Anti-influenza virus composition containing xanthan gum or fucoidan

Abstract

The present invention relates to an antiviral composition against influenza virus and a pharmaceutical composition for preventing or treating influenza virus infection, each comprising xanthan gum or fucoidan as an active ingredient, and a method for preventing or treating influenza virus infection using same.

Description

Composition for anti-influenza virus containing xanthan gum or fucoidan

[0001] The present invention relates to an antiviral composition for influenza virus, comprising xanthan gum or fucoidan as an active ingredient, a pharmaceutical composition for preventing or treating influenza virus infection, and a method for preventing or treating influenza virus infection using the same.

[0002] The influenza virus, a member of the orthologous group, is a representative pathogenic virus that kills thousands to tens of thousands of people each season. Since the 20th century, influenza has mutated across species, leading to pandemics and a high mutation rate that renders existing drugs ineffective. Commonly referred to as pandemic influenza, the disease typically causes chills, fever, sore throat, muscle pain, cough, weakness, and malaise. These nonspecific symptoms are more severe than the common cold but are not life-threatening. However, when accompanied by pneumonia or Reye's syndrome, they can cause fatal complications. Respiratory infections are usually transmitted through the coughing, sputum, and sneezing of an infected person. However, mutations often occur when the virus is transmitted from other animals (especially birds) to humans. There are three types of influenza viruses: influenza A, influenza B, and influenza C. Among these, the influenza that caused a pandemic through animal species transmission, aka swine flu, which was prevalent in 2009, was of the influenza A H1N1 type, which first emerged in Mexico and spread to many countries as several mutations emerged, and the World Health Organization (WHO) defined it as a "global pandemic." In the case of the flu, it is common to prevent and treat it defensively by injecting purified inactivated strains before the common cold season, but in patients who have already developed symptoms during the flu season, prevention is not possible, so the symptoms must be alleviated through treatment. In the case of Tamiflu, which stopped the spread of the swine flu that was spreading helplessly, it is excellent in terms of effectiveness, but cases of side effects such as hallucinations after ingestion are often reported in the media.

[0003] Conventional medications used to treat influenza include oseltamivir (brand name: Tamiflu), zanamivir (brand name: Relenza), peramivir, and amantadine. Oseltamivir, also known as Tamiflu, is currently primarily used to treat H1N1 influenza A. Tamiflu is the world's only exclusive treatment for avian influenza (AI). It is an antiviral drug that works by blocking the enzyme that replicates the virus. It is most effective when taken within 48 hours of the onset of symptoms. Its main therapeutic effects include reducing the worsening of influenza symptoms, reducing the incidence of secondary complications such as bronchitis and pneumonia, and shortening the incubation period. It is also used to treat influenza A and B. Zanamivir, also known as Relenza, is a neuraminidase inhibitor used to treat influenza A and B. However, oseltamivir has the side effect of causing severe vomiting, and although zanamivir has a high antiviral effect, it has the disadvantage of low bioavailability and rapid excretion from the kidneys.

[0004] Against this backdrop, there is a continuous demand for the development of antiviral agents with excellent infection suppression and treatment effects.

[0005] The purpose of the present invention is to provide an antiviral composition against influenza virus, comprising xanthan gum or fucoidan as an active ingredient.

[0006] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a viral infection disease caused by an influenza virus, comprising xanthan gum or fucoidan as an active ingredient.

[0007] Another object of the present invention is to provide a method for preventing or treating influenza virus infection, comprising a step of administering the pharmaceutical composition to a subject.

[0008] Another object of the present invention is to provide a composition comprising xanthan gum or fucoidan as an active ingredient for the prevention or treatment of influenza virus infection.

[0009] Another object of the present invention is to provide a use for the manufacture of a medicament for treating influenza virus infection, comprising a composition comprising xanthan gum or fucoidan as an active ingredient.

[0010] One aspect of the present invention for achieving the above-described purpose relates to an antiviral composition against influenza virus, comprising xanthan gum or fucoidan as an active ingredient.

[0011] In the present invention, the "xanthan gum" is a naturally derived polysaccharide with low toxicity, and is an exocrine polysaccharide produced by the fermentation of Xanthomonas campestris, a bacterium of the genus Xanthomonas. An aqueous solution of xanthan gum has high viscosity at low concentrations and has the characteristic of maintaining the viscosity almost constant over a wide temperature range (10-70°C), pH (pH 6-9), and applied concentration. Due to these rheological properties, xanthan gum is used as a stabilizer, thickener, and thickening agent for suspensions in industries such as food and pharmaceuticals.

[0012] In the present invention, the above "fucoidan" is a type of water-soluble dietary fiber and a polysaccharide contained in brown algae such as wakame and kelp. Depending on the harvesting season and species of brown algae, the average molecular weight of fucoidan varies from 100,000 to 2,000,000 Da, and the fucose content and sulfate group content, which have a significant effect on the physiological activity of fucoidan, also vary.

[0013] In the present invention, the "influenza virus" has two surface antigens, namely hemagglutinin (HA) and neuraminidase (NA), which are glycoproteins on the surface, and eight segmented RNAs exist inside. HA binds to sialic acid residues on the surface of host cells, allowing the virus to attach to the host cells and penetrate them. NA cleaves the α-ketosidic bond between the disaccharide portion of the cell surface and the neuraminic acid residues of the virus that has completed replication in the infected host cell, thereby inducing the virus to replicate in another host cell. The body's virus defense mechanism recognizes HA to prevent binding to the host cell or binds to NA to inhibit the virus in the infected host cell from replicating in nearby cells. However, through various combinations of 16 types of HA and 9 types of NA, the virus can develop numerous mutations and become resistant to defense mechanisms.

[0014] Specifically, the influenza virus may be an influenza A virus, and the influenza A virus may be of the H1N1 type, but is not limited thereto.

[0015] In the present invention, the term "antiviral" refers to all mechanisms and activities that inhibit various subtypes and mutant virus particles from entering a host cell or inhibit replication or proliferation of virus particles within a host cell after infection.

[0016] Specifically, the composition may be used for immunization against endemic, prepandemic or pandemic influenza viruses.

[0017] The above immunization refers to inducing immunity to prevent infectious diseases, and includes all methods of strengthening or acquiring immunity in the body.

[0018] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating a viral infection disease caused by an influenza virus, comprising xanthan gum or fucoidan as an active ingredient.

[0019] In the present invention, the "influenza virus infection" refers to an infection of the lungs and / or respiratory tract by one of the influenza viruses. It is accompanied by respiratory disease, and if the inflammatory response worsens, it can lead to tissue damage.

[0020] In one embodiment of the present invention, it was confirmed that xanthan gum and / or fucoidan had an alleviating effect on inflammation and tissue damage caused by influenza virus infection. Specifically, when administered before / after virus infection, weight loss, one of the symptoms of influenza virus infection, was alleviated (Table 3, Table 8, Figs. 1 and 5), inflammatory cell infiltration was reduced (Table 5, Table 9, Figs. 2 and 6), and viral titers in the lungs and nasal cavity were also confirmed to be reduced (lung: Table 10, Table 11, Figs. 3 and 7, nasal cavity: Table 12, Table 13, Figs. 3 and 8).

[0021] The composition of the present invention exhibits an inhibitory effect against influenza viruses when administered for both preventive and therapeutic purposes, while also alleviating inflammation and reducing damage to lung tissue. In particular, in the case of highly toxic influenza viruses, failure of the anti-inflammatory action can result in a cytokine storm, with a large number of inflammatory cells infiltrating the lungs and excessive inflammatory cytokines being secreted. The pharmaceutical composition of the present invention can effectively counteract this.

[0022] In addition, the pharmaceutical composition of the present invention can be applied for the prevention or treatment of diseases that can be caused by influenza virus infection. For example, the diseases that can be caused by the influenza virus infection can be at least one selected from the group consisting of cold, flu, cough, sneezing, runny nose, muscle pain, pharyngitis, sepsis, pneumonia, nasal congestion, laryngitis, sore throat, hoarseness, headache, pain in the swollen sinuses, rhinitis, pharyngitis, bronchitis, asthma, fever, shortness of breath, general weakness and chills, but is not limited thereto.

[0023] In the present invention, “prevention” means any action by which the composition of the present invention delays the occurrence of infection by an influenza virus.

[0024] In the present invention, “treatment” means any action that causes the composition of the present invention to improve or be beneficial in the symptoms of infection by an influenza virus.

[0025] Specifically, the pharmaceutical composition of the present invention may be a parenteral formulation, and more specifically, may be administered intranasally or intranasally. The intranasal or intranasal administration may be administered in the form of a spray, aerosol, or inhalation.

[0026] In addition, the pharmaceutical composition of the present invention can be applied in a pharmaceutically effective amount, and the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the patient's sex, age, type and severity of the disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0027] In addition, the pharmaceutical composition of the present invention can be applied in a pharmaceutically effective amount, and the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the patient's sex, age, type and severity of the disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0028] Another aspect of the present invention relates to a kit for preventing or treating influenza virus comprising the pharmaceutical composition.

[0029] The kit of the present invention is not particularly limited in type, and a kit of a type commonly used in the relevant technical field can be used. The kit of the present invention may be packaged in a form in which xanthan gum or fucoidan is contained in individual containers, or in a form in which it is contained in a single container divided into one or more compartments with other drugs or excipients, and each active ingredient may be packaged in a unit dosage form for a single administration, but is not limited thereto.

[0030] The xanthan gum or fucoidan in the above kit can be administered at an appropriate time depending on the health condition of the subject to be administered, and can be administered concurrently, sequentially, or in reverse order with other additional drugs as needed.

[0031] Another aspect of the present invention relates to a method for preventing or treating influenza virus infection, comprising administering the pharmaceutical composition to a subject.

[0032] The term "subject" in the present invention encompasses an animal or human with an influenza virus infection whose symptoms can be improved by administration of a pharmaceutical composition according to the present invention. By administering a therapeutic composition according to the present invention to a subject, influenza virus infection can be effectively prevented and treated.

[0033] The term "administration" in the present invention refers to introducing a given substance into a human or animal by any suitable method. The therapeutic composition according to the present invention may be administered orally or parenterally via any common route, as long as it can reach the target tissue. Furthermore, the therapeutic composition according to the present invention may be administered by any device capable of transporting the active ingredient to target cells.

[0034] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition and weight, the degree of disease, the drug form, the route and period of administration, but can be appropriately selected by a person skilled in the art.

[0035] Another aspect of the present invention relates to the use of the pharmaceutical composition for the treatment of influenza virus infection.

[0036] Another aspect of the present invention relates to the use of the antiviral composition for the manufacture of a medicament for treating influenza virus infection. The antiviral composition comprises xanthan gum or fucoidan as an active ingredient.

[0037] The antiviral composition for influenza virus containing xanthan gum or fucoidan of the present invention as an active ingredient exhibits preventive and therapeutic effects against influenza virus and reduces the occurrence of tissue lesions and infiltration of inflammatory cells due to influenza virus infection, and thus can be used in the development of an antiviral agent for preventing or treating infections caused by influenza virus.

[0038] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0039] Figure 1 shows the results of observing changes in body weight when administering a test substance before influenza virus infection.

[0040] Figure 2 shows the results of histopathological evaluation scored upon administration of a test substance prior to influenza virus infection.

[0041] Figure 3 shows the results of measuring the virus titer through the number of M2 and polPA genes copied in the lungs when a test substance was administered before influenza virus infection.

[0042] Figure 4 shows the results of measuring the virus titer through the number of M2 and polPA genes in the nasal cavity when administering a test substance before influenza virus infection.

[0043] Figure 5 shows the results of observing changes in body weight when administering a test substance after influenza virus infection.

[0044] Figure 6 shows the results of histopathological evaluation scored upon administration of a test substance after influenza virus infection.

[0045] Figure 7 shows the results of measuring the virus titer through the number of M2 and polPA genes in the lungs when a test substance is administered after influenza virus infection.

[0046] Figure 8 shows the results of measuring the virus titer through the number of M2 and polPA genes in the nasal cavity when a test substance is administered after influenza virus infection.

[0047] Figure 9 shows the results of observing changes in body weight according to the xanthan gum content when administering a test substance before influenza virus infection.

[0048] Figure 10 shows the results of histopathological evaluation according to the xanthan gum content when administering a test substance before influenza virus infection.

[0049] Figure 11 shows the results of measuring the virus titer in the lungs according to the xanthan gum content when administering a test substance before influenza virus infection.

[0050] Figure 12 shows the results of measuring the virus titer in the nasal cavity according to the xanthan gum content when administering a test substance before influenza virus infection.

[0051] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[0052] Manufacturing Example 1. Preparation of test substance

[0053] Test substances were prepared according to the compositions in Table 1 below. Specifically, benzoic acid, citric acid, and sorbitol (D-sorbitol) were added to approximately 0.8 mL of purified water and stirred to dissolve. Then, according to the compositions of each example shown in Table 1 below, each of the main components, HPMC, xanthan gum, bentonite, and fucoidan, was weighed and added, stirred for more than 2 hours to dissolve, and then purified water was added to make a total amount of 1 mL.

[0054] Ingredients (mg / ml) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 HPMC 4.60------Xanthan gum-0.10--0.250.501.00Bentonite--9.00----Fucoidan---8.00---Benzoic acid 0.600.600.600.600.600.600.600.60Citric acid 0.10-0.400.30---Sorbitol 35.0035.0035.0035.0035.0035.0035.00Purified waterAmountAmountAmountAmountAmountAmountAmountAmountTotal amount1 mL1 mL1 mL1 mL1 mL1 mL1 mL1 mL

[0055] Comparative Example 1. Combination of excipients

[0056] A negative control material was prepared by mixing benzoic acid, citric acid, sorbitol, and purified water with a combination of excipients that did not include the main ingredients HPMC, xanthan gum, bentonite, and fucoidan, and the content of each excipient was the same as that in Table 1 above. The negative control material prepared in this way was applied as Comparative Example 1.

[0057] Comparative Example 2. Oseltamivir

[0058] As one of the positive control substances, oseltamivir was purchased from Sigma-Aldrich and applied as comparative example 2.

[0059] Oseltamivir, brand name Tamiflu, is an antiviral drug used to treat and prevent influenza virus infections. It is particularly effective in suppressing the H1N1 variant, a mutated form of the influenza A virus that causes the novel flu.

[0060] Comparative Example 3. Cold Mask Nasal Spray

[0061] As one of the positive control substances, Cold Mask Nasal Spray (sold by Hanmi Pharmaceutical) was purchased and applied as Comparative Example 3.

[0062] Experimental Example 1. Confirmation of Virus Prevention Effect

[0063] Specific pathogen-free (SPF) female C57BL / 6 mice (7 weeks old, Samtaco, Korea) were used. They were housed in an environment with a temperature of 23±3°C and a relative humidity of 55±15%, with a 12-h light / dark cycle. Each test group was organized so that the average body weight was evenly distributed.

[0064] In addition, influenza A H1N1 virus was inoculated into hatched fertilized eggs, and the culture solution was harvested after 48 hours. The harvested virus was quantified and LD 50 was calculated. The prepared virus solution was 1 LD 50 Diluted with , 30 ul of the diluted solution was used.

[0065] The composition of each test group and the dosage of test substances for the above animal model are as shown in Table 2 below. G1 is the normal control group, G2 is the infected control group, Comparative Examples 1 to 3 are groups G3 to G5, and each substance of Examples 1 to 4 is group G6 to G9. Administration of each substance was started 2 hours before virus infection and administered twice a day (bis in die, BID) at 8-hour intervals for 7 days.

[0066] Comparative Example 2 was administered orally (Per oral, PO), while the remaining Comparative Examples 1, 3, and Examples 1 to 4 were administered intranasally (In). The test animals were fixed using the dorsal skin fixation method, and in the case of Comparative Example 2, the drug was administered directly into the stomach using an oral administration sonde. In the cases of Examples 1, 2, and 4, the drug was administered intranasally via spray, and in the case of Example 3, the drug was shaken well before administration to suspend it, and then administered intranasally by dropwise administration using a micropipette.

[0067]

[0068] 1-1. Check your weight

[0069] Body weight of each individual was measured once a day using an electronic scale. As shown in Table 3 and Figure 1 below, the G4 and G5 groups administered with positive control substances, and the G6 to G9 groups administered with each of the test substances of Examples 1 to 4, showed significantly higher levels than the G3 control group after 3 DPI (3 days post-infection).

[0070] Infection with the influenza virus causes excessive cellular infiltrates in the respiratory tract, producing inflammatory mediators such as INF, leading to weight loss. Administration of the test substances of Examples 1 to 4 of the present invention confirmed that weight loss, a symptom of influenza infection, was attenuated.

[0071] Body weight (Body weight) UNIT: % to 0 DPI (Mean ± SD) DPI Test group G1 G2 G3 0 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 110 1.35 ± 0.69 96.80 ± 1.76*** 96.30 ± 1.85*** 210 4.87 ± 0.9 19 8.72 ± 1.98 *** 95.66 ± 2.44 ***,### 3105.28 ± 1.4397.36 ± 2.71 *** 93.85 ± 2.81 ***,## 4103.97 ± 0.6297.57 ± 2.44 ** 91.83 ± 3.10 ***,### 5104.90 ± 2.3396.17 ± 2.37 *** 92.25 ± 3.31 *** 6-94.44 ± 0.9490.53 ± 3.167-93.44 ± 1.3188.49 ± 3.25DPI test groupG4G5G60100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00196.86 ± 1.40 *** 96.76 ± 1.52 *** 97.02 ± 1.63 *** 298.36 ± 2.41 ***,$ 96.37 ± 2.08 ***,# 96.82 ± 1.67*** 398.71 ± 3.01 ***,$$$ 96.76 ± 2.08 ***,$ 97.14 ± 1.63 ***,$$ 4100.15 ± 2.84 $$$ 96.34 ± 2.52 ***,$$ 96.15 ± 3.00 ***,$$ 599.52 ± 2.15 *,#,$$$ 98.20 ± 3.02 **,$$$ 97.10 ± 1.79 ***,$$ 697.56 ± 1.98 $$ 97.89 ± 2.26 $$ 99.52 ± 3.68 $$$ 794.73 ± 4.14 $ 98.58 ± 2.70 $$$ 99.14 ± 3.76 $$$ DPI test group G7G8G90100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00196.64 ± 2.34 *** 98.02 ± 1.51 ** 97.10 ± 2.42 *** 295.73 ± 2.88 ***,## 99.14 ± 2.47 ***,$$$ 97.04 ± 2.45 *** 396.56 ± 2.92 ***,$ 100.14 ± 2.76 **,#,$$$ 97.89 ± 2.92 ***,$$$ 495.29 ± 3.10 ***,$ 99.68 ± 3.36 $$$ 97.68 ± 2.64 **,$$$ 598.03 ± 3.14 ***,$$$ 99.50 ± 3.75 *,$$$ 99.60 ± 3.02 *,$$$ 6100.14 ± 3.42 #,$$$ 101.13 ± 4.00 ##,$$$ 99.78 ± 2.93 #,$$$ 799.49 ± 3.60 #,$$$ 97.97 ± 4.32 $$$ 98.26 ± 3.60 $$$DPI: days post-infection*** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G1## / # A significant difference at p<0.01 / p<0.05 level compared to the G2$$$ / $ A significant difference at p<0.001 / p<0.05 level compared to the G3

[0072] 1-2. Histopathological examination

[0073] Influenza A virus (H1N1) 1 LD 50 The effects of the test substance of the present invention on influenza-induced inflammation and tissue damage were evaluated in C57BL / 6 mice infected with the virus. The virus infection test design and test groups are as shown in Table 2 above. Except for G1, animals in the remaining test groups were euthanized and necropsied on days 3, 5, and 7 from the date of virus inoculation, with five animals per group per day. In the case of G1, all animals were euthanized and necropsied on day 5 from the date of virus inoculation. Animals other than those on the designated necropsy date were observed until day 7 from the date of virus inoculation.

[0074] At autopsy, the nasal cavity was swabbed using a swab soaked in PBS and stored frozen at -70°C until analysis for virus titer determination.

[0075] The extracted lungs were divided into two parts including the lesion area, and the part for histopathological examination was fixed in 10% neutral buffered formalin (NBF), and the part for virus titer measurement was frozen and stored at -70℃ until analysis.

[0076] Tissues fixed in 10% NBF were prepared for histopathological examination through general tissue processing such as trimming, dehydration, paraffin embedding, and sectioning. Then, hematoxylin & eosin (H&E) staining was performed, and histopathological changes were observed using an optical microscope (Olympus BX53, Japan). Lesions in the lungs were observed and evaluated by scoring according to the degree of inflammatory cell infiltration, and the criteria are shown in Table 4 below.

[0077] Score Score Criteria 0 No lesion or infiltration of inflammatory cells observed (None) 1 Inflammatory cells scattered within the parenchyma (Minimal) 2 Inflammatory cells aggregated within <1 / 3 of the parenchyma (Mild) 3 Inflammatory cells aggregated within 1 / 3 to 2 / 3 of the parenchyma (Moderate) 4 Inflammatory cells aggregated within >2 / 3 of the parenchyma (Severe)

[0078] As a result, as shown in Table 5 and Figure 2 below, the scores of G4 and G7 on the third day post-infection (3 DPI) were significantly lower than those of G2 and G3, and the scores of G4 and G5, which were administered the positive control substance until the seventh day post-infection, and G6 to G9, which were administered each of the test substances of Examples 1 to 4, were confirmed to be lower than those of G2 and G3.

[0079] Histopathological examination UNIT: Score DPI test group G1 G2 G3 3-1.4 ± 0.5 1.2 ± 0.4 5 0.0 ± 0.0 1.8 ± 0.8 2.2 ± 0.4 7-3.4 ± 0.5 3.0 ± 0.0 DPI test group G4 G5 G6 3 0.2 ± 0.4 #,$ 0.6 ± 0.50.6 ± 0.551.0 ± 1.01.4 ± 0.91.6 ± 1.171.4 ± 0.9 ##,$$ 2.0 ± 1.22.2 ± 0.8DPI test groupG7G8G930.0 ± 0.0 #,$ 0.6 ± 0.50.6 ± 0.551.2 ± 0.81.2 ± 1.11.4 ± 1.171.4 ± 0.9 #,$2.2 ± 1.31.2 ± 0.8 ##,$$ DPI: days post-infection## / # A significant difference at p<0.01 / p<0.05 level compared to the G2$$ / $ A significant difference at p<0.01 / p<0.05 level compared to the G3

[0080] These results indicate that when the test substances of Examples 1 to 4 of the present invention are administered for preventive purposes, damage to lung tissue and occurrence of lesions caused by influenza virus can be reduced.

[0081] 1-3. Virus titer measurement

[0082] The amount of virus present in lung tissue was quantified by real-time qRT-PCR using lung tissue collected 5 days after virus infection. The collected lung tissue was analyzed by Wizol TM (WizbioSolution, Seongnam, Korea) A certain amount of reagent was added, and total RNA was extracted from the supernatant using tissue grinding beads. The extracted total RNA was quantified using Nanodrop (NanoDrop2000, Thermo scientific). 1 μg of total RNA was extracted using WizScript TM After synthesizing cDNA using a cDNA synthesis kit (WizbioSolution), WizPure using a certain amount of cDNA reaction product TMReal-time qPCR was performed using qPCR Master-UDG (WizbioSoluton) reagent according to the product instructions. Using primers and probes specific for influenza virus (A / Puerto Rico / 8 / 1934 (H1N1)) M2 (matrix protein 2) and pol PA (polymerase PA), viral genes present in lung tissue were quantified. The amount of viral RNA present in lung tissue was expressed as the number of viral gene copies per ng of total RNA. The sequences of each primer and probe are shown in Table 6 below.

[0083]

[0084] Afterwards, a standard graph was created and the amount of virus remaining in the sample was quantitatively analyzed by inputting each of the above genes.

[0085] As a result, as shown in Fig. 3, when the test substances of Examples 1 to 4 of the present invention were administered, there was no significant decrease in the M2 and polPA genes in the lungs. On the other hand, in the case of the nasal cavity, when the test substances of Examples 1 to 4 of the present invention were administered, as shown in Fig. 4, a decrease in the M2 and polPA genes was observed, confirming the effect of preventing influenza virus infection through the nasal cavity. In particular, it was confirmed that a more significant virus titer reduction effect was observed in the G6, G7, and G9 groups administered with Examples 1, 2, and 4.

[0086] Experimental Example 2. Confirmation of Virus Treatment Efficacy

[0087] An animal model was constructed in the same manner as in Experimental Example 1 above and infected with the influenza A H1N1 virus. However, for confirmation of therapeutic efficacy, the test substance was administered after virus infection.

[0088] Specifically, the composition of each test group and the dosage of the test substance are as shown in Table 7 below. G1 is the normal control group, G2 is the infected control group, Comparative Examples 1 to 3 are groups G3 to G5, and each substance of Examples 1 to 4 is groups G6 to G9. Administration of each substance was initiated 24 hours after virus infection and administered twice a day (bis in die, BID) at 8-hour intervals for 6 days. The administration method according to the administration route is the same as that of Experimental Example 1.

[0089]

[0090] 2-1. Check your weight

[0091] Body weight of each individual was measured once a day using an electronic scale. As shown in Table 8 and Figure 5 below, on the fifth day post-infection (5 DPI), the G6 to G9 groups administered each of the test substances of Examples 1 to 4 showed significantly higher levels than the G3 control group.

[0092] From these results, it was confirmed that weight loss, one of the symptoms of influenza infection, can be alleviated even when the test substances of Examples 1 to 4 of the present invention are administered after infection.

[0093] Body weight (Body weight) UNIT: % to 0 DPI (Mean ± SD) DPI Test group G1 G2 G3 0 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 198.34 ± 1.04 98.13 ± 1.53 97.03 ± 1.09 298.36 ± 1.90 95.85 ± 1.56 96.23 ± 1.78 3 100.29 ± 3.64 96.09 ± 1.85 95.47 ± 3.43 4 101.28 ± 2.05 97.87 ± 1.82 94.88 ± 2.51 * 5104.59 ± 1.7697.33 ± 3.21 * 94.01 ± 2.90 *** 6-95.11 ± 2.43 **93.12 ± 1.76 *** 7-92.53 ± 2.73 ** 89.33 ± 6.35 *** DPI test group G4G5G60100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00196.35 ± 1.8097.40 ± 1.6597.64 ± 3.47295.82 ± 3.0396.90 ± 2.5596.74 ± 4.87395.76 ± 4.7797.10 ± 3.6496.69 ± 4.92493.96 ± 2.64 * 96.40 ± 3.8496.06 ± 5.84594.75 ± 3.47 ** 96.57 ± 4.94 * 95.41 ± 5.53 ** 691.73 ± 1.66 *** 97.13 ± 0.95 * 90.70 ± 5.36 *** 790.01 ± 3.20 ** 93.39 ± 4.26 * 88.52 ± 6.81 *** DPI test group G7G8G90100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00196.87 ± 2.6097.94 ± 2.2797.64 ± 3.47296.33 ± 3.4696.47 ± 2.0096.74 ± 4.87396.97 ± 3.0996.54 ± 3.6096.69 ± 4.92496.51 ± 4.2395.11 ± 3.76 * 96.06 ± 5.84597.53 ± 3.69 * 94.31 ± 4.23 *** 95.41 ± 5.53 ** 696.58 ± 4.64 * 94.61 ± 5.34 ** 90.70 ± 5.36 *** 793.85 ± 6.41 * 92.33 ± 6.37 ** 88.52 ± 6.81 ***DPI: days post-infection*** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G1

[0094] 2-2. Histopathological examination

[0095] In the same manner as in Experimental Examples 1-2 above, 5 animals per group were euthanized on the 3rd, 5th, and 7th days from the date of virus inoculation and autopsied. In the case of G1, all animals were euthanized on the 5th day from the date of virus inoculation, autopsied, and histopathology was observed. Thereafter, they were evaluated using the same scoring criteria as in Table 4 above.

[0096] As a result, as shown in Table 9 and Fig. 6 below, the degree of inflammatory cell infiltration in groups G6 to G9, which were administered each test substance of Examples 1 to 4, tended to be lower in all autopsies compared to G3 overall, and the scores of G4, G7, and G9 on the 5th day (5 DPI) and 7th day (7 DPI) post-infection were significantly lower than those of G2 and G3. In particular, it was confirmed that the degree of inflammatory cell infiltration in G7 and G9 was significantly lower.

[0097] Histopathological examination UNIT: Score DPI test group G1 G2 G3 3-1.6 ± 1.1 1.6 ± 0.5 5 0.0 ± 0.0 2.8 ± 0.4 2.8 ± 0.47-3.6 ± 0.5 3.2 ± 0.4 DPI test group G4 G5 G6 3 0.8 ± 0.4 1.0 ± 0.7 1.0 ± 0.7 5 1.4 ± 0.5 #,$ 2.0 ± 1.01.8 ± 0.871.8 ± 0.8 #,$ 2.6 ± 0.92.4 ± 1.3DPITest groupG7G8G931.2 ± 0.41.0 ± 0.71.0 ± 0.751.4 ± 0.9 #,$ 2.0 ± 1.01.2 ± 0.8 #,$ 71.6 ± 0.5 ##,$$ 2.4 ± 1.31.6 ± 0.5 ##,$$DPI: days post-infection## / # A significant difference at p<0.01 / p<0.05 level compared to the G2$$ / $ A significant difference at p<0.01 / p<0.05 level compared to the G3

[0098] These results indicate that even when the test substances of Examples 1 to 4 of the present invention are administered after influenza virus infection, damage to lung tissue and occurrence of lesions caused by influenza virus can be reduced, indicating that there is a therapeutic effect against influenza virus.

[0099] 2-3. Virus titer measurement

[0100] RNA was extracted from lung and nasal swab samples in the same manner as in Experimental Examples 1-3 above, and the M2 and polPA genes were detected using qRT-PCR to quantitatively analyze the amount of virus residue.

[0101] As a result, as shown in Table 10, Table 11, and Fig. 7 below, in the lung tissues of 7 DPI autopsied individuals, it was confirmed that both the M2 and polPA genes of the G5 positive control group and the G7 and G9 test substance administration groups were reduced compared to the G3 control group. In addition, in the case of the G6 group, it was confirmed that both the M2 and polPA genes were reduced compared to the G3 control group in the lung tissues of 5 DPI autopsied individuals.

[0102] Virus titer (lung) M2 UNIT: copies / ng DPI Test group G1 G2 G3 -78.08 ± 102.34 27.33 ± 30.14 50.00 ± 0.00 37.48 ± 34.7 1 15.52 ± 117.4 27-11.46 ± 9.8 9 4.61 ± 4.87 DPI Test group G4 G5 G6 3 5.02 ± 60.34 25.57 ± 26.40 13.90 ± 11.6 1 5 8.98 ± 10.54 1 0 7.90 ± 176.40 35.34 ± 49.38 78.86 ± 7.6 44.53 ± 6.9 46.38 ± 4.72 DPI Test group G7 G8 G9 36.61 ± 9.1814.52 ± 22.4330.11 ± 25.51543.52 ± 88.4154.11 ± 99.04126.93 ± 155.9273.18 ± 3.1211.40 ± 12.052.41 ± 2.38

[0103] Virus titer (lung) polPA UNIT: copies / ng DPI Test group G1 G2 G3 3-27 15.87 ± 3666.96 1009.60 ± 1413.18 50.00 ± 0.004 91.84 ± 540.21 1242.72 ± 1200.107-174.77 ± 228.20 63.05 ± 63.85 DPI Test group G4 G5 G6 3145 3.46 ± 2714.18 645.15 ± 794.64 432.91 ± 335.67 5132.86 ± 104.09 2282.70 ± 4021.43 465.80 ± 648.05 165.93 ± 182.6153.94 ± 79.5576.27 ± 50.37DPI test group G7G8G9354.76 ± 77.74407.23 ± 767.511050.61 ± 914.0651223.43 ± 1798.12472.83 ± 780.711462.30 ± 1905.12747.74 ± 55.54176.85 ± 152.7229.74 ± 34.23

[0104] In addition, as shown in Table 12, Table 13 and Figure 8 below, in the nasal samples of 7 DPI autopsied individuals, it was confirmed that the M2 and polPA genes of the G5 positive control group, G6, G7 and G9 test substance administration groups were all reduced compared to the G3 control group.

[0105] Virus titer (nasal) M2 UNIT: copies / mLDPI test group G1 G2 G3 3-720.63 ± 1188.14 0.00 ± 0.005 0.00 ± 0.00865847.04 ± 1930483.29825281.04 ± 1845384.517-30780.88 ± 66589.93 28537.64 ± 63675.62DPI test group G4 G5 G6 30.00 ± 0.00327630.74 ± 698396.61 399.55 ± 893.43567612.73 ± 117098.05 8039152.61 ± 10368970.735387.49 ± 11770.657264852.85 ± 236609.52515.45 ± 666.5011598.79 ± 18410.41DPI Test Group G7G8G9 396744.31 ± 208185.08359.83 ± 804.593470.91 ± 6150.445137.75 ± 252.0360885.81 ± 133470.76599.00 ± 1339.41714499.47 ± 32421.8039763.68 ± 87024.283305.15 ± 7390.53

[0106] Virus titer (nasal) polPA UNIT: copies / mLDPI test group G1G2G33-8173.31 ± 7070.2610521.46 ± 19578.4750.00 ± 0.0042551552.83 ± 95009881.85586.32 ± 1077.787-4738095.80 ± 10514434.113314005.76 ± 7390510.19DPI test group G4G5G63687.83 ± 832.9618236216.91 ± 38691441.1811313.29 ± 18427.9751905385.64 ± 2935251.54411088263.44 ± 488467822.79105133.35 ± 226762.09726587812.07 ± 26169008.3649594.24 ± 83919.741523582.19 ± 2434770.92 DPI Test Group G7G8G935222876.41 ± 11628560.1111808.16 ± 21575.74107941.59 ± 196643.6255438.77 ± 6900.603705803.49 ± 8212700.0112887.58 ± 22429.6172069412.39 ± 4607026.073269283.91 ± 7276365.49430285.99 ± 960128.44

[0107] Through the above results, it was confirmed that the virus titer decreased when the test substances of Examples 1, 2 and 4 of the present invention were administered, and thus, they were effective in treating influenza virus infection.

[0108] Experimental Example 3. Confirmation of antiviral effects according to xanthan gum content.

[0109] To determine the antiviral effect according to the xanthan gum content, an animal model was constructed in the same manner as Experimental Example 1 and administered the influenza A H1N1 virus. The specific test group composition and test substance dosage settings are shown in Table 14 below.

[0110] G1 is the normal control group, G2 is the infected control group, and Comparative Example 1 regarding the combination of excipients as the negative control substance and Comparative Example 3 regarding the positive control substance are groups G3 and G4, respectively. Groups G5 to G7 were composed of low to high concentrations according to the xanthan gum content. Administration of each substance was started 2 hours before virus infection and administered twice a day (bis in die, BID) at 8-hour intervals for 7 days. The administration method according to the administration route is the same as Experimental Example 1.

[0111]

[0112] 3-1. Check your weight

[0113] Body weight was measured once daily using an electronic scale. As shown in Table 15 and Figure 9, all experimental groups G5 to G7 administered xanthan gum showed higher levels compared to group G3. This confirms that weight loss, a symptom of influenza infection, can be alleviated even when xanthan gum is administered at low, medium, or high concentrations.

[0114] Body weight (Body weight) UNIT: % to 0 DPI (Mean ± SD) DPI Test group G1 G2 G3 G4 0 100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00100.00 ± 0.001100.14 ± 1.4899.18 ± 1.4499.89 ± 1.33100.99 ± 2.072100.70 ± 2.4498.79 ± 2.0898.24 ± 2.09102.38 ± 2.29 *** 398.60 ± 2.2798.90 ± 2.3195.56 ± 2.06 $ 99.78 ± 2.72 *** 4100.98 ± 2.5798.40 ± 3.8693.18 ± 2.27 $$$ 98.87 ± 3.37 *** 5104.77 ± 2.9296.15 ± 3.2489.82 ± 2.52 $$$ 97.32 ± 4.29*** 6107.01 ± 2.0692.53 ± 5.6186.51 ± 2.70 $$$ 93.43 ± 2.42 ** 7105.75 ± 2.5989.25 ± 6.5483.29 ± 3.52 $$$ 88.38 ± 2.59DPI시험군G5G6G70100.00 ± 0.00100.00 ± 0.00100.00 ± 0.001101.62 ± 1.07 * 100.07 ± 1.83100.12 ± 2.472102.60 ± 1.31 *** 100.58 ± 3.46 * 99.72 ± 2.58399.80 ± 2.04 *** 98.55 ± 2.42 *** 96.87 ± 1.83498.01 ± 1.90 *** 96.48 ± 2.04 * 95.91 ± 3.27 * 597.27 ± 2.48 *** 95.91 ± 2.51 *** 95.56 ± 2.90 *** 696.16 ± 1.97 *** 94.39 ± 3.58 ** 93.18 ± 4.81 ** 795.69 ± 3.07 ***,## 87.97 ± 4.5088.25 ± 7.49DPI: days post-infection$$$ / $ A significant difference at p<0.001 / p<0.05 level compared to the G1*** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G3## A significant difference at p<0.01 compared to the G4

[0115] 3-2. 조직병리학적 검사

[0116] In the same manner as in Experimental Example 1-2 above, six animals per group were euthanized on the 3rd, 5th, and 7th days from the date of virus inoculation and autopsied. In the case of G1, all animals were euthanized on the 5th day from the date of virus inoculation, autopsied, and histopathology was observed. Thereafter, they were evaluated using the same scoring criteria as in Table 4 above.

[0117] As a result, as shown in Table 16 and Figure 10 below, the degree of inflammatory cell infiltration in the G5 to G7 groups administered each test substance of Examples 5 to 7 was lower than that in G3 in all autopsy counts. In particular, it was confirmed that the degree of inflammatory cell infiltration in G5, which was administered a low concentration of xanthan gum, was significantly lower.

[0118] Histopathological examination UNIT: Score DPI test group G1 G2 G3 G4 3-1.3 ± 0.5 1.3 ± 0.5 1.3 ± 0.5 5-2.2 ± 0.4 2.7 ± 0.5 2.2 ± 0.4 70.0 ± 0.03.8 ± 0.4 4.0 ± 0.03.5 ± 0.5 DPI test group G5 G6 G7 3 1.2 ± 0.4 1.2 ± 0.4 1.2 ± 0.4 52.2 ± 0.8 2.2 ± 0.4 2.2 ± 0.8 71.3 ± 0.5 **,## 2.7 ± 0.8 * 2.8 ± 1.0 * DPI: days post-infection** / * A significant difference at p<0.01 / p<0.05 level compared to the G3## A significant difference at p<0.01 compared to the G4

[0119] These results indicate that xanthan gum can reduce influenza virus-induced lung tissue damage and lesion development across all concentration ranges.

[0120]

[0121] 3-3. Virus titer measurement

[0122] RNA was extracted from lung and nasal swab samples in the same manner as in Experimental Examples 1-3 above, and the M2 and polPA genes were detected using qRT-PCR to quantitatively analyze the amount of virus residue.

[0123] As a result, as shown in Table 17, Table 18 and Figure 11 below, it was confirmed that both the M2 and polPA genes of the G5 to G7 groups, which were the test substance administration groups, were reduced compared to the G3 control group in the lung tissues of the 3 DPI and 5 DPI rats.

[0124] Virus titer (lung) M2 UNIT: copies / ng DPI Test group G1 G2 G3 G4 3 -19.89 ± 12.97 23.61 ± 12.85 25.13 ± 7.46 5 -39.19 ± 22.57 42.57 ± 38.62 17.34 ± 15.81 70.00 ± 0.00 16.13 ± 16.04 5.91 ± 5.82 23.22 ± 19.36 DPI Test group G5 G6 G7 3 14.73 ± 11.33 10.09 ± 8.02 31.00 ± 38.36 5 10.86 ± 9.22 38.74 ± 40.44 11.04 ± 10.39 76.45 ± 10.6316.24 ± 13.3116.95 ± 15.56

[0125] Virus titer (lung) polPA UNIT: copies / ng DPI Test group G1 G2 G3 G4 3-857.54 ± 532.069 89.44 ± 478.52 1070.80 ± 394.545-1404.37 ± 1147.112 367.40 ± 2412.48 808.85 ± 742.05 70.04 ± 0.078 05.97 ± 689.61 530.64 ± 606.43 1414.91 ± 718.32 DPI Test group G5 G6 G7 3532.63 ± 426.17*380.05 ± 360.48 1327.49 ± 1632.89 5458.61 ± 415.891711.80 ± 2046.23365.94 ± 359.987340.79 ± 540.49 #1241.74 ± 1057.86808.61 ± 917.61# A significant difference at p<0.05 level compared to G4

[0126] In addition, as shown in Table 19, Table 20 and Figure 12 below, in the nasal samples of 7 DPI autopsied subjects, it was confirmed that both the M2 and polPA genes were reduced in the G5 to G7 groups, which were the test substance administration groups, compared to the G3 control group.

[0127] Virus titer (nasal) M2 UNIT: copies / ngDPI Test group G1 G2 G3 G4 3 -0.25 ± 0.21 0.41 ± 0.32 0.64 ± 1.39 5 -0.01 ± 0.03 0.04 ± 0.06 0.10 ± 0.19 70.01 ± 0.02 0.07 ± 0.10 0.43 ± 0.36 0.11 ± 0.09 * DPI test group G5G6G730.25 ± 0.370.33 ± 0.710.00 ± 0.0050.42 ± 0.740.10 ± 0.200.12 ± 0.2070.06 ± 0.09 ** 0.01 ± 0.01 ** 0.03 ± 0.05 ** ** / * A significant difference at p<0.01 / p<0.05 level compared to the G3

[0128] Virus titer (nasal) polPA UNIT: copies / ng DPI Test group G1 G2 G3 G4 3 -374.78 ± 853.33 29.90 ± 26.57 20.80 ± 40.7 45 -1.08 ± 2.1 14.58 ± 7.6 0 3.59 ± 6.4 27 0.04 ± 0.07 4.97 ± 6.68 44.23 ± 47.4 215.09 ± 10.57 DPI Test group G5 G6 G7 3 24.33 ± 43.38 39.47 ± 92.8 10.06 ± 0.09 5 17.64 ± 24.6 7 5.16 ± 10.3 7 4.96 ± 7.0 17 10.53 ± 8.46 * 2.97 ± 5.49 *3.04 ± 4.80 * * A significant difference at p<0.05 level compared to G3

[0129] Through the above results, it was confirmed that the influenza virus titer was reduced in all concentration ranges of xanthan gum, and thus it is effective in the prevention and / or treatment of influenza virus.

[0130] Experimental Example 4. Statistical Analysis

[0131] The results of each of the above experimental examples were analyzed using parametric multiple comparison procedures or non-parametric multiple comparison procedures, assuming normality of the data.

[0132] For parametric multiple comparisons, normality of data was assumed, and parametric one-way ANOVA was used for testing. If the results were significant, Dunnett's multiple comparison test was used as a post hoc test to analyze the significant differences between test groups. For nonparametric multiple comparisons, Kruskal-Wallis' H-test was used for testing. If the results were significant, Mann Whitney U test was used as a post hoc analysis to analyze the significant differences between test groups. Statistical analysis was performed using Prism 9.4.1 (GraphPad Software Inc., San Diego, CA, USA), and a p value less than 0.05 was considered statistically significant.

[0133] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0134] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Containing xanthan gum or fucoidan as an active ingredient, An antiviral composition against influenza virus.

2. In paragraph 1, An antiviral composition wherein the above influenza virus is an influenza A virus.

3. In paragraph 2, An antiviral composition wherein the above influenza A virus is of type H1N1.

4. In paragraph 1, The above composition is an antiviral composition for use as an immune measure against influenza virus infection.

5. Containing xanthan gum or fucoidan as an active ingredient, A pharmaceutical composition for preventing or treating a viral infection disease caused by an influenza virus.

6. In paragraph 5, A pharmaceutical composition, wherein the viral infection disease caused by the above influenza virus is selected from the group consisting of cold, flu, pharyngitis, laryngitis, pharyngitis, rhinitis, bronchitis, asthma, sepsis, and pneumonia.

7. In paragraph 5, A pharmaceutical composition, wherein the pharmaceutical composition is administered parenterally.

8. In paragraph 7, A pharmaceutical composition characterized in that the above parenteral administration is intranasal or intranasal administration.

9. In paragraph 8, A pharmaceutical composition characterized in that the intranasal or intranasal administration is administered in the form of a spray, aerosol, or inhalation.

10. A method for treating influenza virus infection, comprising administering to a subject the pharmaceutical composition of paragraph 5.

11. Use of the pharmaceutical composition of Article 5 for the treatment of influenza virus infection.

12. Use of the antiviral composition of paragraph 1 for the manufacture of a medicine for treating influenza virus infection.

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