Use of the day bufadienolide in preparing anti-influenza virus products

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of butaclamol in preparation of anti-influenza virus products. The application proves through in-vivo and in-vitro experiments that butaclamol can play an antiviral role by inhibiting replication of H1N1. The in-vitro antiviral effect of butaclamol is superior to that of positive drug oseltamivir, and intragastrically administered butaclamol can reduce the mortality of mice by reducing replication of H1N1 in vivo, and has good practical application value in development of anti-influenza virus drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of bufotalin in the preparation of anti-influenza virus products. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus. It not only causes systemic symptoms such as fever and muscle aches, but also often leads to severe local respiratory lesions.

[0003] Currently, influenza prevention and control measures mainly consist of vaccines and antiviral drugs. Universal influenza vaccines can provide protection and reduce the threat posed by influenza, but their protective efficacy is relatively poor due to antigenic drift and adaptation. Antiviral drugs mainly include four categories: M2 ion channel inhibitors, neuraminidase inhibitors, hemagglutinin inhibitors, and influenza virus RNA polymerase inhibitors. Influenza A viruses have developed high levels of resistance to amantadine, an early approved matrix protein 2 (M2) ion channel inhibitor, and this resistance persists to this day. Neuraminidase inhibitors are the most widely used treatments, but to date, some strains have also developed resistance to them. Hemagglutinin inhibitors and influenza virus RNA polymerase inhibitors have serious drawbacks due to their potential for severe side effects.

[0004] Therefore, developing new anti-influenza drugs has become an urgent problem to be solved. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of bufotalin in the preparation of anti-influenza virus products.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the invention provides the use of bufotalin in the preparation of anti-influenza virus products.

[0008] A second aspect of the present invention provides a method for inhibiting influenza virus infection in vitro, comprising administering an effective dose of bufotalin to an infection system; wherein the influenza virus includes PR8, HSV-1, or SeV.

[0009] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention reveals that bufotalin exerts its antiviral effect by inhibiting H1N1 replication. Cytotoxicity of bufotalin against A549 cells was detected using the CCK8 assay, showing an IC50 of 16.27 nM, indicating superior in vitro antiviral efficacy compared to the positive control drug oseltamivir. Oral administration of bufotalin to mice and subsequent analysis of lung indices and viral RNA levels in the lungs showed that bufotalin continued to reduce H1N1 replication in mice. Therefore, bufotalin possesses significant potential for development into a specific drug for influenza viruses. Attached Figure Description

[0010] Figure 1 This is a schematic diagram illustrating the effect of bufotalin on the cell viability of A549 cells in an embodiment of the present invention.

[0011] Figure 2 The results of the study in this embodiment of the invention show the RNA levels of H1N1 influenza virus M1 and NP, and the protein level of NP, after A549 cells were treated with bufotalin.

[0012] Figure 3 The results show the RNA expression of H1N1 influenza virus NP after A549 cells incubated with different concentrations of bufotalin were infected with PR8 virus in this embodiment of the invention.

[0013] Figure 4 The results of viral RNA expression in A549 cells incubated with different concentrations of bufotalin in this embodiment of the invention are shown; where A represents the result of HSV-1 virus infection and B represents the result of SeV virus infection.

[0014] Figure 5 The following are the results of anti-influenza virus in vivo from toads in this embodiment of the invention; wherein, A is a schematic diagram of the weight changes of mice in different treatment groups; B is the mortality statistics of mice in different treatment groups; C is the lung index results of mice in different treatment groups; D is the RNA expression level of H1N1 NP in mice in different treatment groups detected by qRT-PCR; E is the RNA expression level of H1N1 M1 in mice in different treatment groups detected by qRT-PCR. Detailed Implementation

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0016] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0017] In a typical embodiment of the present invention, the use of bufotalin in the preparation of anti-influenza virus products is provided.

[0018] The structural formula of bufotalin is CAS number 465-11-2.

[0019] In some embodiments, the product is a drug.

[0020] This invention used the CCK8 assay to detect the cytotoxicity of bufotalin against A549 cells, finding that bufotalin had an IC50 of 16.27 nM against A549, demonstrating superior in vitro antiviral efficacy compared to the positive control drug oseltamivir. By administering bufotalin to mice via gavage and detecting lung indices and viral RNA levels in the lungs, it was found that bufotalin could still reduce H1N1 replication in mice. Therefore, bufotalin has great potential for development into a specific drug for influenza viruses.

[0021] In some embodiments, the drug is a single active ingredient drug, the active ingredient of which is bufotalin.

[0022] In some embodiments, the drug is a compound drug whose active ingredient includes bufotalin.

[0023] In some embodiments, the drug also contains at least one other non-pharmaceutical active ingredient.

[0024] In some embodiments, the other non-pharmaceutical active ingredients include pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids, and pharmacologically harmless salts.

[0025] In some embodiments, the dosage form of the drug includes true solutions, colloids, microparticles, emulsions, suspensions, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powder injections, inclusion complexes, implants, patches, and liniments.

[0026] In some implementations, the application achieves its anti-influenza virus function by inhibiting the replication of H1N1.

[0027] In some implementations, the influenza virus includes PR8, HSV-1, or SeV.

[0028] In another typical embodiment of the present invention, a method for in vitro inhibition of influenza virus infection is provided, comprising administering an effective dose of bufotalin to an infected system or infected individual; and, after administration at a predetermined time, significantly reducing or reducing the level of influenza virus in the lungs of the infected system or infected individual to an undetectable level; wherein the influenza virus includes PR8, HSV-1, or SeV.

[0029] As used herein, the term "effective dose" includes, within its meaning, a non-toxic but sufficient amount of bufotalin to provide the desired inhibitory effect. The exact amount required will vary from infection system to infection individual, and therefore it is impossible to specify an exact "effective dose." However, for any given situation, a suitable "effective dose" can be determined by those skilled in the art using only routine experiments.

[0030] Sources of main experimental reagents and materials: A549 cells were purchased from ATCC (CCL-185); bufotalin was purchased from Chem Faces (CFN90212); and the CCK8 kit was purchased from White Shark Biotechnology (BS350B).

[0031] The influenza A virus strain A / PR / 8 / 34, belonging to the influenza virus family, is scientifically named Influenza A virus A / PR / 8 / 34 (H1N1), abbreviated as PR8, and was generously provided by the National Key Laboratory for the Diagnosis and Treatment of Severe Infectious Diseases; Herpes simplex virus type 1, abbreviated as HSV-1, was also generously provided by the National Key Laboratory for the Diagnosis and Treatment of Severe Infectious Diseases; Sendai virus, abbreviated as SeV, was also generously provided by the National Key Laboratory for the Diagnosis and Treatment of Severe Infectious Diseases.

[0032] The primer sequences used in the examples are shown in Table 1.

[0033] Table 1 Primer Sequences

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Example 1: Assay of cell viability of A549 cells by bufotalin A549 cells (ATCC, CCL-185) were administered at a rate of 2 × 10⁻⁶. 5 / wells were seeded in 96-well plates and cultured in a 37°C, 5% CO2 incubator. Bufotalin (ChemFaces, CFN90212) was dissolved in DMSO to prepare a 30 mM stock solution, which was then diluted with DMEM medium supplemented with 10% FBS to different concentrations (5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, and 300 nM). Different concentrations of the drug solution were added to 96-well plates as experimental groups, wells containing untreated normal cells served as control groups, and wells containing only culture medium and no cells served as blank groups. After 12 hours, cell viability was calculated using the CCK8 assay kit according to the manufacturer's instructions. The cell viability calculation formula is: Cell viability = [OD...] 实验组 -OD 空白组 ] / [OD 对照组 )-OD 空白组 ]×100%. The result is as follows: Figure 1 As shown, the IC50 of bufotalin against A549 cells was 16.27 nM.

[0036] Example 2: Detection of the inhibitory effect of bufotalin on PR8 virus A549 cells were administered at a concentration of 1.6 × 10⁻⁶. 6 Cells were seeded in 12-well plates. After 16 hours, they were incubated with 1.5 nM, 3 nM, and 6 nM bufotalin. After 2 hours, PR8 virus was added for infection (MOI=0.1). Cell samples were collected 12 hours after infection. Primers were designed (Table 1) to detect the RNA expression levels of viral matrix protein (M1) and nucleoprotein (NP), and the protein level of NP was detected by Western blot. The results are as follows: Figure 2 As shown, it was found that bufotalin can inhibit H1N1 replication in a dose-dependent manner.

[0037] A549 cells were administered at a concentration of 1.6 × 10⁻⁶. 6 Cells were seeded in 12-well plates. After 16 hours, different concentrations (0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 10 nM) of bufotalin were added for incubation. After 2 hours, PR8 virus was added for infection. Cell samples were collected 12 hours after infection. The RNA level of viral NPs was detected, and the results are as follows: Figure 3 As shown, the EC50 of bufotalin against PR8 was found to be 4.615 nM.

[0038] Example 3: Detection of the inhibitory effects of bufotalin on HSV and SeV viruses. A549 cells were administered at a concentration of 1.6 × 10⁻⁶. 6Cells were seeded in 12-well plates. After 16 hours, 1.5 nM, 3 nM, and 6 nM of bufotalin were added for incubation. After 2 hours, HSV-1 or SeV infection (MOI=0.1) was added. Cell samples were collected 12 hours after infection. Primers were designed (Table 1) to detect the RNA expression levels of HSV-1 or SeV. The results are as follows: Figure 4 As shown, it was found that bufotalin can inhibit the replication of HSV-1.

[0039] Example 4: Detection of anti-influenza virus in bufotalin Five- to six-week-old male BALB / c mice were randomly divided into a control group, a model group, an oseltamivir group, a low-dose bufotalin group (Gam-L), and a high-dose bufotalin group (Gam-M). Animal models were established by anesthetizing and intranasally infecting mice with a PR8 virus dilution containing two LD50 concentrations. The control group consisted of uninfected normal mice, treated daily by gavage with sterile water (100 μL). The model group consisted of virus-infected mice, treated daily by gavage with sterile water (100 μL). The oseltamivir group received 70 mg / kg / day via gavage (100 μL). The low-dose bufotalin group (Gam-L) received 0.525 μg / kg / day via gavage (100 μL). The high-dose bufotalin group (Gam-M) received 1.05 μg / kg / day via gavage (100 μL). Monitor the body weight of mice in each group. Mice were considered dead when their body weight dropped to 75%. The results are as follows: Figure 5 As shown in A and B, this indicates that bufotalin can reduce the mortality rate of mice. Mice were sacrificed on day 6, and lungs were collected to calculate the lung index, as shown in Figure 6. Figure 5 As shown in C, it was found that bufotalin did not affect the lung index. RNA levels of NP and M1 were detected by qRT-PCR, as shown... Figure 5 As shown in E and F, bufotalin can reduce H1N1 replication and decrease mortality in vivo.

[0040] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of bufotalin in the preparation of anti-influenza virus products.

2. The application as described in claim 1, characterized in that, The product is a medicine.

3. The application as described in claim 2, characterized in that, The drug is a single active ingredient drug, and its active ingredient is bufotalin.

4. The application as described in claim 2, characterized in that, The drug is a compound drug, and its active ingredient includes bufotalin.

5. The application as described in claim 2, characterized in that, The drug also contains at least one other non-pharmaceutical active ingredient.

6. The application as described in claim 5, characterized in that, Other non-pharmaceutical active ingredients include pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids, and pharmacologically harmless salts.

7. The application as described in claim 2, characterized in that, The dosage forms of the drugs include true solutions, colloids, microparticles, emulsions, suspensions, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powder injections, inclusion complexes, implants, patches, and liniments.

8. The application as described in claim 1, characterized in that, The application achieves its anti-influenza virus function by inhibiting the replication of H1N1.

9. The application as described in claim 1, characterized in that, The influenza viruses mentioned include PR8, HSV-1, or SeV.

10. A method for inhibiting influenza virus infection in vitro, characterized in that, This includes administering an effective dose of bufotalin to the infection system; the influenza virus includes PR8, HSV-1, or SeV.

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