears, weakness, and loss of appetite. Although the mortality rate is not very high at 1-4%, it impairs pig growth and causes damage to farmers. Therefore, there is a trend to vaccinate pigs recently, but the virus mutates so frequently that it is difficult to expect complete preventive efficacy, and control is necessary in parallel with treatment. As a zoonotic disease, swine influenza type A virus H1N1 infected humans in 2009, causing a widespread outbreak of new influenza worldwide. In general, there are four types of influenza virus, including viruses A, B, C, and D, and viruses belonging to type A are the main cause of disease in pigs. There are H1 to H18 and N1 to N11 depending on the structure (sequence) of the hemagglutinin and neuraminidase proteins that are present in the viral envelope and play an important role in penetration and budding out of infected cells, and there are a total of more than 198 viral subtypes depending on the combination of these two types of proteins.

[0028] Along with swine influenza virus disease, avian influenza viruses are classified into highly pathogenic viruses (H5N1, H5N6, H5N8, H7N9, etc.) with a mortality rate of 100% and low pathogenic viruses (H9N2, etc.) with a low mortality rate but a decrease in production performance (growth and egg laying rate), causing great damage to farmers. As it is an RNA virus like swine influenza, it mutates frequently and recombinant viruses are frequently generated, making it difficult to expect the effectiveness of vaccines, and it is unclear what kind of serious damage a new recombinant virus will cause (as it is a zoonotic disease, it can cause great disasters for humans as well), so measures other than vaccines (securing treatments) must be taken to make it possible to control the disease.

[0029] The pharma- ceutically acceptable salt is preferably an acid addition salt formed with a pharma- ceutically acceptable free acid. The free acid may be an inorganic acid or an organic acid. The inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc., and the organic acid may be citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, etc. In addition, the pharmaceutical composition containing the nucleoside represented by Chemical Formulas 1 to 6 of the present application may include not only pharma- ceutically acceptable salts but also all salts, hydrates, and solvates that can be prepared by conventional methods.

[0030] The addition salt according to the present application can be prepared by a conventional method, for example, by dissolving a compound selected from the group consisting of the compounds represented by Chemical Formulas 1 to 6 in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and then adding an excess amount of an organic acid or an aqueous solution of an inorganic acid, followed by precipitation or crystallization. The solvent or excess acid is then evaporated from the mixture, followed by drying to obtain the addition salt, or the precipitated salt can be filtered by suction.

[0031] The composition may also be selected from a pharmaceutical composition or a health food composition.

[0032] In a specific embodiment, the antiviral composition may further include one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions. Specifically, the carrier, excipient and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspensions, liquids for internal use, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used.

[0033] In another embodiment of the present application, the pharmaceutical composition can be formulated and used as granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, suspensions, emulsions, drops or liquids by a conventional method. According to one embodiment of the present application, the pharmaceutical composition can be administered to a subject in a conventional manner, for example, via oral, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes, which are well known in the art, but are not limited thereto.

[0034] The specific dosage of the compounds represented by the chemical formulas 1 to 6 or pharma- ceutically acceptable salts thereof may vary depending on the condition and weight of the subject, the type and severity of the disease, the drug form, the administration route and period, and may be appropriately selected by those skilled in the art.

[0035] According to one embodiment of the present application, when an appropriate amount for each livestock species is orally administered to animals (intragastric administration) as described below, but is not limited thereto, it is absorbed through intestinal epithelial cells, flows into the bloodstream, and diffuses to each organ tissue, and in particular, is delivered / absorbed by cells present in tissues where viruses grow, thereby suppressing viral growth in infected cells due to viral challenge inoculation, and as a result, it can be used as a therapeutic agent for animals infected with viruses.

[0036] -Dogs under 6 months: 100 mg / kg, dogs over 6 months: 200 mg / kg, fed twice daily, fed daily for 4 weeks or more - Cats under 6 months: 100 mg / kg, Cats over 6 months: 200 mg / kg, fed twice daily, fed daily for 4 weeks or more -Chickens under 1 week old: 20mg / kg, chickens under 2 weeks old: 50mg / kg, chickens over 3 weeks old: 100mg / kg, fed twice a day or mixed according to daily feed intake, daily for 4 weeks or more Pigs under 4 weeks of age: 100 mg / kg, Pigs under 8 weeks of age: 200 mg / kg, Pigs over 8 weeks of age: 300 mg / kg, Pigs over 50 kg of age: 400 mg / kg, fed twice a day or mixed according to daily feed intake, daily for 4 weeks or more Calves under 2 months: 200 mg / kg, cattle under 6 months: 300 mg / kg, cattle over 6 months: 400 mg / kg, fed twice a day or mixed according to daily feed intake, daily for 4 weeks or more

[0037] However, this is merely an example and does not limit the scope of the present application.

[0038] In the present application, the subject may be, but is not limited to, a mammal, a bird, a fish, or an arthropod. The mammal subject may be a pig, a cow, a dog, a cat, or a chicken. The fish subject may be a flounder, a salmon, a sea bream, an eel, a rockfish, or a trout. The arthropod subject may be a shrimp or a lobster.

[0039] In still another embodiment of the present application, the health food may contain, relative to 100 parts by weight of the total health food, 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 parts by weight, 0.01 to 5 parts by weight, 0.01 to 1 parts 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 parts 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, but is not limited thereto. In another embodiment of the present application, the health food may further include one or more additives selected from the group consisting of organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar, and sorbitol. The organic acids may be, but are not limited to, citric acid, malic acid, adipic acid, lactic acid, or malic acid, the phosphates may be, but are not limited to, sodium phosphate, potassium phosphate, acid pyrophosphate, or polyphosphate (polymerized phosphate), and the antioxidants may be, but are not limited to, natural antioxidants such as polyphenols, catechin, α-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid, or phytic acid. In another embodiment of the present application, the health food may contain, in addition to the active ingredients, various nutrients, probiotics, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. According to one embodiment of the present application, the dosage form of the health food may be, but is not limited to, a solid, powder, granule, tablet, capsule, liquid or beverage form.In addition, the health food may be used to manufacture foods such as, but not limited to, confectionery, sugar, ice cream products, dairy products, meat products, fish products, tofu or soybeans, edible oils and fats, noodles, tea, beverages, special nutritional foods, dietary supplements, seasonings, ice, ginseng products, kimchi pickles, dried seafood, fruits, vegetables, dried fruit or vegetable products, cut products, fruit juice, vegetable juice, mixed juices thereof, chips, noodles, processed livestock foods, processed seafood foods, processed dairy foods, fermented milk foods, bean foods, grain foods, microbial fermented foods, confectionery and bread, seasonings, processed meat products, acidic drinking water, licorice, and herbs.

[0040] The present application also provides a feed additive comprising, as an active ingredient, a compound selected from the group consisting of the compounds of Chemical Formulas 1 to 6 and pharma- ceutically acceptable salts thereof.

[0041] In one embodiment, the feed additive of the present application may be applied in various forms, similar to the antiviral composition. Examples of formulation forms include, but are not limited to, liquids, suspensions, powders, granules, tablets, capsules, pills, etc. In addition, in order to formulate the feed additive in the above form, one or more additives and excipients that can be generally contained in feed additives other than the active ingredient, such as diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, can be selected and used, and flavors, immune enhancers, etc. for providing additional functions can be mixed and used. Specifically, the diluent can be lactose, corn starch, soybean oil, amorphous cellulose, or mannitol, the lubricant can be magnesium stearate or talc, and the binder can be polyvinylpyrrolidone or hydroxypropyl cellulose. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, polacrilin potassium, or crospovidone; the sweetener may be sucrose, fructose, sorbitol, aspartame, or nucleic acid (IMP, GMP); the stabilizer may be sodium carboxymethylcellulose, β-cyclodextrin, white beeswax, or xanthan gum; and the preservative may be methyl parahydroxybenzoate, propyl parahydroxybenzoate, or potassium sorbate.

[0042] The feed additive may be selected from the group consisting of mammals, fish, birds or arthropods, specifically pigs, cows, chickens, goats, sheep, horses, fish, shrimp, insects, dogs and cats, specifically but not limited to pigs, cows, chickens, dogs and cats. The feed additive may be any of a variety of viruses, including influenza viruses such as Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Porcine Epidemic Diarrhea virus (PEDV), Swine Influenza virus (SIV) or Avian Influenza virus (AIV), Porcine Rotavirus (PRV), Porcine Circovirus (PCV), Infectious Bronchitis virus (IBV), Chicken Newcastle Disease virus (NDV), Bovine Viral Diarrhea virus (BVDV), Bovine Rotavirus (BRV) and Canine Parvovirus. The activity of a virus selected from the group consisting of rabies, bronchitis, bronchitis, and chronic mycobacterial virus (CPV) can be suppressed.

[0043] The feed additive may be fed to subjects such as mammals, fish, birds, or arthropods, specifically pigs, cows, chickens, goats, sheep, horses, fish, shrimp, insects, dogs, or cats, in the same manner and dosage as the antiviral composition of the present application, and may be fed orally in a manner generally known in the art, for example, by mixing with feed, but is not limited thereto.

[0044] In one embodiment, the feed additive of the present application may be added in various ratios such as, for example, 0.01 to 300 g, 1 g to 200 g, or 10 g to 100 g per 1 kg of feed based on the dry weight (i.e., 0.001 wt % to 30 wt %, 0.1 wt % to 20 wt %, or 1 wt % to 10 wt % based on the total dry weight of the feed), depending on the dosage and administration described above, but is not limited thereto.

[0045] The antiviral material selected from the compounds of Formulae 1 to 6 and pharma- ceutical acceptable salts thereof contained in the antiviral composition, pharmaceutical composition or feed additive according to the present application effectively inhibits viral infection and proliferation by inhibiting the function of inosine monophosphate dehydrogenase (IMP Dehydrogenase), which produces the necessary GMP when a virus invades a cell and replicates its genes, thereby causing a shortage of sources for viral gene replication and inhibiting smooth viral gene replication, or by causing an abnormality in the functional protein of the virus that is generated as the final product by being inserted into the replication process as a guanosine analog in the viral gene replication process, thereby reducing infectivity.

[0046] Well-known efficacy mechanisms known for some nucleoside analogues, such as immunomodulatory effects, increased expression of interferon stimulating factors, and inhibition of viral RNA polymerase, may also be selected from the compounds of Chemical Formulae 1 to 6 and their pharma- ceutical acceptable salts contained in the antiviral composition, pharmaceutical composition, or feed additive of the present application.

[0047] In one embodiment, in order to confirm the effect of the nucleosides of formulas 1 to 6 according to the present application in suppressing infection and proliferation of various viruses in each livestock species, the antiviral efficacy was evaluated through an in vitro cell evaluation method, and it was confirmed that the nucleosides exhibited excellent antiviral effects against all the viruses in each livestock species specified above. In addition, the nucleosides were challenged with influenza virus (2009 Pandemic strain, CA04) in mice, and then orally fed for 5 days to confirm the in vivo antiviral effect, and it was confirmed that the nucleosides exhibited excellent antiviral effects. In addition, in order to confirm the antiviral efficacy in target animals, the nucleosides were challenged with PRRSV (NA strain) in piglets, and then orally fed for 14 days to evaluate the viral disease alleviation effect and the virus reduction effect in blood and lung tissue, and it was confirmed that the nucleosides exhibited excellent antiviral effects.

[0048] The present application will be described in detail below with reference to examples for better understanding. However, the following examples are merely illustrative of the contents of the present application, and the scope of the present application is not limited to the following examples. The examples of the present application are provided to those having average knowledge in the art to more completely explain the present application. EXAMPLES

[0049] Example 1: Manufacturing method of nucleoside analogues using IMP, XMP, and GMP

[0050] 1-1. Method for producing dialdehyde nucleosides For oxidative cleavage, IMP, XMP, or GMP (manufactured and supplied by CJ CheilJedang) dissolved in water is prepared using periodate (NaIO4) as a catalyst and an organic solvent, then filtered using filter paper, and the permeate is purified using anion exchange resin (WA30) and freeze-dried to obtain the final product (Maria Meurillon et al., 2014 Eur. J. Med. Chem. 77:18-37). Figure 1a shows the chemical structures of the dialdehyde forms of inosine, xanthosine, and guanosine, which are nucleoside analogs.

[0051] 1-2. Method for producing acyclic diol nucleosides For oxidative cleavage, IMP, XMP, or GMP (manufactured and supplied by CJ CheilJedang) dissolved in water is prepared using periodate (NaIO4) as a catalyst and an organic solvent, then filtered through filter paper, and the permeate is 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). Figure 1b shows the chemical structures of the acyclic diol forms of inosine, xanthosine, and guanosine, which are nucleoside analogs.

[0052] Example 2: Verification of efficacy and safety of PRRS virus infection suppression based on in vitro cell evaluation To evaluate the efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine in inhibiting viral infection and proliferation against PRRS virus, monkey kidney cells (Marc-145 cell line) were cultured at 2 × 10 4 After seeding at 100 cells / well, the cells were cultured for one day. The next day, 100 TCID50 The cells were simultaneously treated with 1000ml PRRSV (NA type, VR-2332 strain) and serial dilutions of inosine, xanthosine, and guanosine in the aldehyde and acyclic diol forms, and after 3 days, the cells were fixed with a 1:1 mixture of MeOH / acetone, treated with a PRRSV N (Nucleocapid) protein binding antibody SDOW-17 as a primary antibody and reacted with a secondary antibody that emits fluorescence in response to the primary antibody, and the number of fluorescent cells was measured to analyze whether the virus infection was suppressed. In addition, the number of cells was counted using DAPI (4',6-diamidino-2-phenylindole), and the number of infected cells was calculated from the total number of cells, and the efficacy of the materials was compared based on the infection level quantified.

[0053] As shown in Figures 2, 3, 4, 5, 6 and 7, the efficacy of each material in inhibiting PRRSV infection and proliferation at each concentration was evaluated. In the case of dialdehyde-type guanosine, 50% infection inhibition was observed at a low concentration (40μM), and more than 90% infection inhibition was observed at 100μM, showing the best antiviral efficacy. As can be seen through DAPI staining at the concentration showing efficacy, no cytotoxicity was observed up to 1000μM, confirming that antiviral efficacy can be demonstrated within a very safe concentration range. In addition, in the cytotoxicity evaluation using the WST-1 experimental method, no cytotoxicity was observed at 6000 or 10000μM or more, confirming that the material is very safe [Table 1].

[0054] In conclusion, it was confirmed that the selectivity index, as shown in Table 1, was highest for dialdehyde guanosine. The compound with the second highest efficacy after dialdehyde guanosine was dialdehyde xanthosine, and although the inosine-based analogue showed efficacy at the highest concentration, it was determined that the concentration was not high enough to be inappropriate for animal application. In addition, the acyclic diol forms of inosine, xanthosine, and guanosine were treated at concentrations of 500, 1000, 2000, 4000, and 8000 micromolar (μM) to evaluate their efficacy levels and cytotoxicity, and they showed higher IC2 concentrations than the dialdehyde forms of inosine, xanthosine, and guanosine. 50 It was confirmed that the compound had antiviral efficacy against PRRSV at concentrations of 400-500 μM.

[0055] [Table 1]

[0056] Example 3: Verification of efficacy and safety of PED virus infection suppression based on in vitro cell evaluation To evaluate the efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine in inhibiting viral infection and proliferation against PED virus, monkey kidney cells (Vero cell line) were cultured at 4 × 10 4 After seeding, cells / well were cultured for one day. The next day, 200 TCID 50The cells were simultaneously treated with 100 / ml PEDV (G1a type, SM98 strain) and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and after 2 days, RNA was isolated using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754). The isolated RNA was used to verify the antiviral efficacy of the material using a commercially available PEDV detection kit (MEDIAN Diagnostics, NS-PED-31).

[0057] As can be seen from Table 2, dialdehyde guanosine showed the best efficacy, followed by dialdehyde xanthosine and inosine. In addition, acyclic diol forms inosine, xanthosine, and guanosine also showed antiviral efficacy against PEDV, but at higher IC concentrations than the dialdehyde form. 50 The values ​​were shown.

[0058] [Table 2]

[0059] Example 4: Verification of the efficacy of in vitro cell evaluation to suppress infection with porcine circovirus type 2 (PCV2) To evaluate the viral infection and inhibitory efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine against PCV2, porcine kidney cells (PK15 cell line) were cultured at 4 × 10 4 After seeding at 100 cells / well, 200 TCID 50The cells were simultaneously treated with 1 / well of PCV2 and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and after 4 days, DNA was isolated using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754).

[0060] The isolated DNA was used to verify the antiviral efficacy of the material using PCV2-specific primers (F-AGGAGGGCGTTCTGACTGTG, R-ACCGCTACCGTTGGAGAAGG) and real-time RT-qPCR (1 cycle of 95°C, 30 seconds, 40 cycles of 95°C, 20 seconds, 56°C, 60 seconds).

[0061] As can be seen from Table 3, dialdehyde guanosine showed overwhelmingly good efficacy, followed by dialdehyde xanthosine and inosine. In addition, acyclic diol inosine and xanthosine also showed antiviral efficacy against PCV2, and acyclic diol guanosine showed an IC at relatively high concentrations. 50 The values ​​were shown.

[0062] [Table 3]

[0063] Example 5: Verification of efficacy and safety of suppressing porcine rotavirus (PRV) infection based on in vitro cell evaluation To evaluate the efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine in inhibiting viral infection and proliferation against PRV, monkey kidney cells (Vero cell line) were cultured at 4 × 10 4After seeding, cells / well were cultured for one day. The next day, 200 TCID 50 The cells were simultaneously treated with 1 / well of PEDV (G1a type, SM98 strain) and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and after 3 days, RNA was isolated using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754). The isolated RNA was used to verify the antiviral efficacy of the material using a commercially available PRV detection kit (Ingenetix, DHUV02053).

[0064] As can be seen from Table 4, the dialdehyde forms of inosine and xanthosine showed similar levels (IC at concentrations of 100 μM or less). 50 The IC value was 177.4 μM for guanosine. 50 In addition, the acyclic diol form had a higher IC value than the dialdehyde form. 50 The values ​​shown were: inosine, guanosine, xanthosine in that order of effectiveness.

[0065] [Table 4]

[0066] Example 6: Verification of infection inhibition and safety against influenza virus (IV) based on in vitro cell evaluation To evaluate the viral infection and inhibitory efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine against SIV (Swine Influenza Virus), canine kidney cells (MDCK cell line) were cultured at 5 × 10 4After seeding, cells / well were cultured for one day. The next day, 200 TCID 50 The cells were simultaneously treated with SIV at 1 / well and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and RNA was isolated after 4 days using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754).

[0067] The isolated RNA was used to verify the antiviral efficacy of the material using SIV-specific primers (F-GATGGTAGATGGATGGTACGGTTAT, R-TTGTTAGTAATCTCGTCAATGGCATT) and real-time RT-qPCR (1 cycle of 95°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec).

[0068] As can be seen from Table 5, when cells were treated with dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, it was confirmed that SIV infection and proliferation were inhibited. Dialdehyde form of guanosine had the best efficacy (IC 50 The results showed that the antiviral activity of the acyclic diol was higher than that of the inosine, but was higher at very high concentrations.

[0069] [Table 5]

[0070] Example 7: Verification of infection inhibition and safety against infectious bronchitis virus (IBV) based on in vitro cell evaluation To evaluate the viral infection and inhibitory efficacy of the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine against IBV, monkey kidney cells (Vero cell line) were cultured at 4 × 10 4 After seeding at 100 cells / well, the cells were cultured for one day. The next day, 200 TCID 50 The cells were simultaneously treated with IBV at 1 / well and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and RNA was isolated after 4 days using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754).

[0071] The isolated RNA was used to evaluate the antiviral efficacy of each material using IBV-specific primers (F-ATGCTCAACCTTGTCCCTAGCA, R-TCAAACTGCGGATCATCACGT) and real-time RT-qPCR (1 cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 45 s, and 72°C for 30 s).

[0072] As can be seen from Table 6, the inhibitory effects of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine on IBV infection and proliferation were confirmed. The dialdehyde form of inosine showed the best efficacy (IC 50 : 76.4 μM), and the order of antiviral effects was xanthosine, followed by guanosine. In addition, in the acyclic diol form, xanthosine and guanosine had similar levels of IC 50 In comparison, inosine showed a relatively high IC 50 The efficacy was shown by value.

[0073] [Table 6]

[0074] Example 8: Verification of efficacy and safety of suppressing infection with Chicken Newcastle Disease Virus (NDV) based on in vitro cell evaluation To evaluate the efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine in inhibiting viral infection and proliferation against NDV, monkey kidney cells (Vero cell line) were cultured at 4 × 10 4 After seeding, cells / well were cultured for one day. The next day, 200 TCID 50 The cells were simultaneously treated with 100 / well of NDV and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and after 3 days, RNA was isolated using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754). The isolated RNA was used to verify the antiviral efficacy of the material using a commercially available NDV detection kit (iNtRON, IP15456).

[0075] As can be seen from Table 7, dialdehyde-type inosine had the best efficacy (IC 50 The IC value of xanthosine and guanosine was 469.4μM, and the inhibitory effect on viral infection and proliferation was confirmed in that order. Inosine and guanosine in the acyclic diol form had a higher IC value than the dialdehyde form. 50 The antiviral efficacy was shown by the concentration, and xanthosine showed 39.4% (±0.97) inhibitory efficacy against NDV infection at a concentration of 8000 μM.

[0076] [Table 7]

[0077] Example 9: Verification of infection inhibition and safety against bovine viral diarrhea virus (BVDV) based on in vitro cell evaluation To evaluate the viral infection and inhibitory efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine against BVDV, bovine kidney (MDBK) cell line was cultured at 4 × 10 4 After seeding, cells / well were cultured for one day. The next day, 200 TCID 50 The cells were simultaneously treated with 1 / well of BVDV and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and RNA was isolated after 4 days using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754).

[0078] The isolated RNA was subjected to real-time RT-qPCR (1 cycle of 94°C for 10 minutes, 35 cycles of 94°C for 10 seconds, 53°C for 20 seconds, and 72°C for 30 seconds) to confirm the antiviral efficacy of each material.

[0079] As can be seen from Table 8, when cells were treated with dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, it was confirmed that the infection and proliferation of BVDV was inhibited. The dialdehyde form of xanthosine had the best efficacy (IC 50 The IC value was 97.8 μM, with inosine being the most effective, followed by guanosine. The acyclic diol form had a higher IC value than the dialdehyde form.50 The efficacy was confirmed by the value (log 10 (Efficacy confirmed as reducing virus titer by 1 or more).

[0080] [Table 8]

[0081] Example 10: Verification of infection inhibition and safety against bovine rotavirus (BRV) based on in vitro cell evaluation To evaluate the viral infection and inhibitory efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine against BRV, bovine kidney (MDBK) cell line was cultured at 4 × 10 4 After seeding, cells / well were cultured for one day. The next day, 200 TCID 50 The cells were simultaneously treated with 1 / well of BRV and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and RNA was isolated after 4 days using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754).

[0082] The isolated RNA was subjected to real-time RT-qPCR (95°C, 1 cycle for 10 minutes, 95°C, 15 seconds, 60°C, 15 seconds, 72°C, 20 seconds) using BRV-specific primers (F-TCATTTCAGTTGATGAGACCACC, R-ATTCAATTCTAAGCGTGAGTCCTAC) to confirm the antiviral efficacy of each material.

[0083] As can be seen from Table 9, the dialdehyde form of xanthosine has excellent infection and proliferation inhibitory activity (IC 50The IC value was 50.5μM, with inosine being the most effective, followed by guanosine. The acyclic diol forms showed antiviral efficacy against BRV in the order of xanthosine, guanosine, and inosine, but had a slightly higher IC value than the dialdehyde form. 50 The values ​​were shown.

[0084] [Table 9]

[0085] Example 11: Verification of the efficacy of suppressing infection with canine parvovirus (CPV) based on in vitro cell evaluation To evaluate the viral infection and inhibitory efficacy of dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine against CPV, MDCK cell line cells were cultured at 5 × 10 4 After seeding at 100 cells / well, 200 TCID 50 The cells were simultaneously treated with 1 / well of CPV and the dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, and after 4 days, DNA was isolated using a commercially available Viral gene (DNA / RNA) extraction kit (iNtRON, 101410754).

[0086] The isolated DNA was used to verify the antiviral efficacy of the material using CPV-specific primers (F-AGCTAGTTTCATCTTAACTATCAAATT, R-TTAACTCTCCAGAAAACTCATGC) and realtime T-qPCR (1 cycle of 95°C for 5 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds).

[0087] As can be seen from Table 10, when cells were treated with dialdehyde and acyclic diol forms of inosine, xanthosine, and guanosine, it was confirmed that the infection and proliferation of CPV was inhibited. The dialdehyde form of xanthosine had the best efficacy (IC 50 In the acyclic diol form, inosine and xanthosine had almost the same IC 50 The antiviral efficacy of guanosine at 8000 μM was 18.0% (±0.68) in inhibiting CPV infection (log 10 (Efficacy confirmed to reduce virus titer by 1 or more).

[0088] [Table 10]

[0089] Example 12: Verification of efficacy and safety of influenza virus (IV) infection suppression based on in vivo mouse challenge evaluation For the in vivo efficacy analysis of dialdehyde-type xanthosine and guanosine analogs using mice, a simplified pharmacokinetics (PK) analysis was performed to determine the inoculation method, frequency, and concentration before the challenge inoculation evaluation using influenza virus. When comparing two inoculation methods, one involved forced oral inoculation of 200 ul at a concentration of 250 mg / kg via the intragastric route, and then 10 blood samples were taken to analyze the changes in blood concentration. The other inoculation method involved inoculation of 100 ul at a concentration of 50 mg / kg via the intraperitoneal route using a syringe, and then blood samples were taken in the same manner and analyzed. In [Figure 8] and [Figure 9], the remaining concentration values ​​at 10 time points are shown, and the trend line of the values ​​is drawn and analyzed. Based on the AUC (Area under the curve) value, the time when the material concentration is halved is estimated. As a result, for dialdehyde-type guanosine, 50% of the material remaining amount is measured at about 9 hours when administered via the intragastric route and about 6 hours when administered via the intraperitoneal route. For dialdehyde-type xanthosine, it is about 6 hours for both the intragastric route and the intraperitoneal route. The half-life, which is the time when the concentration drops to less than half of the maximum concentration in plasma, is 1.3 to 3.1 hours, and the dialdehyde xanthosine, which has a slightly higher water solubility, shows a tendency for the half-life to be shorter. When comparing the maximum concentrations, the intraperitoneal injection administration was higher than the oral administration, and based on this, it can be seen that a mechanism that can increase the absorption rate when administered orally may be necessary.

[0090] Based on the above results, a material inoculation plan was established for evaluating the challenge inoculation of mice with influenza virus (2009 pandemic strain, CA04 (virus isolated from a patient infected with swine-origin influenza virus)), and the design and schedule of the test treatment groups are shown in [Figure 10] and [Table 11].

[0091] [Table 11]

[0092] Exam Content -Lung tissue extraction by time point (virus titration) - Observation of lesions in lung tissue at autopsy - Daily clinical observation - Observation of damage to each organ and tissue during autopsy - Check your weight every day -3 and 5 dpi, one animal per treatment group was necropsied (7 dpi, all animals were necropsied at the end of the study)

[0093] As shown in Figure 10, after a one-week acclimation period, 100 MLD 50 SIV (2009 pandemic strain CA04) (50% mouse lethal dose) was administered intranasally before and after the challenge inoculation 3 hours later, and the material was administered intragastricly as shown in Table 11. The material was administered once a day from the day after the virus challenge inoculation until the 5th day. Monitoring was continued until the 7th day when the final necropsy was performed, and clinical symptoms were observed and body weight (BW) was measured every day during the progression period. On the 3rd and 5th days after infection, necropsies were performed on one animal from each treatment group to observe lung lesions caused by the virus and abnormal lesions in the liver, gastrointestinal tract, spleen, kidney, heart, small intestine, and large intestine to verify the safety of the material. Total RNA was later extracted from the excised lungs to trace the amount of virus remaining in the lung tissue and measure the efficacy of the antiviral material.

[0094] As a result, the in vivo anti-influenza virus efficacy of orally administered dialdehyde-type xanthosine and guanosine was shown to be similar to that confirmed in vitro, and no abnormal lesions were observed due to the materials fed, proving their safety. However, although the efficacy was superior to that of the control group (a group fed only PBS (phosphate buffered saline)), there was a difference in efficacy between the two groups, dialdehyde-type xanthosine and guanosine, and due to the high solubility and good absorption rate of dialdehyde-type xanthosine, it was more effective than dialdehyde-type guanosine, contrary to the cell-based in vitro anti-viral efficacy evaluation. In detail, as shown in Figure 11, the change in body weight during the 7-day SIV challenge inoculation evaluation period was observed. The mice administered only PBS showed weight loss due to the virus from the 3rd day, while the mice administered dialdehyde-form xanthosine and guanosine analogs intragastricly (oral inoculation method) showed only very mild symptoms and very mild weight loss during the period when the material was administered until the 5th day and remained in the body until the 6th day, but weight loss was observed on the 7th day when the material was not fed for 2 days. However, the level of weight loss was mild, and it was confirmed that the antiviral efficacy of oral feeding of the material was very excellent until the end of the test compared to the control group. By comprehensively analyzing the rate of change in weight loss, it was possible to obtain information that feeding of the material must be administered until the virus is completely eradicated, and it was possible to prove that feeding of the material is generally effective in minimizing viral symptoms.

[0095] The survival rate can be explained based on the above results, and as shown in Figure 12, when the survival rate on the 8th day was predicted based on all the data up to the 7th day, the control group, which had a very severe weight loss, showed 100% death (80% survival rate on the 7th day), and the groups orally fed dialdehyde-type xanthosine and guanosine showed the same weight loss pattern, with the dialdehyde-type xanthosine group showing 100% survival (100% survival rate on the 7th day) and the dialdehyde-type guanosine group showing 60% survival (80% survival rate on the 7th day). Dialdehyde-type xanthosine, which has high water solubility and high absorption rate (see PK results), showed high plasma concentration, and it can be assumed that the above results were obtained from the weight change and survival rate.

[0096] The same pattern of results was observed for lung lesions observed through autopsy, but as shown in [Figure 13], the lesions on the 3rd day, when the virus proliferation peaks, were slight, but the lung lesions on the 5th day of the control group were very severe due to the aftereffects of the virus proliferation. On the other hand, the dialdehyde forms of xanthosine and guanosine showed milder lung lesions compared to the control group, and showed the same results as the clinical symptoms observed from the appearance. In the same context, the material was not fed after the 5th day, and the evaluation period was short, only 7 days, so the results of complete eradication of the virus could not be seen, and the difference in the symptom level between the control group and the 7th day in the observation of lung lesions was seen to be very narrow. Nevertheless, a comparative analysis of the observation results on the 5th day can clearly confirm the antiviral efficacy of the material fed. In addition, as shown in Figure 14, the amount of virus remaining in the lung tissue was measured lower when the material was fed, and the lowest virus titer was observed with dialdehyde-type xanthosine, which was more effective than dialdehyde-type guanosine. As a result, it can be said that the material inhibited virus proliferation, leading to the clinical symptoms and autopsy results described above. In addition, Figure 15 shows photos of lung tissues removed at autopsy showing representative lung lesions in each group. In the control group, blood components were filled at a very severe level and tissue necrosis was observed, while in the dialdehyde-type xanthosine and guanosine, only mild lesions were observed compared to the control group, which can be said to be macroscopic observation results that support the above results.In order to confirm the actual effectiveness against specific viruses by livestock species, the dosage and frequency of the material must be derived for each livestock species targeted by the virus, and information on the duration of administration must be obtained. However, since the dialdehyde forms of xanthosine and guanosine effectively demonstrate antiviral efficacy (alleviating disease symptoms and inhibiting viral growth) in vivo, the in vivo efficacy was confirmed first. This provides a basis for claiming the antiviral efficacy for each virus in the in vitro examples shown above.

[0097] For reference, the influenza virus used in all the clinical evaluations using small animals mentioned above is a virus that can infect and cause symptoms in both chickens and pigs. When chickens and pigs are infected with the influenza virus, the dialdehyde forms of xanthosine and guanosine alleviate symptoms and increase survival rates, proving that they can be used as antiviral therapeutic agents in chickens and pigs.

[0098] Example 13: Verification of efficacy and safety of Porcine reproductive and respiratory syndrome (PRRS) virus infection suppression based on in vivo piglet challenge evaluation To analyze the in vivo antiviral efficacy of dialdehyde-form XMP using young piglets, a challenge inoculation evaluation using PRRS virus was performed. An isolator was used to prevent airborne transmission of PRRSV, and 3-week-old piglets were used to ensure the discrimination of viral clinical symptoms. Treatment groups were allocated as described in [Table 12], and the evaluation was performed as described in [Figure 16]. After a 3-day acclimation period after introducing 18-day-old piglets, they were challenged with PRRSV (NA strain, more pathogenic than EU strain) via the intranasal route, and the material or PBS (phosphate buffered saline) was orally fed using a catheter every morning and afternoon. After the challenge, the body weight was measured at intervals of 3 to 4 days, and clinical symptoms were monitored. Two weeks after the start of the challenge, the lesions in the lung tissue were confirmed through necropsy to verify the antiviral efficacy of dialdehyde-form xanthosine.

[0099] [Table 12]

[0100] As shown in [Figure 17], the positive control group inoculated with the virus and fed PBS showed very slow weight gain due to damage caused by the virus infection, but the piglets orally fed the material showed no weight gain delay due to the virus infection and showed the same weight gain pattern as the positive control group that was not treated. This is a very important result, and it shows that when xanthosine in the form of dialdehyde is fed with feed, economic losses due to PRRSV infection can actually be prevented in livestock farms. In addition, the virus titer in the lung tissue (the tropism of PRRSV is macrophages present in the lungs) was measured at the time of autopsy, and as shown in [Figure 18], the virus amount was confirmed to be reduced in the material-treated group compared to the positive control group. This proves that the efficacy of alleviating clinical symptoms actually reduces the amount of virus remaining in the host's body and inhibits viral proliferation, thereby demonstrating antiviral efficacy. [Figure 19] is a photograph of the lungs removed at the time of autopsy, and the interstitial pneumonia lesions were observed with the naked eye. Unlike the positive control group, which showed lesions throughout the lungs, the negative control group and the material-treated group showed relatively intact lungs. Finally, the removed lungs were sliced ​​and immunohistochemistry was performed using an antibody that specifically detects the PRRS virus. As shown in [Figure 20], unlike the positive control group, where a large amount of virus was present, only a very small amount of virus was detected in the material-treated group. Taking all the experimental results together, it can be concluded that it is possible to treat PRRS virus infection, which causes chronic economic damage to pig farms, by orally feeding the dialdehyde form of xanthosine.

Claims

1. An antiviral composition comprising one or more nucleoside analogs selected from the group consisting of nucleoside analogs of the following formulas 1 to 6 and pharma- ceutically acceptable salts thereof: [Formula 1] [ka]

2. The viruses include Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Porcine Epidemic Diarrhea virus (PEDV), Swine Influenza virus (SIV, Avian Influenza virus (AIV)), Porcine Rotavirus (PRV), Porcine Circovirus (PCV), Infectious Bronchitis virus (In ...

2. The composition according to claim 1, wherein the virus is one or more selected from the group consisting of: avian viral diarrhea virus (IBV), avian Newcastle disease virus (NDV), bovine viral diarrhea virus (BVD), bovine rotavirus (BRV) and canine parvovirus (CPV).

3. An immunomodulatory composition comprising one or more nucleoside analogs selected from the group consisting of nucleoside analogs of the following formulas 1 to 6 and pharma- ceutically acceptable salts thereof: [chemical 2] [ka]

4. 3. The composition according to claim 1 or 2, characterized in that it is a pharmaceutical composition or a food composition.

5. A feed additive comprising one or more nucleoside analogs selected from the group consisting of nucleoside analogs of the following chemical formulas 1 to 6 and pharma- ceutically acceptable salts thereof. [C3] [ka]

6. 6. The feed additive according to claim 5, which is for mammals, birds, fish or arthropods.

7. A feed comprising one or more nucleoside analogs selected from the group consisting of nucleoside analogs of the following chemical formulas 1 to 6 and pharma- ceutically acceptable salts thereof. [C4] [ka]

8. 8. The feed of claim 7, which is for mammals, birds, fish or arthropods.

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