KR20200026390A - Adjuvant for preventing or treating of viral diseases comprising ionophore as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for supporting antiviral activity comprising ionophore as an active ingredient. Specifically, salinomycin according to the present invention exhibits antiviral activity by inhibiting endosomal acidification and M2 proton channel activity in influenza virus-infected cell lines. Also, monensin and nigericin, other polyether ionophores, exhibit antiviral activity in infected cell lines. In addition, since salinomycin significantly increases the antiviral effect of OSV against influenza virus and OSV resistant influenza virus, polyether ionophores comprising salinomycin, monensin, and nigericin can be usefully used as an adjuvant for preventing or treating viral diseases.

Description

Adjuvant for preventing or treating of viral diseases comprising ionophore as an active ingredient}

The present invention relates to an agent for the prevention or treatment of viral diseases comprising ionophore as an active ingredient.

Many existing infectious diseases are caused by viruses and cause many serious diseases such as rabies, smallpox, myelitis, hepatitis, immunodeficiency, encephalitis and AIDS. Infectious diseases caused by these viruses are very contagious, such as colds, measles, mumps, and chickenpox, and may indicate respiratory or digestive disorders, or measles causes congenital abnormalities. Tumor viruses cause tumors and cancers in the human body. do.

Influenza viruses are one of the most common viruses in the world, affecting both humans and animals, causing 30 to 500,000 deaths annually worldwide and causing severe national economic losses. Influenza virus is an RNA enveloped virus belonging to Orthomyxoviridae , which is a ribonucleic acid (RNA) bound to a nucleoprotein (NP) surrounded by a viral envelope with lipid bilayer structure and foreign glycoproteins. It consists of an internal nucleocapsid or core. The inner layer of the viral envelope consists mainly of matrix proteins, and the outer layer consists mostly of host-derived lipid material.

Influenza viruses that cause human infections are type A and B, and viral antigen mutations are determined by hemaglutinin (HA) and neuraminidase (NA) on the surface of the virus, and new antigenic strains appear every year. . In particular, avian influenza virus, which has been a problem until recently, infects various kinds of birds such as chickens, turkeys, ducks, and wild birds due to stool, and kills more than 80% when chickens are infected due to rapid spread. It has been reported to cause great damage and threats to the poultry industry worldwide, and it has been reported to cause infections in humans by infecting the human body (Gubareva, LV et al, Lancet 355, 2000). After the pandemic of the Spanish flu (H1N1) in 1918, the pandemic of influenza virus infections occurred in several decades, including the Asian flu (H2N2) in 1957 and the Hong Kong flu (H3N2) in 1968. I have declared a pandemic.

In order to prevent or treat such viral infection, antiviral agents are targeted to inhibit adsorption into epithelial cells, inhibit invasion into cells, inhibit transcription and replication of genes, inhibit protein synthesis, and inhibit release from cells. There are currently two classes of drugs targeting the influenza virus proteins NA and M2, which are approved by the US FDA as antiviral agents for influenza viruses. Proton channel M2 inhibitors, amantadine and rimantadine, are effective only against influenza virus type A and have been reported to develop resistant viruses and severe side effects in the nervous system and stomach during use for 40 years. . The oseltamivir phosphate (OSV-P, Tamiflu) and zanamivir, NA inhibitors, are effective against both influenza viruses A and B, but zanamivir has low bioavailability and fast kidney Disadvantages of emissions, OSV-P has the side effect of severe vomiting.

Ionophores are small molecules that move specific ions into and out of a lipid bilayer membrane and are classified as either electrogenic or electroneutral ionophores. Electrogenic ionophores such as valinomycin, carbonyl cyanide m- chlorophenylhydrazone (CCCP) or carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) transport net charge into and out of the membrane. On the other hand, electrically neutral ionophores such as monensin, A23187, nigericin or salinomycin form zwitter ion complexes with cations to electrically neutral cation exchange diffusion ( exchange diffusion).

The inventors have derived salinomycin, a monovalent cationic ionophore, as an antiviral candidate for influenza virus through a high-throughput screening method, and then polyether ionopores containing salinomycin are influenza The present invention was completed by showing antiviral activity in virus infected cell lines and confirming that salinomycin significantly increased the antiviral effect of OSV against influenza viruses and OSV resistant influenza viruses.

An object of the present invention is to provide an adjuvant for the prevention or treatment of viral diseases comprising ionophore as an active ingredient.

In order to achieve the above object, the present invention provides an agent for the prevention or treatment of viral diseases comprising ionophore as an active ingredient.

In addition, the present invention comprises a first component containing a pharmaceutically treatable amount of salinomycin as an active ingredient; And it provides a pharmaceutical kit for the prevention or treatment of viral diseases comprising a second component containing a pharmaceutically treatable amount of oseltamivir phosphate as an active ingredient.

Salinomycin according to the present invention exhibits antiviral activity by inhibiting endosomal acidification and M2 proton channel activity in influenza virus infected cell lines, and another polyether ionophores, monensin and nigerycin, also in influenza virus infected cell lines Antiviral activity. In addition, since salinomycin significantly increases the antiviral effect of OSV on influenza viruses and OSV resistant influenza viruses, polyether ionophores including salinomycin, monensin, and nigerin are used to prevent or treat viral diseases. It can be usefully used as a supplement for.

1 shows two influenza virus type A (PR8, A / Puerto Rico / 8/1934, H1N1; HK, A / Hong Kong / 8/1968, H3N2) and one type B (Lee) of salinomycin in MDCK cells. , B / Lee / 1940) is a result graph showing the cytoprotective effect against infection.
Figure 2 Expression of viral proteins (NP, nucleoprotein; HA, hemagglutinin; M, matrix protein) of salinomycin and oseltamivir carboxylate (OSV-C) in PR8 infected MDCK cells It is a result figure which showed the inhibitory effect.
Figure 3 is a graph showing the effect of reducing the number of virus plaques of salinomycin and oseltamivir carboxylate (OSV-C) in PRCK-infected MDCK cells.
Figure 4 is a graph showing the effect of reducing the number of virus plaques according to the treatment time of salinomycin or EGCG (epigallocatechin gallate) in PR8 infected MDCK cells (A, virus infection and drug treatment time; B, virus plaque count measurement results ).
5 is a confocal fluorescence microscope showing the result of staining viral nucleoprotein (NP, green) and cell nucleus (blue) after treatment with salinomycin, EGCG (epigallocatechin gallate) or ribavirin (RBV) in PR8-infected MDCK cells It is a photograph.
FIG. 6 is a confocal fluorescence micrograph showing the results of staining with acridine orange after treatment with salinomycin, batillomycin A1 or chloroquine in MDCK cells (red, low pH signal).
7 shows after treatment with salinomycin in PR549-infected A549 cells, early endosome antigen 1 (green), LAMP1 (lysosomal-associated membrane protein 1, green), viral nucleoprotein (NP, red) and cell nucleus (blue). ) Is a confocal fluorescence micrograph showing the result of staining (A, NP distribution distribution in EEA1; B, NP distribution in LAMP1 results).
FIG. 8 shows the results of dynamic light scattering analysis of empty VLPs (virus like particles) (A), PR8M2-S VLPs (B), and PR8M2-R VLPs (C).
9 shows microchip gel electrophoresis analysis (A) and empty VLPs, PR8M2-S VLPs, PR8M2-R VLPs and LeeM2 of empty VLPs (virus like particles), PR8M2-S VLPs and PR8M2-R VLPs. Western blot analysis (B) results of VLPs (p30 (CA), MVL Gag-derived p30 capsid).
10 is a graph showing the results of measuring M2 proton channel activity after treatment with amantadine (AMT) or salinomycin in each VLPs (A, empty VLPs (virus like particles); B, FCCP VLPs; C, PR8M2) -S VLPs; D, PR8M2-R VLPs; E, PR8M2-S VLPs and PR8M2-R VLPs; and F, LeeM2 VLPs)
FIG. 11 is a micrograph showing the results of staining with a gizo after treatment with salinomycin or anti-HA (hemagglutinin) antibody to Vero E6 cells infected with PR8.
FIG. 12 shows antiviral synergistic effect against recombinant PR8 virus (C and D) induced wild type PR8 (A and B) and H275Y mutations according to the mixing ratio of salinomycin and oseltamivir carboxylate (OSV-C). It is a graph of the measurement result.
13 is a result graph showing the antiviral effect of the combination of salinomycin, oseltamivir phosphate (OSV-P) or salinomycin with oseltamivir phosphate in an influenza virus A infected mouse model (A, drug administration) Constant; B, mouse weight change; and C, mouse survival rate).
14 is a graph showing the antiviral effect of oseltamivir phosphate (OSV-P) or a combination of salinomycin and oseltamivir phosphate in OSV resistant influenza virus A infected mouse model (A, mouse weight change; And B, survival rate of mice).

Hereinafter, the present invention will be described in detail.

The present invention provides an agent for the prevention or treatment of viral diseases comprising ionophore as an active ingredient.

The ionophore may be a polyether ionophore, and specifically, salinomycin, monensin, nigerin, pyrithione, hinikitiol, A23187 or ionomycinyl Can be.

The salinomycin may be a compound represented by Formula 1:

[Formula 1]

.

.

The adjuvant may be to enhance antiviral activity against RNA viruses, and specifically, influenza virus, Tamiflu resistant influenza virus, avian influenza virus, adenovirus, adenovirus, rhinovirus, hepatitis A virus virus, hepatitis C virus, polio virus, measles virus, Ebola virus, Coxsackie virus, yellow fever virus, Dengue Fever virus, lasa virus, lymphocytic choriomeningitis virus, Junin virus, machuppo virus, guanaito virus, Hantavirus, Rift Valley Fever virus, La Crosse v irus, California encephalitis virus, Crimea-Congo virus, Marburg virus, Japanese encephalitis virus, severe acute respiratory syndrome (SARS) virus (severe acute) group consisting of respiratory syndrome virus, parainfluenza virus, respiratory syncytial virus, Punta Toro virus, Takacaribe virus and Pichinde virus It may be to enhance the antiviral activity against any one or more viruses selected from. More specifically, it may be to enhance antiviral activity against influenza virus type A or B.

The adjuvant is selected from the group consisting of amantadine, rimantadine, favipiravir, baloxabir marboxil, oseltamivir phosphate and zanamivir. It may be to enhance the activity of one or more antiviral agents.

In a specific embodiment of the present invention, the inventors derived salinomycin through screening antiviral activity against influenza virus, and salinomycin suppressed viral component protein expression and reduced virus plaque count in influenza virus infected cell lines. Was confirmed (see FIGS. 1 to 4).

In addition to salinomycin, other polyether ionopores, monensin and nigerycin, also showed antiviral activity in influenza virus infected cell lines (see Table 6), with salinomycin endosomal acidification and M2 protons. It was confirmed that inhibiting channel activity inhibits migration of viral proteins into the cell nucleus (see FIGS. 5-11).

Salinomycin has also been shown in cell and mouse models to significantly increase the antiviral effect of OSV on influenza viruses and OSV resistant influenza viruses (see FIGS. 12-14). Therefore, polyether ionophores including salinomycin, monensin and nigerin can be usefully used as an adjuvant for the prevention or treatment of viral diseases.

The adjuvant according to the present invention may include 10 to 95% by weight of ionophore as an active ingredient based on the total weight of the composition. In addition, the adjuvant of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the active ingredient.

Adjuvants of the present invention may include carriers, diluents, excipients or mixtures thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers can be used as long as they are suitable for delivery of the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added as necessary.

When formulating the adjuvant, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used may be added.

Adjuvants of the present invention may be formulated as oral or parenteral preparations. Oral formulations may include solid and liquid formulations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and such solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or mixtures thereof. In addition, the solid preparation may include a lubricant, for example magnesium styrate, talc and the like. On the other hand, the liquid formulation may be a suspension, a liquid solution, an emulsion or a syrup. At this time, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, preservatives and the like.

The parenteral preparations may include injections, suppositories, respiratory inhalation powders, spray aerosols, powders and creams, and the like. The injection may include a sterile aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. In this case, as the non-aqueous solvent or the suspension solvent, vegetable oil such as propylene glycol, polyethylene glycol, olive oil, or injectable ester such as ethyl oleate may be used.

Adjuvants of the present invention can be administered orally or parenterally according to the desired method. Parenteral administration can include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection modes.

The adjuvant may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, the drug being used simultaneously, and the like. However, for the desired effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration can be from one to several times a day.

In addition, the present invention comprises a first component containing a pharmaceutically treatable amount of salinomycin as an active ingredient; And it provides a pharmaceutical kit for the prevention or treatment of viral diseases comprising a second component containing a pharmaceutically treatable amount of oseltamivir phosphate as an active ingredient.

In the pharmaceutical kit of the present invention, the first component and the second component may be administered sequentially or simultaneously to prevent or treat a viral disease. When the first component and the second component are sequentially administered, the second component may be administered about 30 minutes to 180 minutes, 60 minutes to 120 minutes, 80 minutes to 100 minutes before or after administration of the first component.

Concomitant administration of the first component and the second component included in the pharmaceutical kit can increase antiviral activity against viral diseases.

The pharmaceutical kit can be administered to a mammal, specifically a human. In addition, it may be administered to birds such as poultry, and specifically may be administered to chicken, duck, goose, turkey, quail, pheasant and the like.

The viral disease may be a disease caused by an RNA virus infection, and specifically, influenza virus, Tamiflu-resistant influenza virus, avian influenza virus, adenovirus, adenovirus, rhinovirus, hepatitis A virus , Hepatitis C virus, polio virus, measles virus, Ebola virus, Coxsackie virus, yellow fever virus, dengue virus Dengue Fever virus, lassa virus, lymphocytic choriomeningitis virus, Junin virus, machuppo virus, guanaito virus, hantavirus (hantavirus), Rift Valley Fever virus, La Crosse virus, California Encephalitis virus (California encephalitis virus), Crimea-Congo virus, Marburg virus, Japanese encephalitis virus, severe acute respiratory syndrome virus (SARS) virus , Parainfluenza virus, respiratory syncytial virus, Punta Toro virus, Takacaribe virus and Picinde virus The disease may be caused by one or more viral infections. More specifically, it may be a disease caused by influenza virus type A or B infection.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1. Preparation of Viruses

Influenza virus type A (A / Puerto Rico / 8/1934, PR8, H1N1; A / Hong Kong / 8/1968, HK, H3N2) and type B (B / Lee / 1940, Lee) Was purchased and mouse-adapted PR8 (maPR8) was obtained from Chung-Ang University HJ Kim. PR8, HK and maPR8 were amplified by infecting 10-day-old embryonated SPF chicken eggs for 3 days at 37 ° C. Lee was amplified by infecting MDCK cells at 35 ° C. for 3 days in serum-free MEM (minimum essential medium, Hyclone, USA) medium containing 2 μg / mL TPCK-trypsin (tosyl phenylalanyl chloromethyl ketone-trypsin). Virus was obtained by centrifugation of allantoic fluid or cell culture medium at 1,300 xg for 10 minutes and stored at -70 ° C.

Experimental Example 1. Derivation of salinomycin through screening antiviral activity against influenza virus

To elicit candidates with antiviral activity against influenza viruses, the antiviral effects of influenza virus type A (PR8 and HK) and type B (Lee) of 2,000 compounds were analyzed by MTT (3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltertrazolium) assay was used.

Specifically, MDCK (Madin-Darby canin kidney) cells (American Type Culture Collextion (ATCC), USA) were incubated in 96 × well plates at 3 × 10 ⁴ / well and influenza virus PR8, HK or Lee with a MOI of 0.001 (multiplicity of infection, inoculated virus amount / cell count) at 35 ° C. for 1 hour. After washing with phosphate-buffered saline (PBS), each of 2,000 compounds diluted to a final concentration of 20 μM was treated in MEM containing 2 μg / mL of TPCK-trypsin. After culturing for 3 days, the cell viability was measured by adding 2.5 mg / mL of MTT, and cell viability was calculated as a ratio of viability of normal cells not infected with the virus. Nine compounds with cell viability of 80% or more were treated by concentration, and antiviral effects were measured in the same manner as above. 50% cytotoxic concentration (CC ₅₀ ) and 50% effect concentration (EC ₅₀ ) were calculated with GraphPad Prism 6 software (GraphPad, USA).

Antiviral Effects of Atobacuon, Evans Blue and Salinomycin Against Influenza Viruses compound CC ₅₀ ^a for MDCK cells
(μM) EC ₅₀ ^b (μM) for influenza virus (index ^c ) PR8 ^d HK ^e Lee ^f Atobacuon > 100.0 2.4 ± 0.2 (> 41.7) 2.1 ± 0.1 (> 47.6) 2.0 ± 0.1 (> 50.0) Evans Blue > 100.0 10.6 ± 1.9 (> 9.4) 5.4 ± 0.5 (> 18.5) 1.5 ± 0.2 (> 66.7) Salinomycin 35.6 0.7 ± 0.1 (48.3) 0.4 ± 0.1 (84.5) 0.8 ± 0.1 (42.3) Amantadine ^* > 100.0 > 100.0 (nd ⁱ ) 0.9 ± 0.2
(> 111.1) > 100.0 (n.d.) Ribavirin ^{g *} > 100.0 18.8 ± 1.8 (> 5.3) 12.8 ± 3.2 (> 7.8) 13.5 ± 0.5 (> 7.4) OSV-C ^{h *} > 100.0 0.02 ± 0.01
(> 5,000) <0.005 (> 20,000) 0.13 ± 0.03
(> 769)

^a concentration at which cell viability is reduced by 50%

^b the concentration required to recover the survival rate of cells infected with influenza virus by 50%

^c ratio of CC ₅₀ / EC ₅₀ (selectivity index)

^d A / Puerto Rico / 8/1934

^e A / Hong Kong / 8/1968

^f B / Lee / 1940

^g ribavirin

^h oseltamivir carboxylate

ⁱ not detected

^* Positive control

The screening resulted in three compounds showing concentration-dependent antiviral activity (atovaquone, Evans blue and salinomycin) among 9 compounds with cell viability of 80% or higher. Among the three compounds, salinomycin (Sigma-Aldrich, USA), EC ₅₀ was 0.4 to 0.8 μM and CC ₅₀ was 35.6 μM for all the tested viruses, showing the strongest antiviral activity (Table 1 and 1). Therefore, salinomycin was derived as an antiviral candidate.

Experimental Example 2 Confirmation of Wide Range Antiviral Activity of Salinomycin Against Various Influenza Viruses

In order to confirm whether the antiviral effect of salinomycin identified in Experimental Example 1 also appeared for a variety of influenza viruses, the antiviral effect against 13 types of influenza viruses A and 4 types B was investigated by MTT assay.

Specifically, a total of 17 influenza viruses of Table 2 were obtained from the ATCC, Korea Disease Control Center and Agriculture, Forestry and Livestock Quarantine Headquarters. Influenza virus was used in the same manner as described in Experiment 1 except that the viruses of Table 2 were used and the compounds to be measured for antiviral activity were salinomycin, amantadine, ribavirin and OSV-C. Antiviral activity was measured.

number Classification Virus One Influenza A / H1N1 Virus A / Brisbane / 59/2007 2 A / California / 7/2009 3 A / Korea / 01/2009 4 rgA / Korea / 09 / 2009Δ53-60 (H275Y) ^a 5 A / Korea / 2785/2009 ^b 6 rgA / Puerto Rico / 8/1934 (H275Y) ^c 7 A / Taiwan / 1/1986 8 Influenza A / H3N2 Virus A / Brisbane / 10/2007 9 A / Perth / 16/2009 10 A / Seoul / 11/1988 11 A / Victoria / 361 / 2011-like 12 Avian Influenza Virus A / duck / Korea / GJ79 / 2007 (H3N8) 13 A / chicken / Korea / MS96 / 1996 (H9N2) 14 Influenza B Virus B / Florida / 4/2006 15 B / Panama / 45/1990 16 B / Taiwan / 2/1962 17 B / Wisconsin / 1 / 2010-like

^a reverse genetics such that there is a truncation between the 53rd and 60th amino acids and ^a point putation of H275Y in the neuraminidase of rgA / Korea / 09 / 2009Δ53-60 Generated, mouse-adapted, OSV-resistant strains

^b. OSV-resistant strain isolated from Korean patients

OSV- the resistant strain generated by the reverse genetics to have a point mutation in the ^c H275Y The lamina of claim PR8 New

compound EC ₅₀ ^a (μM) for influenza virus (index ^b ) A / Brisbane / 59/2007 A / California / 7 /
2009 A / Korea / 01/2009 rgA / Korea / 09 / 2009Δ53-60
(H275Y) A / Korea / 2785/2009 rgA / Puerto Rico / 8 /
1934
(H275Y) A / Taiwan / 1/1986 Salinomycin 1.4 ± 0.3
(41.6) 1.9 ± 0.2
(30.3) 1.2 ± 0.0
(48.8) 1.1 ± 0.2
(53.4) 1.7 ± 0.0
(34.0) 0.7 ± 0.1
(86.3) 3.5 ± 0.1
(16.3) Amantadine 0.1 ± 0.0
(> 90.0) > 100.0
(ND) 100.0
(ND) 100.0
(ND) 100.0
(ND) 100.0
(ND) 10.5 ± 2.6
(> 9.6) Ribavirin 28.2 ± 6.9
(> 3.6) 19.3 ± 0.3
(526.3) 13.1 ± 2.0
(> 7.6) 6.5 ± 2.5
(> 15.5) 38.7 ± 4.5
(> 2.6) 24.6 ± 0.2
(> 4.1) 50.2 ± 1.9
(> 2.0) OSV-C 0.14 ± 0.02
(> 740.7) 0.19 ± 0.06
(> 526.3) <0.005
(> 20,000) 4.45 ± 1.97
(> 22.5) 2.95 ± 0.55
(33.9) 1.41 ± 0.03
(> 70.9) 0.99 ± 0.21
(101.5)

^a concentration necessary to restore the survival rate of cells infected with influenza virus by up to 50%

^b Selectivity index of CC ₅₀ / EC ₅₀ , CC ₅₀ values are calculated from the values shown in Table 1

compound EC ₅₀ ^a (μM) against influenza A / H3N2 virus
(Selection index ^b ) EC ₅₀ ^a (μM) against avian influenza virus
(Selection index ^b ) A / Brisbane / 10/2007 A / Perth / 16/2009 A / Seoul / 11/1988 A / Victoria / 361 / 2011-like A / duck / Korea / GJ79 / 2007
(H3N8) A / chicken / Korea / MS96 / 1996
(H9N2) Salinomycin 2.5 ± 0.1
(22.4) 2.6 ± 0.1
(21.6) 1.3 ± 0.4
(43.2) 1.7 ± 0.3
(34.0) 1.6 ± 0.5
(36.2) 4.2 ± 0.1
(13.5) Amantadine > 100.0
(ND) > 100.0
(ND) 0.1 ± 0.0
(> 1,000) > 100.0
(ND) 0.5 ± 0.1
(> 200.0) 0.6 ± 0.3
(> 181.8) Ribavirin 6.8 ± 0.8
(> 14.7) 53.5 ± 1.0
(> 1.9) 12.5 ± 3.5
(> 8.0) 17.3 ± 3.0
(> 5.8) 18.5 ± 0.7
(> 5.4) 76.8 ± 4.7
(> 1.3) OSV-C 0.28 ± 0.10
(> 357.1) 0.02 ± 0.01
(> 5,714) 0.01 ± 0.00
(10,000) <0.005
(> 20,000) 0.01 ± 0.00
(> 13,333) 0.27 ± 0.01
(> 377.4)

^a concentration necessary to restore the survival rate of cells infected with influenza virus by up to 50%

^b Selectivity index of CC ₅₀ / EC ₅₀ , CC ₅₀ values are calculated from the values shown in Table 1

compound EC ₅₀ ^a (μM) for influenza B virus (index ^b ) B / Florida / 4/2006 B / Panama / 45/1990 B / Taiwan / 2/1962 B / Wisconsin / 1 /
2010-like Salinomycin 0.4 ± 0.1
(160.3) 0.9 ± 0.1
(29.5) 2.5 ± 0.1
(22.9) 3.8 ± 0.4
(15.0) Amantadine > 100.0
(ND) > 100.0
(ND) > 100.0
(ND) > 100.0
(ND) Ribavirin 6.7 ± 2.7
(> 15.0) 14.6 ± 2.2
(> 6.8) 16.3 ± 1.1
(> 6.2) 8.3 ± 0.1
(> 12.0) OSV-C 0.11 ± 0.04
(> 909.1) 0.03 ± 0.01
(> 4,000) 0.28 ± 0.04
(> 357.1) 0.19 ± 0.01
(> 540.5)

^a concentration necessary to restore the survival rate of cells infected with influenza virus by up to 50%

^b Selectivity index of CC ₅₀ / EC ₅₀ , CC ₅₀ values are calculated from the values shown in Table 1

As a result, salinomycin showed antiviral effects against a total of 17 influenza viruses, including amantadine or OSV resistant influenza viruses, and EC ₅₀ was 0.4 to 4.3 μM (Tables 3 to 5).

Experimental Example 3. Confirmation of antiviral effect of polyether ionophore

The antiviral effects of monensin and nigerin, compounds belonging to polyether ionophores other than salinomycin, were investigated by MTT assay.

Specifically, except for the treatment of monensin (Cat. #. M5273, Sigma-Aldrich) and Nigerycin (Cat. #. N7143, Sigma-Aldrich), the anti-virus in the same manner as described in Experiment 1 The effect was measured.

compound CC ₅₀ ^a (μM) on MDCK cells EC ₅₀ ^b (μM) for influenza virus (index ^c ) PR8 ^d HK ^e Lee ^f Monensin > 100.0 2.0 ± 1.1
(> 50.0) 1.6 ± 0.9
(> 64.5) 29.0 ± 4.3
(> 3.4) Nigerine 18.8 0.04 ± 0.02
(470.0) <0.005
(> 3,760) 0.37 ± 0.03
(51.5)

^a concentration at which cell viability is reduced by 50%

^b the concentration required to recover the survival rate of cells infected with influenza virus by 50%

^c ratio of CC ₅₀ / EC ₅₀ (selectivity index)

^d A / Puerto Rico / 8/1934

^e A / Hong Kong / 8/1968

^f B / Lee / 1940

As a result, monensin and nigerycin also showed excellent antiviral effects against influenza virus, and EC ₅₀ of monensin was 2.0 to 29.0 μM, CC ₅₀ of 100 μM or more, and NI ₅₀ of 0.37 μM at 0.005 μM or less. , CC ₅₀ was found to be 18.8 μM (Table 6). This suggests that compounds belonging to the polyether ionophores, including salinomycin, have overall antiviral efficacy.

Experimental Example 4. Confirmation of antiviral effect of salinomycin in influenza A virus infected cell line

In order to verify the antiviral effect of salinomycin derived in Experimental Example 1, the following experiment was performed on MDCK cells infected with PR8 with MOI 0.001.

4-1. Confirmation of Salinomycin Inhibitory Effect on Viral Constituent Protein Expression

In order to confirm the effect of inhibiting viral protein expression of salinomycin, Western blot analysis was performed. MDCK cells infected with PR8 were treated with salinomycin (0.1, 1.0 or 10.0 μM) or 0.1 μM of OSV-C (prodrug of OSV-P, US Biological, USA), and cells were obtained and lysed one day later. . Cell lysates were loaded onto 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel at 30 μg per well, followed by electrophoresis, and the separated proteins were transferred to PVDF (polyvinylidene difluoride) membranes. Since, anti-NP (Cat. # .11675-MM03, Sino Biological, China) anti-HA (Cat. #. 86001-RM01, Sino Biological), anti-M1 (Cat. #. Sc-57881, Santa Cruz Biotechnology , USA), anti-M2 (Cat. #. Sc-32238, Santa Cruz Biotechnology) or anti-β-actin (β-actin, Cat. #. A1987, Sigma-Aldrich) and horseradish peroxidase (HRP) Reacted with the bound secondary antibody. Subsequently, HRP conjugates were detected using SuperSignal West Pico chemiluminescent substrates (Pierce, USA), and blot images were analyzed by LAS-4000 image analysis equipment (Fujifilm, Japan).

As a result, salinomycin inhibited the expression of the viral proteins NP, HA, M1 and M2 in a concentration-dependent manner, and when treated with 10.0 μM of salinomycin, the treatment with OSV-C 0.1 μM was positive. (FIG. 2).

4-2. Confirmation of Salinomycin Virus Plaque Reduction

MDCK cells infected with PR8 were treated with salinomycin (0.1, 1.0 or 10.0 μM) or OSV-C (0.1 μM) and cell cultures were obtained 1 or 2 days later. To titrate infected virus particles, MDCK cells were cultured in 48-well plates and the following day, the virus infected cell cultures obtained were serially diluted from 10 ⁻¹ to 10 ⁻⁶ times and treated for 1 hour. . After washing the cells with PBS, cells monolayers were added with MEM medium containing 0.5% CMC (carboxymethylcellulose) and 2 μg / mL TPCK-trypsin as overlay medium for 3 days at 33 ° C. Incubated. Viral plaques were visualized by staining with crystal violet. Thereafter, the number of plaques visually observed in one well of the plate was measured.

As a result, salinomycin treatment reduced the number of infectious influenza virus particles in cell cultures on days 1 and 2 after infection, and the effect was dependent on the concentration of salinomycin. In addition, the effect of salinomycin 1.0 to 10.0 μM was shown to be similar to the effect of OSV-C 0.1 μM positive control (Fig. 3).

Example 2 Preparation of PR8M2 Virus-like Particles (VLPs)

2-1. Preparation of Plasmids

Synthesis of PR8M2 cDNA (GenBank accession No. EF467824) encoding two amantadine resistant amino acid sequences A27 and N31 (Bioneer Corp. South Korea), pcDNA 3.1 / myc -His (-) A (Invitrogen, And cloned into the NheI and BamHI sites of USA) and named pcDNA-PR8M2-R. PcDNA-PR8M2-S expressing amantadine sensitive PR8M2 having amino acid sequences of V27 and S31 instead of the amino acid sequence was prepared in the same manner as above. In addition, LeeM2 cDNA (GenBank accession No. DQ79290) was synthesized (Bioneer Corp.) to prepare a plasmid in the same manner as described above. PCgp, a retrovirus packaging plasmid expressing murine leukemia virus (MLV) Gag-Pol, was obtained from Paula M. Cannon of the University of Southern California.

2-2. Manufacturing of VLPs

VLPs comprising PR8M2-S or PR8M2-R with MLV Gag were prepared. Specifically, HEK293T cells (human embryonic kidney (ATCC) is incubated in a 150 mm cell culture dish and co-transformation of plasmid pCgp 15 μg and pcDNA-PR8M2-S, pcDNA-PR8M2-R or pcDNA-LeeM2 45 μg. co-transfection). Empty VLPs (null VLPs) as a control was prepared by transforming the cells with only pCgp. After 2 days of transformation, a total of 80 mL of cell culture was centrifuged for 2 h at 16,500 × g at 4 ° C. with 20% sucrose cushion (SW-32Ti rotor, Beckmann Instruments, USA). The pellet was resuspended in 1 mL of PBS, followed by ultracentrifugation at 82,000 × g for 1 hour at 4 ° C. (SW-60Ti rotor, Beckmann Instruments). The pellet was resuspended in 150 μl of 10 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer (pH 7.5) and then used for subsequent analysis.

Experimental Example 5. Confirmation of antiviral mechanism of salinomycin in influenza virus infected cell line

In order to confirm the mechanism of action of the antiviral effect on the influenza virus of salinomycin identified in Experimental Examples 1 to 4, the following experiment was performed.

5-1. Comparison of antiviral activity according to treatment time of salinomycin

To investigate which stage of the virus life cycle acts, the effect of reducing virus plaques on the treatment time of salinomycin in PR8-infected MDCK cells was compared.

Specifically, MDCK cells were cultured in 48-well plates, then infected with PR8 at 4 ° C. for 1 hour, washed with PBS, and further cultured at 35 ° C. for 4 hours. 10 μM salinomycin or 30 μM EGCG (epigallocatechin gallate, Sigma-Aldrich) were treated concurrently with virus infection or 1, 2 or 4 hours after infection (FIG. 4A). Five hours after the virus infection, the cell monolayers were washed with PBS, and the medium plaques were added to quantify the virus plaques in the same manner as described in Experimental Example 2-2. The number of virus plaques in each drug treatment group was expressed as a percentage of the number of plaques in the non-drug control group.

As a result, when the drug was treated for 1 hour to infect the virus at 4 ° C, EGCG, an entry blocker, inhibited viral infection by 92.3%, while salinomycin had no antiviral effect. However, treatment with salinomycin until the beginning of the life cycle of the virus at 35 ° C showed antiviral effects and was 0-5 hours, 1-5 hours, 2-5 hours or 4-5 hours after the start of infection. The virus plaque reduction when treated to up to 83.2%, 79.5%, 60.8% or 53.4%, respectively (Fig. 4B). This means that salinomycin does not act at the stage of attachment of the virus to its host cells, but at an early stage of the virus's life cycle.

5-2. Confirmation of Salinomycin Inhibition of Viral Protein Transfer into the Nucleus

The viral ribonucleoprotein (vRNP) completely migrates into the nucleus 4 hours after infection. In order to investigate the antiviral mechanisms acting at the early stage of the virus life cycle of salinomycin, confocal fluorescence microscope was used to examine the intracellular distribution of NP, a representative viral protein constituting vRNP 4 hours after virus infection. Take a picture to observe.

Specifically, MDCK cells were infected with 2.5 MOI of PR8 in 4-well chamber slides, treated with 10 μM salinomycin, 100 μM EGCG or 100 μM ribavirin, and incubated at 37 ° C. for 4 hours. The cells were fixed with 4% paraformaldehyde and increased permeability with 0.1% triton X-100. After blocking with 1% Bovine serum albumin and 10% normal goat serum dissolved in PBS, slides were washed with anti-influenza A NP antibodies (Cat. #. Sc-80481, Santa Cruz). Biotechnology) at 4 ° C. overnight. Subsequently, the mixture was reacted at room temperature with an Alexa Fluor 488 conjugated anti-mouse antibody (Invitrogen, USA) for 1 hour, and then mounted with a VAPI containing shield mounting medium containing DAPI (Vector Laboratories, USA). Counterstaining. Stained cells were observed by photographing with a Zeiss LSM 700 confocal microscope.

As a result, the salinomycin treated group expressed NP only in the cytoplasm, the EGCG treated group which was a positive control did not express NP at all, and the ribavirin treated group expressed weakly NP in the nucleus (FIG. 5). This is because EGCG inhibits the cellular influx of viruses, ribavirin only inhibits the synthesis of viral RNA, does not participate in vRNP migration, and salinomycin inhibits the migration of vRNP into the nucleus, thus acting differently from these drugs. Suggest mechanism.

5-3. Confirmation of Salinomycin-induced Acidification Inhibition of Endosomes-Lysosomes

Salinomycin is a monovalent cation ionophore and is expected to inhibit the formation of proton gradients of vacuolar ATPase (V-ATPase). Therefore, MDCK cells were stained with acridine orange to determine whether the electrically neutral ionophore salinomycin could neutralize acidic organs such as lysosomes, endosomes or Golgi in the cells. Confocal fluorescence microscopy was photographed and observed.

Specifically, MDCK cells were cultured at 37 ° C. by dispensing 8 × 10 ⁴ / well into 35 mm glass bottom dishes (glass-bottom dishes, Greiner Bio-One, Germany). The next day, 100 μM of salinomycin, 100 nM of Bafilomycin A1 (bafilomycin A1, V-ATPase inhibitor, Sigma-Aldrich) or 100 μM of chloroquine (pH neutralizer, Sigma-Aldrich) were treated for 1 hour. The acridine orange was then added at a final concentration of 4 μg / mL for 10 minutes, followed by an excitation wavelength of 488 mm, an emission wavelength of 493/560 nm (green) and 590/720 nm ( Cells were observed under a confocal fluorescence microscope.

As a result, the control group showed red fluorescence showing low pH, whereas the salinomycin treated group did not show red fluorescence similarly to the positive control bacillomycin A1 group, and the chloroquine treated group showed yellow / green fluorescence. Appeared (FIG. 6). This means that salinomycin increases the pH of organelles in the cytoplasm, suggesting that salinomycin can act as a competitive inhibitor of cell proton channels, resulting in inhibition of acidification of endosome-lysosomes.

5-4. Inhibition of vRNP Release in Endosomes by Salinomycin

NP of A549 cells (human lung adenocarcinoma, ATCC) infected with PR8 to confirm whether vRNP release from the endosome is inhibited by the endosome-lysosomal acidification inhibitory effect of salinomycin confirmed in Experimental Example 3-3 Staining was performed by taking a picture with a confocal fluorescence microscope.

Specifically, A549 cells were cultured by dispensing 8 × 10 ⁴ cells / well in 4-well chamber slides, and then PR8 was infected with 10 MOI at 4 ° C. for 30 minutes. After washing with PBS, salinomycin was dissolved in MEM containing protein synthesis inhibitor CHX (cycloheximide, Sigma-Aldrich) at 10 μg / mL, and the cells were treated with 10 μM at 37 ° C. for 8 hours. The cell lysate was either antibody to NP and early endosome antigen 1 (early endosomal marker) (Cat. #. Sc-33585, Santa Cruz Biotechnology) or NP and LAMP1 (lysosomal-associated membrane protein 1, late endosomal marker ) (Cat. #. 9691, Cell Signaling Technology). NP was detected with an anti-mouse antibody conjugated with Alexa Fluor 633 (Invitrogen, USA) (red), and EEA1 and LAMP1 were detected with an anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Nuclei were counterstained with DAPI (blue).

As a result, in the control group, all of the NP migrated into the nucleus, whereas in the salinomycin treated group, NP appeared in the cytoplasm, and NP was present in the early or late endosomes around the nucleus (FIG. 7). This means that the replication of the virus is inhibited by CHX, so that the NP in the cytoplasm is not the NP of the replicated virus, but the NP of the virus that has entered the cell. Therefore, this result means that the salinomycin treatment prevents vRNP from being released from the endosome and therefore cannot migrate into the nucleus.

5-5. Confirmation of Salinomycin Inhibitory Effect on the Proton Channel M2 Activity

According to the results confirmed in Experimental Example 3-4, using the VLPs prepared in Example 1 to investigate the effect of salinomycin on the function of the viral protein M2 involved in the release of vRNP from the endosomes The same experiment was performed.

5-5-1. Characterization of VLPs (1)

In order to confirm homogeneity of the VLPs prepared in Example 1, dynamic light scattering analysis was performed (Zetaxizer Nano series, Malvern Instruments).

As a result, the average diameters of the blank VLPs, PR8M2-S VLPs, and PR8M2-R VLPs were 268.3 ± 8.3 nm, 256.8 ± 2.5 nm, and 238.3 ± 5.9 nm, respectively. It was confirmed (FIG. 8).

5-5-2. Characterization of VLPs (2)

In order to confirm the expression of MVL Gag-derived p30 capsid (CA) protein and M2 protein in the VLPs prepared in Example 1, microchip gel electrophoresis and Western blot analysis were performed. Was performed.

Specifically, 2 μl of the VLPs prepared in Example 1 was loaded onto a microchip and electrophoresed and analyzed by an Agilent 2200 TapeStation system (including P200 Screen Tape, Agilent Technologies, USA). In addition, the VLPs prepared in Example 1 were loaded onto a 12% SDS-PAGE gel at 2 μl / well and electrophoresed. The CA protein was reacted with an anti-Gag primary antibody (Cat. # .R187, ATCC) and then detected with an HRP conjugated anti-rat secondary antibody, and the M2 protein fused with myc-His tag (taq) Anti-6 × His tack antibody (Cat. #., Ab8184, Abcam) and HRP conjugated anti-mouse secondary antibody were detected.

As a result, CA protein was expressed in each VLPs in the same manner, M2 protein was expressed in PR8M2-S, PR8M2-R and LeeM2 VLPs except the empty VLPs (Fig. 9).

5-5-3. Confirmation of Salinomycin Inhibitory Effect on Virus Proton Channel M2 Activity

Each VLPs prepared in Example 1 was prepared by dissolving in 10 mM HEPES (pH 7.0) and 150 mM sodium chloride buffer containing 1% FMP-Blue dye. As a positive control, FCCP VLPs were prepared by mixing empty VLPs with 5 μM of FCCP (Sigma-Aldrich), an ionophore that transports protons from the membrane. To measure proton channel activity, each of the mixtures was placed in a 96-well black plate with a protein amount of 1 μg and treated with 100 μM of amantadine or 100 μM of salinomycin at room temperature for 1 hour. 20 μl of 150 mM MES (2- (N-morpholino) ethanesulfonic acid, pH 4.5, Sigma-Aldrich) was added to expose each VLPs to acidic conditions, followed by fluorescence at SpectraMax M3 micro for 6 minutes at a total of 5 minutes. Plate reader (Molecular Devices, USA) measured at excitation wavelength 530 nm and emission wavelength 565 nm.

As a result, in acidic conditions, empty VLPs were found to slowly move protons in the membrane, with no effect from drug treatment (FIG. 10A). VLPs prepared by adding FCCP as a positive control showed a sharp increase in the activity of proton channels at 36 seconds after exposure to acidic conditions, an equilibrium decrease, and no effect of amantadine treatment, but treated with salinomycin. The abrupt increase in proton channel activity of the FCCP VLPs was alleviated (FIG. 10B). This means that when the electrically neutral ionophore and the electrogenic ionophore are present at the same time, the action of the electrically neutral ionophore is more prevalent.

In the PR8M2-S VLPs and PR8M2-R VLPs, the proton channel activity increased rapidly after exposure to acidic conditions. The increase in the proton channel activity of PR8M2-S VLPs was alleviated by amantadine treatment, but the proton channel activity of PR8M2-R VLPs increased. Was not affected by amantadine treatment. In addition, treatment with salinomycin mitigates increased proton channel activity of both PR8M2-S VLPs and PR8M2-R VLPs (FIGS. 10C and 10D), and the inhibitory effect of salinomycin on each of these VLPs was in acidic conditions. Five minutes after exposure were identified as 54% and 72%, respectively (FIG. 10E). This means that salinomycin can inhibit M2 proton channel activity whether or not it is resistant to amantadine.

In LeeM2 VLPs, proton channel activity was rapidly increased after exposure to acidic conditions, but there was no effect of amantadine treatment, but the increase in proton channel activity was alleviated by salinomycin treatment (FIG. 10F).

In conclusion, it was confirmed that salinomycin could inhibit the proton channel activity of VLPs against influenza viruses A and B.

5-6. Identifying the Effect of Salinomycin on Membrane Fusion Activity

To confirm whether salinomycin acts on HA2, another target involved in the release of vRNP from the endosome, membrane fusion activity of PR8-infected Vero E6 cells (African green monkey kidney epithelial cells (ATCC)) was measured.

Specifically, Vero E6 cells were incubated in 12-well plates at 3 × 10 ⁵ cells / mL, and then PR8 was infected with 0.5 MOI at 37 ° C. without TPCK-trypsin treatment. The next day, TPCK-trypsin was dissolved in DMEM in virus infected cells and treated at 37 ° C. for 5 minutes at a concentration of 5 μg / mL, followed by 2 μM of salinomycin or 0.5 μg / mL of anti-HA2 antibody (Sino Biological). Dissolved in DMEM, treated and incubated for 15 minutes. Each drug was washed with PBS (PBS-CM) containing 1 mM magnesium chloride (MgCl ₂ ) and 0.1 mM calcium chloride (CaCl ₂ ) and then dissolved in acidic (pH 5.6) or neutral (pH 7.0) PBS-CM. Incubated for 15 minutes further treatment, the acidity was adjusted with citric acid (citric acid). After the intercellular fusion for 3 hours, the cells were fixed with 96% ethanol, stained with Kimja (Giemsa, Sigma-Aldrich) and photographed under a microscope.

As a result, large fusion cells with multiple nuclei were observed by influenza virus infection under acidic conditions, and intercellular fusion was suppressed in the anti-HA antibody-administered group, which was a positive control, whereas salinomycin treatment affected intercellular fusion. Not given (FIG. 11). This means that HA2, which functions as an intercellular fusion protein, is not a target of salinomycin.

Experimental Example 6. Confirmation of antiviral synergistic effect of salinomycin and OSV-C in influenza virus A infected cell line

In order to confirm the antiviral synergistic effects of salinomycin and OSV-C, isobologram analysis (Quinton L. Fivelman et al , Antimicrobial Agents and Chemotherapy, 2004: 4097-4102, 2004) was performed.

Specifically, the antiviral activity against the recombinant PR8 virus induced with the wild type PR8 of the salinomycin and OSV-C and the H275Y point mutation of the neuraminidase was measured in the same manner as described in Experimental Example 1 to determine the EC ₅₀ value. Calculated. The EC ₅₀ for wild-type PR8 virus of salinomycin and OSV-C was 1.81 and 0.07 μM, respectively, and the EC ₅₀ for recombinant PR8 virus was 2.37 and 3.52 μM, respectively. Thereafter, salinomycin and OSV-C were prepared by mixing in ratios of 5: 0, 4: 1, 3: 2, 2: 3, 1: 4, and 0: 5, and each mixture was serially diluted two-fold ( serial dilutions), and the concentration of each mixture was such that the EC ₅₀ value of each single compound was in the middle of the concentration range. The EC ₅₀ values for the antiviral activity were measured in the same manner as in the case of each single compound using the samples of the concentrations of the six mixtures prepared by mixing ratio, and FIC ₅₀ (50% split inhibition concentration, fractional inhibitory) The sum of the values of concentraion 50) was calculated and plotted against the mixing ratio of each compound:

ΣFIC ₅₀ = (mixture of Reno in live azithromycin EC ₅₀₎ / (live Reno azithromycin alone in EC ₅₀₎ + (mixture of C in OSV-EC ₅₀₎ / (OSV-C alone in EC _50).

As a result, the average ∑FIC ₅₀ value for the wild-type PR8 virus of the mixture of salinomycin and OSV-C was 0.79, and the average ∑FIC ₅₀ value for the recombinant PR8 virus was 0.63. It was confirmed that an elevated antiviral effect was shown for the viruses (FIG. 12).

Experimental Example 7. Confirmation of antiviral effect by co-administration of salinomycin and OSV-P in influenza virus A infected mouse model

Influenza virus A infected mouse model to verify the antiviral effect of salinomycin at the cellular level confirmed in the above experimental examples in an animal model and to confirm the antiviral synergistic effect by the combined administration of salinomycin and OSV-P. Was used.

7-1. Influenza Virus A Infected Mouse Model Fabrication and Drug Dosing Schedule

Female BALB / c mice from 6 to 7 weeks of age were purchased from Orient Biotech (Korea) and sorted 5 per group to 5 times 50% mouse lethal doses (MLD ₅₀ ) into the nasal cavity (90 plaques per horse). MaPR8 was administered in plaque forming units (PFU). Antiviral drugs include OSV-P (0.1 or 10 mg / kg, Hanmi Pharmaceutical, Korea), Salinomycin (10 mg / kg) and a mixture of both drugs (OSV-P 0.1 mg / kg and Salinomycin 10 mg / kg) was prepared by dissolving in 0.5% CMC. The drug was orally administered 4 hours before virus infection and 4 hours after infection, and then orally twice a day for the next 5 days (FIG. 13A).

7-2. Confirmation of weight recovery effect of mice by co-administration of salinomycin and OSV-P

Changes in the weight of the mice were observed up to 14 days after the virus infection, and the subjects whose weight was reduced by more than 30% were euthanized according to the guidelines approved by the Animal Experimental Ethics Committee of the Korea Research Institute of Chemical Technology.

As a result of measuring the change in the weight of the mouse, the weight of the mouse continued to decrease after the virus infection, and decreased by 70% on the 8th day. The OSV-P high dose group showed a significant recovery from the negative control on the 3rd day of the infection. There was no weight recovery effect in the -P low dose group and salinomycin-treated group. The combination of OSV-P and salinomycin showed significant recovery compared to the negative control from day 4 of infection (FIG. 13B).

7-3. Confirmation of increased survival rate of mice by co-administration of salinomycin and OSV-P

Survival rates of mice are shown by plotting Kaplan-Meier survival curves using GraphPad Prism 6 software.

As a result of measuring the survival rate of the mice, the survival rate of the negative control group began to decrease from the 6th day of infection, and died on the 9th day of the infection. . On the other hand, the OSV-P high concentration group was maintained at 100% survival rate, the combined survival group of OSV-P and salinomycin was 80% final survival rate (Fig. 13C).

In conclusion, the salinomycin and OSV-P low concentration group did not show an antiviral effect, but the combination of salinomycin and OSV-P at the same concentration showed an excellent antiviral effect according to the synergistic effect. This suggests that salinomycin can be used as an adjuvant to lower the effective dose of antiviral agents such as OSV-P.

Experimental Example 8. Confirmation of antiviral effect by combination of salinomycin and OSV-P in mouse model of OSV-P resistant influenza virus A infection

In order to confirm whether the antiviral synergistic effect of the salinomycin and OSV-P combination administration confirmed in Experimental Example 8 is reproduced in the OSV-P resistant influenza virus A infected mouse model, the following experiment was performed.

8-1. OSV-P Resistant Influenza Virus A Infected Mouse Model Fabrication and Drug Dosing Schedule

Instead of maPR8 virus, RGA / Korea / 09 / 2009Δ53-60 virus, OSV-P resistant influenza virus A, was administered 5 times MLD ₅₀ (corresponding to 1.5 × 10 ⁵ PFU per horse), and the drug was administered OSV-P (10 mg / kg or 100 mg / kg), except with OSV-P (10 mg / kg or 100 mg / kg) + salinomycin (10 mg / kg), or salinomycin (10 mg / kg) A mouse model was prepared and the drug was administered in the same manner as described in Experimental Example 7-1.

8-2. Confirmation of mouse weight recovery effect by combined administration of salinomycin and OSV-P

Changes in the weight of the mice were observed up to 14 days after the virus infection, and the subjects whose weight was reduced by more than 30% were euthanized according to the guidelines approved by the Animal Experimental Ethics Committee of the Korea Research Institute of Chemical Research.

As a result of measuring the change in the weight of the mouse, the weight of the mouse continued to decrease after the virus infection, and decreased to 70% on the 8th day. There was no significant difference in the OSV-P low-dose group according to the combination with salinomycin. , OSV-P high concentration group showed a significant weight recovery effect by salinomycin combined administration compared to OSV-P alone administration (Fig. 14A).

8-3. Confirmation of increased survival rate of mice by co-administration of salinomycin and OSV-P

Survival rates of mice are shown by plotting Kaplan-Meier survival curves using GraphPad Prism 6 software.

As a result of measuring the survival rate of the mice, the negative control group began to decrease the survival rate from the 7th day of infection, and died on the 8th day of the infection, and the OSV-P high concentration alone or the OSV-P and salinomycin combination group received 100% survival rate. In the OSV-P low concentration alone group, the final survival rate was 60%, whereas the OSV-P low concentration and salinomycin combination group showed a 100% survival rate, confirming the effect of increasing the mouse survival by the salinomycin combination. (FIG. 14B).

In conclusion, OSV-P resistant influenza virus A infected mouse model, unlike the results of Experiment 7 above, OSV-P required a higher concentration to exhibit an antiviral effect, and this model was administered by salinomycin alone. Had no antiviral effect. However, compared to the administration of OSV-P alone, the combination of OSV-P and salinomycin was found to further increase the weight recovery effect and survival rate of the mouse. This suggests that salinomycin can be used as an adjuvant to lower the effective dose of antiviral agents such as OSV-P even against OSV-P resistant influenza virus A.

Claims (11)

Adjuvant for the prevention or treatment of viral diseases comprising ionophore as an active ingredient.
The adjuvant for preventing or treating a viral disease according to claim 1, wherein the ionophore is a polyether ionophore.
The method of claim 2, wherein the polyether ionophore is salinomycin, monensin, nigericin, pyrithione, hinikitiol, A23187 or ionoma. An adjuvant for the prevention or treatment of a viral disease, which is an isinomycin.
The adjuvant of claim 1, wherein the adjuvant enhances antiviral activity against an RNA virus.
The method of claim 4, wherein the RNA virus is influenza virus, Tamiflu resistant influenza virus, avian influenza virus, adenovirus (adenovirus), rhinovirus (rhinovirus), hepatitis A virus (hepatitis A virus, hepatitis C virus (hepatitis) C virus, polio virus, measles virus, Ebola virus, Coxsackie virus, yellow fever virus, Dengue Fever virus, Lhasa Virus, lymphocytic choriomeningitis virus, Junin virus, machuppo virus, guanaito virus, hantavirus, Rift valley fever Virus Valley Fever virus, La Crosse virus, California encephalitis virus, Crimea-Congo virus, Marburg virus, Japanese encephalitis virus, severe acute respiratory syndrome virus (SARS) virus, parainfluenza virus ), One or more selected from the group consisting of respiratory syncytial virus, Punta Toro virus, Takacaribe virus, and Pichinde virus. Prophylactic or therapeutic supplements.
The agent for preventing or treating a viral disease according to claim 5, wherein the influenza virus is influenza virus type A or B.
The method of claim 1, wherein the adjuvant is amantadine, rimantadine, favipiravir, baloxabir marboxil, oseltamivir phosphate and zanamivir. An agent for preventing or treating a viral disease, which promotes one or more activities selected from the group consisting of.
A first component containing a pharmaceutically treatable amount of salinomycin as an active ingredient; And
A pharmaceutical kit for preventing or treating a viral disease comprising a second component containing a pharmaceutically treatable amount of oseltamivir phosphate as an active ingredient.
The pharmaceutical kit of claim 8, wherein the first component and the second component are administered sequentially or simultaneously.
The pharmaceutical kit for preventing or treating a viral disease of claim 8, wherein the viral disease is a disease caused by an RNA virus infection.
The method of claim 10, wherein the RNA virus is influenza virus, Tamiflu-resistant influenza virus, avian influenza virus, adenovirus (adenovirus), rhinovirus (rhinovirus), hepatitis A virus (hepatitis A virus, hepatitis C virus (hepatitis) C virus, polio virus, measles virus, Ebola virus, Coxsackie virus, yellow fever virus, Dengue Fever virus, Lhasa Virus, lymphocytic choriomeningitis virus, Junin virus, machuppo virus, guanaito virus, hantavirus, Rift valley fever Virus Valley Fever virus, La Crosse virus, California encephalitis virus, Crimea-Congo virus, Marburg virus, Japanese encephalitis virus, severe acute respiratory syndrome virus (SARS) virus, parainfluenza virus ), One or more selected from the group consisting of respiratory syncytial virus, Punta Toro virus, Takacaribe virus, and Pichinde virus. Prophylactic or therapeutic pharmaceutical kits.

Images