Pharmaceutical composition for preventing or treating flavivirus infections

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating flavivirus infections. The invention provides a pharmaceutical composition. The pharmaceutical composition comprises at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, triflurazine, dihydrostreptomycin, hydroxychloroquine, thiolidazine hydrochloride, efavirenz, mitefonew, nystatin, micafungin, bleomycin, remistat, montelukast, norfloxacin, nedaplatin and cephalosporin, and the pharmaceutical composition is prepared from the following raw materials in parts by weight. It is possible to exhibit an excellent growth inhibition or inactivation effect on different species of flavivirus.

Description

[0001] This application is a divisional application of an application filed on April 24, 2019, with application number 201980028691.7 and invention name “Pharmaceutical composition for preventing or treating flavivirus infection”. Technical Field

[0002] The present invention relates to a pharmaceutical composition for preventing or treating flavivirus infection. Background Art

[0003] Flaviviruses are small, enveloped, positive-strand RNA viruses, some of which currently pose or have the potential to threaten human health worldwide. For example, yellow fever virus has become the cause of epidemics in certain jungles of parts of sub-Saharan Africa and South Africa. While many yellow fever infections are mild, they can cause severe and life-threatening illness. These illnesses develop in two phases. The early, acute phase is characterized by high fever, chills, headache, back pain, myalgia, anorexia, nausea, and vomiting. These symptoms resolve after three to four days. In some patients, these symptoms may recur as the disease enters the so-called virulence phase (the "toxic phase"). During the toxic phase, high fever returns and can lead to shock, bleeding (for example, from the mouth, nose, eyes, and / or stomach), kidney failure, and liver failure. Liver failure is often referred to as "yellow fever" because it causes jaundice, a yellowing of the skin and discoloration of the whites of the eyes. Half of patients who enter the toxic phase die within 10 to 14 days. However, those who recover from yellow fever have lifelong immunity to reinfection. The re-emergence of yellow fever virus poses a serious public health threat, as infection with the virus has increased steadily over the past two decades, with approximately 200,000 cases and 30,000 deaths annually.

[0004] Dengue (DEN) virus is another example of a flavivirus. Dengue viruses are transmitted to humans by mosquitoes, primarily Aedes aegypti, and are a growing public health problem worldwide. Approximately 50 of every 100 million people are infected with dengue virus. In some areas, infection rates as high as 6% have been observed, and four antigenic dengue virus types (dengue serotypes 1–4) are found throughout the Caribbean, Asia, and the Americas. DEN infection (dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS)) is an immunopathological condition that occurs in individuals who are sequentially infected with different antigenic types of DEN: dengue hemorrhagic fever (DHF) is responsible for over 3.6 million deaths, with 58,000 deaths reported between 1980 and 1995. Due to the pathogenicity of DHF / DSS, it is believed that an optimal dengue vaccine would provide immunity to all four antigenic types simultaneously and confer long-term immunity. Despite vigorous efforts to develop an effective dengue vaccine since World War II, there is currently no approved and available dengue vaccine.

[0005] Flaviviruses, including yellow fever virus and dengue virus, have two important biological characteristics that cause disease in humans and animals. One of these two characteristics is neural tissue affinity, or "neurotropism," which refers to the tendency of the virus to invade and infect the host's neural tissue. Nerve tissue-friendly (i.e., neurotropic) flavivirus infection can lead to infections and damage to the brain and spinal cord (i.e., encephalitis), confusion, paralysis, and convulsions. The second characteristic of flavivirus infection is visceral organ affinity, which means that the virus tends to invade and infect important internal organs, including the liver, kidneys, and heart. Visceral organ-friendly flavivirus infection can cause infections and damage to the liver (hepatitis), kidneys (nephritis), and myocardium (myocarditis), inhibiting the normal functioning of these organs. Nerve tissue affinity and visceral organ affinity are considered to be unique and different characteristics of flaviviruses.

[0006] Some flaviviruses have essentially neural tissue affinity (e.g., West Nile virus), others have visceral organ affinity (e.g., yellow fever virus and dengue virus), while others exhibit both characteristics (e.g., Kyasanur Forest Disease Virus). However, all flaviviruses exhibit both neural tissue affinity and visceral organ affinity to a greater or lesser extent. An interaction between neural tissue affinity and visceral organ affinity can occur in the host because visceral organs are infected before attacking the central nervous system. Therefore, neural tissue affinity depends on the ability of the virus to replicate in extraneural organs (intestines). This extraneural replication causes viremia, which can attack the brain and spinal cord.

[0007] One attempt to develop vaccines against flaviviruses is to alter the virulence properties of the virus, thereby causing the vaccine virus to lose its affinity for nerve tissue and internal organs in humans and animals. Taking yellow fever virus as an example, two vaccines have been developed: yellow fever 17D and the French nerve-affinity vaccine. The yellow fever 17D vaccine was developed by using continuous inoculation, or "serial passage," in chicken embryo tissue to produce a virus with significantly reduced nerve tissue affinity and internal organ affinity. The French nerve-affinity vaccine was developed by continuous passage in mouse brain tissue to completely eliminate internal organ affinity while retaining nerve tissue affinity. Use of the French vaccine is associated with a high incidence of neurological events (post-vaccination encephalitis). Currently, no other approved vaccines are commercially available against clinically important flaviviruses with internal organ affinity, such as dengue fever, West Nile virus, and Omsk hemorrhagic fever virus. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] The object of the present invention is to provide a composition for preventing or treating flavivirus infection, which is effective against various flaviviruses.

[0010] Furthermore, another object of the present invention is to provide a composition capable of inhibiting the growth of or inactivating various flaviviruses.

[0011] Solutions for solving problems

[0012] 1. A pharmaceutical composition for preventing or treating flavivirus infection, comprising: at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoroperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine HCl, efavirenz, miltefosine, nystatin, micafungin, bleomycin, resminostat, montelukast, norfloxacin, nedaplatin, and cephalothin; or at least one of pharmaceutically acceptable salts of the foregoing compounds.

[0013] 2. The composition according to item 1 above, wherein the flavivirus infection is an infection caused by Apoi virus, Aroa virus, Bagaza virus, Banzivirus, Bouboui virus, Bukarasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, dengue virus, Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus virus), Kadamvirus, Kedougou virus, Kokobera virus, Koutango virus, Khosanur Forest disease virus, Langat virus, Louping ill virus, Meaban virus, Modoc virus, Montana myotis leukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, Rio Bravo virus, Royal Farmvirus, Saboya virus, Saint Louis encephalitis virusencephalitisvirus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Sepik virus, Tembusuvirus, tick-borne encephalitis virus, Tyuleniyvirus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaounde virus, yellow fever virus, Yokose virus, or Zika virus.

[0014] 3. The composition according to item 1 above, wherein the flavivirus infection is an infection caused by dengue virus.

[0015] 4. The composition according to item 1 above, comprising: at least one of tobramycin, trifluoroperazine 2HCl, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz, and miltefosine; or at least one of the pharmaceutically acceptable salts of the foregoing compounds.

[0016] 5. A composition for inhibiting the growth of flavivirus or inactivating it, comprising: at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, efavirenz, miltefosine, nystatin, micafungin, bleomycin, resminostat, montelukast, norfloxacin, nedaplatin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the foregoing compounds.

[0017] 6. The composition according to the above 5, wherein the flavivirus is Apo virus, Aroa virus, Bagza virus, Banchi virus, Bobo virus, Bukalasa bat virus, Kasipakoli virus, Carey Island virus, Bovine Ridge virus, Dakar bat virus, Dengue virus, Eje Mountain virus, Entebbe bat virus, Gadgezi Valley virus, Ilheus virus, Israeli Turkish meningoencephalomyelitis virus, Japanese encephalitis virus, Jugla virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobel virus, Kutango virus, Kosanur Forest disease virus, Langat virus, Jump virus, Leap virus, Miban virus, Modok virus, Montana myotis bat leukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, pink bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboa virus, St. Louis encephalitis virus, Salviejo virus, Santa Pareta virus, Somali Reef virus, Sepik virus, Tembusu virus, tick-borne encephalitis virus, Chouleni virus, Uganda S virus, Usutu virus, Wessels-Brown virus, West Nile virus, Yaoundé virus, yellow fever virus, Yokosuka virus, or Zika virus.

[0018] 7. The composition according to 5 above, wherein the flavivirus is dengue virus.

[0019] 8. The composition according to 5 above, comprising: at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz and miltefosine; or at least one of the pharmaceutically acceptable salts of the foregoing compounds.

[0020] Effects of the Invention

[0021] The composition of the present invention exhibits excellent effects in suppressing the proliferation of viruses of the genus Flavivirus and increasing the survival rate of infections caused by the viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1 to 3 A graph illustrating the inhibitory effect on dengue virus RdRp activity.

[0023] Figure 4 A graph illustrating the inhibitory effect on dengue virus helicase activity.

[0024] Figure 5 A graph illustrating the effect of reducing dengue viremia.

[0025] Figure 6Graph illustrating the results of FFA showing dengue virus reduction.

[0026] Figure 7 Graph illustrating the inhibitory effect on dengue virus growth and the increased survival rate of infection.

[0027] Figure 8 A diagram illustrating tissue-specific anti-dengue virus effects.

[0028] Figure 9 A diagram illustrating confirmation of viral growth inhibition by histological analysis. DETAILED DESCRIPTION

[0029] Hereinafter, the present invention will be described in detail.

[0030] The present invention relates to a pharmaceutical composition for preventing or treating flavivirus infection.

[0031] Flavivirus is the first genus of the Togaviridae family and has common antigens within the genus due to its agglutination with erythrocytes.

[0032] As used herein, flavivirus refers to any species of virus known in the art, including but not limited to its corresponding genus, and flavivirus infection refers to a disease caused by viral infection with the corresponding virus or a virus-carrying condition.

[0033] The composition of the present invention can show an inactivation and / or growth inhibition effect on different species of the genus Flavivirus. For example, the virus can be Apo virus, Aroa virus, Bagza virus, Banchi virus, Bobo virus, Bukarassa bat virus, Kasipakoli virus, Carey Island virus, Bovine Ridge virus, Dakar bat virus, Dengue virus, Mount Edge virus, Entebbe bat virus, Gadgezi Valley virus, Ilheus virus, Israeli Turkish meningoencephalomyelitis virus, Japanese encephalitis virus, Jugla virus, Jutiapa virus, Kadam virus, Keddugu virus, Kokobel virus, Kutango virus, Kosanur forest disease virus, Langat virus, jumping disease virus, Miban virus, Modoc virus, Montana mouse-eared bat white cell virus, Cellular encephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, pink bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, St. Louis encephalitis virus, Salbieho virus, Santa Parita virus, Somalia reef virus, Sepik virus, Tembusu virus, tick-borne encephalitis virus, Qiuleni virus, Uganda S virus, Usutu virus, Wessels-Brown virus, West Nile virus, Yaounde virus, yellow fever virus, Yokosuka virus, and Zika virus, etc., specifically, it can be dengue virus, and more specifically, it can be dengue virus serotype 2, but is not limited thereto.

[0034] The pharmaceutical composition of the present invention may include, for example, at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, efavirenz, miltefosine, nystatin, micafungin, bleomycin, resminostat, montelukast, norfloxacin, nedaplatin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0035] According to a specific embodiment, the composition of the present invention may include at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, resminostat, montelukast, norfloxacin and nedaplatin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0036] Furthermore, according to a specific embodiment, the composition of the present invention may include at least one of efavirenz, miltefosine, nystatin, micafungin, bleomycin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0037] In addition, according to a specific embodiment, the composition of the present invention may include at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz and miltefosine; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0038] Pharmaceutically acceptable salts may include salts of acidic or basic groups that may be present in the above-mentioned drugs (compounds). For example, pharmaceutically acceptable salts used herein may include sodium salts, calcium salts, potassium salts, hydrochlorides, sulfates, and hydrates, and may be prepared by conventional methods known in the art for preparing salts, but are not limited thereto.

[0039] The following table shows the current applications of each of the above drugs, the inhibitory target enzyme in flaviviruses, and the IC 50 , but not limited to this.

[0040] [Table 1]

[0041]

[0042] The substances listed above are drugs that have been previously approved by the FDA but have not been used for flavivirus infections.

[0043] The above substances can be used as enzyme inhibitors, for example, they inhibit the activities of helicase and RNA-dependent RNA polymerase (RdRp), which are enzymes required for viral replication, thereby exhibiting efficacy against flavivirus infections.

[0044] Here, it is known that RNA-dependent RNA polymerase (RdRp) in the genus Flavivirus has a high genetic conservation rate at the gene level and also shows a high conservation rate at the protein level (polypeptide motif level) in various RNA virus families including Flavivirus (Viruses. 2018 Feb;10(2):76.). Therefore, the pharmaceutical composition of the present invention can exhibit efficacy against different species of Flavivirus.

[0045] In the case of helicases, the amino acid sequences at the active sites of the enzymes in flaviviruses are very similar and conserved, and these sequences are known to help recognize viral RNA. In addition, it is known that conserved sequences have similar functions in all flaviviruses (contributing to hydrogen bonds or hydrophobic interactions with adjacent specific sequences) and contribute to the activity of the enzyme. For example, it has been found that the sites of dengue virus helicase and Zika virus helicase coupling to ssRNA substrates are very similar (12904-12920 Nucleic Acids Research, 2017, Vol.45, No.22).

[0046] The pharmaceutical composition of the present invention can be formulated while containing the above-mentioned substances.

[0047] The pharmaceutical compositions of the present invention can be formulated for delivery by any route of administration."Route of administration" may refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, transmucosal, transdermal or parenteral routes, or enteral.

[0048] The pharmaceutical composition of the present invention may also include any pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or vehicle for carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may include a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. The components of the carrier should be "pharmaceutically acceptable," i.e., compatible with the other ingredients of the formulation. When the carrier contacts any tissue or organ that the carrier may contact, the carrier should also be suitable for use, particularly without risk of toxicity, irritation, allergic reaction, and / or any other complications that exceed immunogenicity or its therapeutic advantage.

[0049] The composition of the present invention can be administered to a subject in whom a flavivirus infection is to be prevented or to a subject suffering from a flavivirus infection. The subject can be a mammal including a human, in particular a human.

[0050] The pharmaceutical composition of the present invention can be delivered in a therapeutically effective amount. Definite therapeutically effective amount is the amount of the composition that obtains the most effective results in terms of therapeutic efficacy in a given subject. This amount can be determined based on the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics and biological activity), the subject's physiological condition (age, sex, disease type and stage, general physical health, response to a given dose and type of drug), the characteristics of one or more pharmaceutically acceptable carriers in the preparation, and the route of administration, but is not limited thereto. In fact, the definite therapeutically effective amount depends on many factors, and is not limited to the above requirements. The technical staff of clinical and pharmacological fields determines the therapeutically effective amount by routine experiments, such as by monitoring the reaction of the subject to the compound administration and adjusting the dosage accordingly.

[0051] The present invention also relates to compositions for inhibiting the growth of or inactivating flaviviruses.

[0052] The composition of the present invention may comprise at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, efavirenz, miltefosine, nystatin, micafungin, bleomycin, resminostat, montelukast, norfloxacin, nedaplatin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0053] According to a specific embodiment, the composition of the present invention may comprise at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, resminostat, montelukast, norfloxacin and nedaplatin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0054] Furthermore, according to a specific embodiment, the composition of the present invention may comprise at least one of efavirenz, miltefosine, nystatin, micafungin, bleomycin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0055] In addition, according to a specific embodiment, the composition of the present invention may comprise at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz and miltefosine; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0056] The composition of the present invention can show an inactivation and / or growth inhibition effect on different species of the genus Flavivirus. For example, the virus can be Apo virus, Aroa virus, Bagza virus, Banchi virus, Bobo virus, Bukarassa bat virus, Kasipakoli virus, Carey Island virus, Bovine Ridge virus, Dakar bat virus, Dengue virus, Mount Edge virus, Entebbe bat virus, Gadgezi Valley virus, Ilheus virus, Israeli Turkish meningoencephalomyelitis virus, Japanese encephalitis virus, Jugla virus, Jutiapa virus, Kadam virus, Keddugu virus, Kokobel virus, Kutango virus, Kosanur forest disease virus, Langat virus, jumping disease virus, Miban virus, Modoc virus, Montana mouse-eared bat white cell virus, Cellular encephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, pink bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, St. Louis encephalitis virus, Salbieho virus, Santa Parita virus, Somalia reef virus, Sepik virus, Tembusu virus, tick-borne encephalitis virus, Qiuleni virus, Uganda S virus, Usutu virus, Wessels-Brown virus, West Nile virus, Yaounde virus, yellow fever virus, Yokosuka virus, and Zika virus, etc., specifically, it can be dengue virus, and more specifically, it can be dengue virus serotype 2, but is not limited thereto.

[0057] The composition of the present invention can be formulated according to the pharmaceutical compositions listed above, but is not limited thereto.

[0058] In addition, the present invention also relates to the use of preparing a composition for preventing or treating flavivirus infection, wherein the composition comprises: at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, efavirenz, miltefosine, nystatin, micafungin, bleomycin, resminostat, montelukast, norfloxacin, nedaplatin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0059] According to a specific embodiment, the composition of the present invention may comprise at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, resminostat, montelukast, norfloxacin and nedaplatin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0060] Furthermore, according to a specific embodiment, the composition of the present invention may comprise at least one of efavirenz, miltefosine, nystatin, micafungin, bleomycin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0061] In addition, according to a specific embodiment, the composition of the present invention may comprise at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz and miltefosine; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0062] The flavivirus infection in the use of the present invention may be caused by the above-mentioned viruses.

[0063] As described above, the compound (drug) or a pharmaceutically acceptable salt thereof can exhibit excellent effects of inhibiting the growth of flavivirus and inactivating it, and thus can be used to prepare a composition for preventing or treating flavivirus infection.

[0064] In addition, the present invention also relates to a method for preventing or treating flavivirus infection by administering to a subject at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, efavirenz, miltefosine, nystatin, micafungin, bleomycin, resminostat, montelukast, norfloxacin, nedaplatin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the foregoing compounds.

[0065] According to a specific embodiment, the composition of the present invention may comprise at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, resminostat, montelukast, norfloxacin and nedaplatin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0066] Furthermore, according to a specific embodiment, the composition of the present invention may comprise at least one of efavirenz, miltefosine, nystatin, micafungin, bleomycin and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0067] In addition, according to a specific embodiment, the composition of the present invention may comprise at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz and miltefosine; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0068] The subject may be any subject potentially infected with a flavivirus infection, and specifically, may be a mammal including a human, more specifically, may be a human, but is not limited thereto.

[0069] Flavivirus infections can be caused by the above viruses.

[0070] Hereinafter, the present invention will be described in detail with reference to Examples.

[0071] Example

[0072] 1. Methods

[0073] (1) Evaluation of cell survival rate

[0074] CCK-8 assay was performed according to the manufacturer's instructions.

[0075] Before the experiment, 10.5×10 4 cells / cm 2 and 7.0×10 4 cells / cm 2 VeroE6 cells were seeded at a density of 100 μg / ml. After incubation for 24 hours at 37°C and 5% CO2, montelukast was treated with serially diluted concentrations for 48 hours. Each group was treated with CCK-8 reagent at a concentration of 10% v / v and incubated for 1-4 hours under normal cell culture conditions. Quantitative analysis of colorimetric intensity changes was performed by measuring the optical density of formazan at 450 nm using a microplate reader (Molecular Devices, Inc., USA). The experiment was performed three times and the data are expressed as mean ± SEM.

[0076] (2) Experimental groups for intracellular viral infection experiments

[0077] Before the experiment, 10.5×10 4 cells / cm2 and 7.0×10 4 cells / cm 2 VeroE6 cells were inoculated at a density of . After incubation for 24 hours at 37°C and 5% CO2, dengue virus serotype 2 was inoculated in each cell culture at an MOI of 0.1, 0.5 and 2.5 in serum-free medium for 2 hours. The culture plate was gently shaken once every 30 minutes to evenly distribute the virus in the cells. Montelukast solution was prepared at a serial dilution concentration in each complete culture medium containing 0.75% methylcellulose. After incubation, the viral culture medium was removed and the cells were washed once with sterile PBS and then treated with compound solution. The cells were incubated in the culture chamber for 48 hours. After incubation, each group was prepared for focus formation analysis (FFA) and relative viral RNA expression analysis. For FFA, the compound solution was removed and the cells were washed once with sterile PBS and then fixed with 4% paraformaldehyde. The cells were washed with TRIZOL for viral RNA expression analysis and stored at -70°C for further analysis.

[0078] (3) Focus Formation Analysis (FFA)

[0079] FFA was performed on the NS1 protein of dengue virus (DV) serotype 2. Cells fixed with 4% paraformaldehyde were washed three times with PBS. Blocking solution was incubated for 1 hour at room temperature. After washing with PBS, each group was treated with primary antibody (mouse anti-flavivirus NS1, 1:2000) at room temperature for 2 hours or overnight at 4°C. After washing with PBS, secondary antibody (anti-mouse IgG-FITC, 1:500) was applied to each group for 1 hour at room temperature. After rinsing, each group was treated with PBS containing 10 μg / ml HOECHST 33342 for nuclear staining. Fluorescently stained viral focus-forming units were observed under a microscope (Olympus, Japan) and analyzed.

[0080] (4) Animal experiments

[0081] AG129 mice (129 / Sv IFN-α / β, -γ receptor-deficient) were purchased from Marshall BioResources (Aldbrough, UK). All experimental procedures were pre-approved by the Jeonbuk National University IACUC and performed in accordance with the assessment guidelines and recommendations of the Korean Association for Laboratory Animal Research. Because DV is transferred via mosquitoes, cages with filter lids were used to avoid accidental contact and contamination.

[0082] (5) Experimental groups for in vivo viral infection experiments

[0083] To determine the appropriate titer of infectious virus, 10 6 , 5×10 6 and 10 7 To evaluate the antiviral activity of montelukast, AG129 mice (10-12 weeks) were first injected with 10 7 FFU (IP), followed by injection of 200 μl of montelukast (IP, 10 mg / kg / day) or PBS. Dosage was once daily. The negative control (uninfected group) was administered with 300 μl of PBS (IP) instead of virus. For the drug control group, 200 μl of montelukast (IP, 10 mg / kg / day) was administered alone to uninfected AG129 mice.

[0084] (6) Analysis of mouse weight loss and survival rate

[0085] The status of weight loss and mortality was monitored daily. In the survival analysis, euthanized mice that showed severe disease-related symptoms or rapid weight loss were recorded as currently dead after display. The relative percentage of each body weight relative to day 0 was analyzed.

[0086] (7) Viremia analysis, whole blood RNA expression analysis

[0087] On day 3, blood was collected from each mouse and transferred to an EDTA-coated anticoagulant tube. After shaking for 10 minutes and gently mixing, 20 μl of each sample was analyzed by complete blood count using a HEMAVET 950FS (Drew Scientific). The measured values ​​of each factor were semi-quantitative relative to those of the uninfected control. Plasma was separated by centrifugation at 8000 g and 4 ° C for 15 minutes to analyze cytokine congestion and expression changes. Plasma virus titer (viremia) and viral RNA expression in whole blood were analyzed by quantitative real-time PCR (qRT-PCR).

[0088] All samples were analyzed according to the manufacturer's instructions (BD Biosciences, USA).

[0089] (8) Mouse autopsy

[0090] On the 3rd day, mice in each group were sacrificed (n=6). Organ samples (spleen, liver, lung, small intestine and large intestine) were collected and prepared for viral RNA expression analysis and histopathological analysis. The spleen was weighed to measure splenomegaly. Half of the spleen, liver and lung were homogenized for viral RNA expression analysis and treated with TRIZOL reagent and then stored at -70°C for further analysis. The same steps were applied to the whole sample of the small intestine and large intestine. The other half of the spleen, liver and lung were immersed in 4% paraformaldehyde for fixation and stored at 4°C.

[0091] (9) Analysis of relative viral RNA expression levels

[0092] To investigate changes in viral RNA expression in collected samples, RNA was isolated from cell-based and animal-based samples treated with TRIZOL reagent and then analyzed using MLVRT according to the manufacturer's instructions. TM cDNA synthesis was performed using reverse transcriptase.

[0093] For target gene amplification, primers were designed to account for the overlap between two exons of the target gene with a GC content of less than 50%. The expected amplicon size was approximately 100 bp. Primer sequences were identified as shown in Table 2. To quantify viral RNA, quantitative real-time PCR (qRT-PCR) was performed. Each reaction was performed in a 20 μL volume using SYBR Green Master Mix. Quantstudio 3 TM (Applied Biosystems Inc., USA) was used for the two-step amplification process in this study. To analyze viremia, a linear correlation standard curve was established between the virus titer (FFU / mL) and the corresponding threshold cycle (CT) value.

[0094] Virus in plasma was calculated according to the standard curve equation. A standard curve of serially diluted virus titers was derived. For cell-based panels and mouse organ samples (including whole blood), semi-quantitative viral RNA was analyzed for GAPDH as an endogenous control.

[0095] [Table 2]

[0096]

[0097] (10) Pathological analysis

[0098] Organ samples fixed with 4% paraformaldehyde were subjected to a series of procedures such as paraffin embedding, sectioning, and hematoxylin and eosin staining.

[0099] (11) Statistical analysis

[0100] Data plots using concentration-dependent analysis were analyzed using OriginPro 8. Briefly, log S curves were used for each data set. Unless otherwise indicated, error bars represent ±SEM. Statistical significance of in vitro and in vivo antiviral efficacy assays was evaluated by a two-tailed t-test. Statistical significance of survival was analyzed by the log-rank test using the R Foundation for Statistical Computing, Austria.

[0101] 2. Results

[0102] (1) Confirmation of inhibition of dengue virus enzyme activity

[0103] It was confirmed that the degree of inhibition of dengue virus RdRp enzyme activity depends on the concentration of 14 compounds (given IC 50 value, Figures 1 to 3 ).

[0104] Reference Figures 1 to 3 , it can be seen that 14 compounds efficiently inhibited dengue virus RdRp.

[0105] (2) Confirmation of helicase activity inhibition

[0106] It was confirmed that the degree of inhibition of dengue virus helicase activity depends on the concentration of the six compounds (given IC 50 value, Figure 4 ).

[0107] Reference Figure 4 , it can be seen that the six compounds effectively inhibited the dengue virus helicase.

[0108] (3) Confirmation of the effect of reducing dengue viremia

[0109] The dengue infection model (AG129 mice) was used to confirm the reduction of DENV viremia in the blood of mice after drug treatment ( Figure 5 Micafungin sodium was introduced via intraperitoneal (IP) injection, miltefosine was introduced via oral injection, and vehicle control was introduced via PBS.

[0110] Reference Figure 5 , it can be seen that micafungin sodium and / or miltefosine treatment significantly reduced dengue viremia.

[0111] (4) Confirmation of dengue virus reduction effect

[0112] Cell-based evaluation was performed to evaluate the anti-dengue virus activity of each compound. In vitro viral focus formation assay was performed using VeroE6 cells to study the concentration-dependent antiviral effect of each compound. Viral focus images are shown in Figure 6.

[0113] Reference Figure 6 , it can be seen that the virus was significantly reduced during the treatment with 11 compounds.

[0114] (5) Confirmation of viral growth inhibition and survival rate increase

[0115] DENV2 (dengue virus serotype 2)-infected AG129 mice (10-12 weeks, n=10) were treated once daily with 10 mg / kg montelukast. PBS was injected as a vehicle. After infection, body weight was measured daily, and the survival rate and congestion in plasma were determined on the third day after infection. Figure 7 ).

[0116] Reference Figure 7 , it can be seen that the montelukast-administered group had a low degree of weight loss, a high survival rate, and low congestion.

[0117] (6) Confirmation of tissue-specific antiviral effects

[0118] Tissue-specific antiviral effects of montelukast have been established for each major organ.

[0119] AG129 mice (n=6) infected with DENV2 (dengue virus serotype 2) were treated daily with 10 mg / kg montelukast. Mice were sacrificed on the third day after treatment. Whole blood, spleen, liver, lungs, small intestine, and large intestine were collected, and half of the spleen, liver, and lungs were preserved in paraformaldehyde. The remaining half was used for RNA isolation and viral titer analysis.

[0120] Relative dengue virus RNA expression was determined from each tissue sample and the results are shown in Figure 8 .

[0121] Reference Figure 8 , it can be seen that the expression of dengue virus RNA in the montelukast-treated group was significantly low.

[0122] (7) Histological analysis of viral proliferation inhibition

[0123] Mice infected with dengue virus were treated with montelukast to obtain histological data of the treated and untreated groups. After removing the spleen and liver of each group, H&E staining was performed. The results are shown in Figure 9 .

[0124] Reference Figure 9 , it can be seen that montelukast treatment reduced viral replication.

Claims

1. A pharmaceutical composition for preventing or treating flavivirus infection, comprising: Efavirenz or at least one of a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1, wherein the flavivirus infection is caused by the following viruses: Apo virus, Aroa virus, Bagza virus, Banchi virus, Boboi virus, Bukalasa bat virus, Kasipakoli virus, Carey Island virus, Bovine Ridge virus, Dakar bat virus, Dengue virus, Mount Edge virus, Entebbe bat virus, Gadgezi Valley virus, Ilheus virus, Israeli Turkish meningoencephalomyelitis virus, Japanese encephalitis virus, Jugla virus, Jutiapa virus, Kadam virus, Keddugu virus, Kokobel virus, Kutango virus, Kyosanur Forest disease virus, Langate virus, jumping disease virus, Miban virus, Modoc virus, Montana myotis bat leukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, pink bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboa virus, St. Louis encephalitis virus, Salviejo virus, Santa Pareta virus, Somalia reef virus, Sepik virus, Tembusu virus, tick-borne encephalitis virus, Chouleni virus, Uganda S virus, Usutu virus, Wessels-Brown virus, West Nile virus, Yaoundé virus, yellow fever virus, Yokosuka virus, or Zika virus.

3. The composition of claim 1, wherein the flavivirus infection is a dengue virus-derived infection.

4. The composition according to claim 1, further comprising: at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, micafungin sodium, cephalosporin, miltefosine, netilmicin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine and micafungin; or at least one of the pharmaceutically acceptable salts of said compounds.

5. A composition for inhibiting the growth of or inactivating a flavivirus, the composition comprising: Efavirenz or at least one of a pharmaceutically acceptable salt thereof.

6. The composition of claim 5, wherein the flavivirus is Apo virus, Aroa virus, Bagza virus, Banchi virus, Bobo virus, Bukalasa bat virus, Kasipakoli virus, Carey Island virus, Bovine Ridge virus, Dakar bat virus, Dengue virus, Mount Edge virus, Entebbe bat virus, Gadgezi Valley virus, Ilheus virus, Israeli Turkish meningoencephalomyelitis virus, Japanese encephalitis virus, Jugla virus, Jutiapa virus, Kadam virus, Keddugu virus, Kokobel virus, Kutango virus, Kyosanur Forest disease virus, Langat disease virus. virus, jumping disease virus, Miban virus, Modok virus, Montana myotis bat leukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, pink bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboa virus, St. Louis encephalitis virus, Salviejo virus, Santa Pareta virus, Somalia reef virus, Sepik virus, Tembusu virus, tick-borne encephalitis virus, Choleni virus, Uganda S virus, Usutu virus, Wessels-Brown virus, West Nile virus, Yaoundé virus, yellow fever virus, Yokosuka virus, or Zika virus.

7. The composition of claim 5, wherein the flavivirus is dengue virus.

8. The composition according to claim 5, further comprising: at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, resminostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, micafungin sodium, cephalosporin, miltefosine, netilmicin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine and micafungin; or at least one of the pharmaceutically acceptable salts of said compounds.

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