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 with an application date of April 24, 2019, an application number of 201980028691.7, and an invention title of "Drug composition for preventing or treating flavivirus infections". Technical Field

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

[0003] Flaviviruses are small enveloped positive-strand RNA viruses, some of which currently or in the future threaten or potentially threaten the health of humans globally. For example, the yellow fever virus has been the cause of certain jungle epidemics in parts of sub-Saharan Africa or South Africa. Many yellow fever infections are not very severe, but they can lead to serious and life-threatening diseases. The conditions of these diseases are divided into the following two stages. The early acute stage is characterized by high fever, chills, headache, backache, myalgia, anorexia, nausea, and vomiting. After 3 to 4 days, such symptoms disappear. In some patients, when the disease enters the so-called virulent phase (i.e., the "toxic phase"), these symptoms may recur. In the toxic phase, the high fever reappears and may lead to shock, bleeding (e.g., bleeding from the mouth, nose, eyes, and / or stomach), renal failure, and liver failure. In fact, liver failure is usually referred to as "yellow fever" because it causes jaundice, which causes the skin to turn yellow and the eyes to turn white. Half of the patients who enter the toxic phase die within 10 to 14 days. However, people who recover from yellow fever have lifelong immunity to reinfection. In the past two decades, the number of yellow fever virus infections has gradually increased, with approximately 200,000 cases of yellow fever infection per year and approximately 30,000 deaths per year. Therefore, the re-emergence of the yellow fever virus poses a serious threat to public health.

[0004] Dengue (DEN) virus is another example of flavivirus. Dengue viruses are transmitted to humans by mosquitoes, mainly Aedes aegypti, and cause an increasingly serious public health problem worldwide. Approximately 50 out of 100 million people are infected with dengue virus. In some regions, infection rates as high as 6% have been observed. Four antigenic dengue viruses (types 1-4 dengue) are found throughout the Caribbean, Asia, and the Americas. DEN infection [dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS)] is an immunopathological disease that occurs in individuals who are sequentially infected with different antigenic types of DEN: more than 3.6 million people have died from dengue hemorrhagic fever (DHF), and between 1980 and 1995, 58,000 deaths from DHF were reported. Due to the pathogenicity of DHF / DSS, the optimal dengue vaccine is thought to provide immunity against all four antigenic types simultaneously and confer long-term immunity. Since World War II, despite active efforts to develop an effective dengue vaccine, no approved and available dengue vaccine exists yet.

[0005] Flaviviruses, including yellow fever virus and dengue virus, have two important biological characteristics that cause diseases in humans and animals. One of these two characteristics is neurotropism, which means the tendency of the virus to invade and infect the host's nervous tissue. Neurotropic (i.e., nerve-friendly) flavivirus infections can lead to infections and damage (i.e., encephalitis) of the brain and spinal cord, confusion, paralysis, and convulsions. The second characteristic of flavivirus infection is viscerotropism, which means the virus tends to invade and infect important internal organs including the liver, kidneys, and heart. Viscerotropic flavivirus infections can cause infections and damage to the liver (hepatitis), kidneys (nephritis), and myocardium (myocarditis), inhibiting the normal functioning of these organs. Neurotropism and viscerotropism are considered unique and distinct characteristics of flaviviruses.

[0006] Some flaviviruses are essentially neurotropic (e.g., West Nile virus), some are viscerotropic (e.g., yellow fever virus and dengue virus), while others exhibit both characteristics (e.g., Kyasanur Forest Disease Virus). However, both neurotropism and viscerotropism exist to some extent in all flaviviruses. The interaction between neurotropism and viscerotropism can occur in the host because the internal organs are infected before the central nervous system is attacked. Therefore, neurotropism depends on the virus's ability to replicate in extra-neural organs (intestines). This extra-neural replication causes viremia, which can invade the brain and spinal cord.

[0007] One attempt to develop a vaccine against flaviviruses is to modify the virulence characteristics of the virus so that the vaccine virus loses its tropism for neural and visceral tissues of humans and animals. Taking yellow fever virus as an example, two vaccines have been developed: yellow fever 17D and the French neurotropic vaccine. The yellow fever 17D vaccine was developed by generating a virus with significantly reduced tropism for neural and visceral tissues by serial passage, i.e., "continuous passage", in chick embryo tissue. The French neurotropic vaccine was developed by serial passage in mouse brain tissue to completely remove visceral tropism while retaining neural tropism. The use of the French vaccine is associated with a high incidence of neurological accidents (post-vaccination encephalitis). Currently, there is no approved vaccine on the market for clinically important flaviviruses with visceral tropism such as dengue virus, West Nile virus, Omsk hemorrhagic fever virus, etc. Summary of the Invention

[0008] Problems to be Solved by the Invention

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

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

[0011] Solutions for Solving the Problems

[0012] 1. A pharmaceutical composition for preventing or treating flavivirus infection, the composition 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 above compounds.

[0013] 2. The composition according to 1 above, wherein the flavivirus infection is an infection caused by the following viruses: 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, GadgetsGully virus, Ilheus virus, Israelturkey meningoencephalomyelitis virus, Japanese encephalitisvirus, Jugra virus, Jutiapa virus, Kadamvirus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur 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 Louisencephalitisvirus), 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 1 above, wherein the flavivirus infection is a dengue virus-derived infection.

[0015] 4. The composition according to 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, remisostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz, and miltefosine; or at least one of the pharmaceutically acceptable salts of the above compounds.

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

[0017] 6. The composition according to 5 above, wherein the flavivirus is Apoi virus, Aroa virus, Baguio virus, Banzi virus, Bobo i virus, Bukalasa bat virus, Kasokeri virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadget's Valley virus, Ilheus virus, Israel Turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Kutingo virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Mbandza 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 Farm virus, Saboya virus, Saint Louis encephalitis virus, Sarbeho virus, San Palita virus, Somaliland reef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaounde 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, which comprises: at least one of tobramycin, trifluoperazine dihydrochloride, amikacin hydrate, hydroxychloroquine sulfate, thioridazine hydrochloride, dihydrostreptomycin sulfate, gentamicin sulfate, netilmicin sulfate, capreomycin sulfate, paromomycin sulfate, remisostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, cephalosporin, micafungin sodium, efavirenz, and miltefosine; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0020] Effects of the Invention

[0021] The composition of the present invention shows excellent effects in inhibiting the proliferation of viruses of the flavivirus genus and increasing the survival rate of infectious diseases caused by the above viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1 to 3 A figure for illustrating the inhibitory effect on Dengue virus RdRp activity.

[0023] Figure 4 A figure for illustrating the inhibitory effect on Dengue virus helicase activity.

[0024] Figure 5 A figure for illustrating the effect of reducing Dengue virus viremia.

[0025] Figure 6A figure for illustrating the FFA results showing the reduction effect of dengue virus.

[0026] Figure 7 A figure for illustrating the growth inhibitory effect of dengue virus and the increase in infection survival rate.

[0027] Figure 8 A figure for illustrating the tissue-specific anti-dengue virus effect.

[0028] Figure 9 A figure for illustrating the growth inhibitory effect of virus confirmed by histological analysis. Detailed implementation mode

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

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

[0031] Flavivirus is the first genus of Togaviridae and has a common antigen in this genus due to its agglutination with red blood cells.

[0032] As used herein, flavivirus refers to any virus of any species known in the art that is included in its corresponding genus without limitation, and flavivirus infection refers to a disease or a virus-carrying condition caused by a viral infection of the corresponding virus.

[0033] The composition of the present invention can show an inactivating and / or growth inhibitory effect on different species of the genus Flavivirus. For example, the virus can be Apoi virus, Aroa virus, Baguio virus, Banzi virus, Boboí virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadget's Valley virus, Ilheus virus, Israel Turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Mbandza 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 Farm virus, Saboya virus, Saint Louis encephalitis virus, Sarbecovirus, San Perlita virus, Somaliland reef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundé virus, Yellow fever virus, Yokose virus, and Zika virus, etc. Specifically, it can be Dengue virus, and more specifically, it can be Dengue virus serotype 2, but 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, remisostat, 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, remisostat, montelukast, norfloxacin, and nedaplatin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0036] In addition, 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, remisostat, 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, etc., and can be prepared by conventional methods known in the art for preparing salts, but are not limited thereto.

[0039] The following table shows the existing applications of the above-mentioned drugs, the inhibitory target enzymes in flaviviruses, and IC 50 , but not limited thereto.

[0040] [Table 1]

[0041]

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

[0043] The above-mentioned substance can be used as an enzyme inhibitor. For example, it inhibits the activities of helicase and RNA-dependent RNA polymerase (RdRp), which are enzymes required for virus replication, thereby showing efficacy against flavivirus infections.

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

[0045] Regarding helicase, the amino acid sequences at the enzyme active sites in flaviviruses are very similarly conserved, and these sequences are known to contribute to the recognition of viral RNA. In addition, it is known that the conserved sequences have similar functions (contributing to hydrogen bonding or hydrophobic interactions with adjacent specific sequences) in all flaviviruses and contribute to the enzyme activity. For example, it has been found that the sites where dengue virus helicase and Zika virus helicase couple with 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 substance.

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

[0048] The pharmaceutical composition of the present invention may also contain any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or vehicle that carries or transports 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 liquid or solid fillers, diluents, excipients, solvents or encapsulating materials or combinations thereof. Each component of the carrier should be "pharmaceutically acceptable", that is, compatible with the other components of the formulation. When the carrier comes into contact with any tissue or organ with which it can come into contact, the carrier should also be suitable for use, especially not involving risks such as toxicity, irritation, allergic reactions, and / or any other complications such as immunogenicity or its therapeutic advantages exceeding.

[0049] The compositions of the present invention can be administered to a subject attempting to prevent flavivirus infection or a subject suffering from flavivirus infection. The subject can be a mammal including a human, particularly a human.

[0050] The pharmaceutical compositions of the present invention can be delivered in a therapeutically effective amount. The exact therapeutically effective amount is the amount of the composition that achieves the most effective result 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 physiological condition of the subject (age, gender, type and stage of disease, general health status, response to a given dose and type of drug), the characteristics of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration, but is not limited thereto. In fact, the exact therapeutically effective amount depends on many factors and is not limited to the above requirements. Those skilled in the clinical and pharmacological arts determine the therapeutically effective amount through routine experimentation, such as by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly.

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

[0052] The compositions of the present invention can comprise at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, efavirenz, miltefosine, nystatin, micafungin, bleomycin, remisostat, 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 compositions of the present invention can comprise at least one of gentamicin sulfate, netilmicin, tobramycin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, thioridazine hydrochloride, remisostat, 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 compositions of the present invention can 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, remestemcel-L, 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 may exhibit an inactivating and / or growth inhibitory effect on different species of the genus Flavivirus. For example, the virus may be Apoi virus, Aroa virus, Bagaza virus, Banzi virus, Boboí virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadget's Valley virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Mbandza 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 Farm virus, Saboya virus, Saint Louis encephalitis virus, Sarbejo virus, San Perlita virus, Sombre Reef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundé virus, Yellow fever virus, Yokose virus, and Zika virus, etc. Specifically, it may be Dengue virus, and more specifically, it may be Dengue virus serotype 2, but not limited thereto.

[0057] The composition of the present invention may be formulated as the pharmaceutical composition listed above, but not limited thereto.

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

[0060] In addition, 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, remisostat, 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 virus.

[0063] As described above, the compound (drug) or its pharmaceutically acceptable salt may 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, remisostat, montelukast, norfloxacin, nedaplatin, and cephalosporin; or at least one of the pharmaceutically acceptable salts of the above 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, remisostat, montelukast, norfloxacin, and nedaplatin; or at least one of the pharmaceutically acceptable salts of the above compounds.

[0066] In addition, 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, remestemcel-L, 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 can be any subject potentially infected with flavivirus infection, and specifically, can be a mammal including a human, more specifically, can be a human, but is not limited thereto.

[0069] The flavivirus infection can be caused by the above virus.

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

[0071] Embodiment

[0072] 1. Method

[0073] (1) Evaluation of cell viability

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

[0075] Before the experiment, Vero E6 cells were inoculated at densities of 10.5×10 4 cells / cm 2 and 7.0×10 4 cells / cm 2 . After incubation at 37 °C and 5% CO 2 for 24 hours, montelukast was treated at serially diluted concentrations for 48 hours. Each group was treated with CCK-8 reagent at a concentration of 10% v / v and incubated under normal cell culture conditions for 1 - 4 hours. Quantitative analysis of the change in colorimetric intensity was performed by measuring the optical density of formazan salt at 450 nm using a microplate reader ((Molecular Devices, Inc., USA)). The experiment was performed three times, and the data were expressed as mean ± SEM.

[0076] (2) Experimental groups for intracellular virus infection experiment

[0077] Before the experiment, 10.5×104 cells / cm 2 and 7.0×10 4 cells / cm 2 Vero E6 cells were inoculated at a density of. After incubation at 37 °C, 5% CO 2 for 24 h, dengue virus serotype 2 was inoculated into each cell culture at an MOI of 0.1, 0.5, and 2.5 for 2 h in serum-free medium. The culture plates were gently shaken every 30 min to evenly distribute the virus in the cells. Montelukast solutions were prepared at serially diluted concentrations in each complete medium containing 0.75% methylcellulose. After incubation, the virus medium was removed, and the cells were washed once with sterile PBS and then treated with the compound solution. The cells were incubated in the culture chamber for 48 h. After incubation, each group was prepared for focus formation assay (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 assay (FFA)

[0079] FFA was performed on the NS1 protein of dengue virus (DV) serotype 2. The cells fixed with 4% paraformaldehyde were washed 3 times with PBS. The blocking solution was applied for 1 h at room temperature. After washing with PBS, each group was treated with the primary antibody (mouse anti-flavivirus NS1, 1:2000) for 2 h at room temperature or overnight at 4 °C. After washing with PBS, the secondary antibody (anti-mouse IgG-FITC, 1:500) was applied to each group for 1 h at room temperature. After rinsing, each group was treated with PBS containing 10 μg / ml HOECHST 33342 for nuclear staining. The fluorescently stained viral focus-forming units were observed and analyzed under a microscope (Olympus, Japan).

[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 were conducted in accordance with the evaluation guidelines and recommendations of the Korean Association for Laboratory Animal Research. Since DV is transferred by mosquitoes, cages with filter lids were used to avoid accidental contact and contamination.

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

[0083] To determine the appropriate virus titer for infection, various virus titers of 10 6 , 5×10 6 and 10 7 FFU were inoculated into AG129 mice (10 - 12 weeks old) via intraperitoneal injection. To evaluate the antiviral property of montelukast, AG129 mice (10 - 12 weeks old) were first injected with 10 7 FFU (IP), and then injected with 200 μl of montelukast (IP, 10 mg / kg / day) or PBS. The drug was administered once a day. The negative control (non - infected group) was administered with 300 μl of PBS (IP) instead of the virus. For the drug control group, 200 μl of montelukast ((IP, 10 mg / kg / day) was administered alone to non - infected 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 showing severe disease - related symptoms or rapid weight loss were recorded as currently dead after being shown. The relative percentage of each 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 HEMAVET 950FS (Drew Scientific). The measured values of each factor were semi - quantified relative to those of the non - infected 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 virus 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 day 3, the 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, treated with TRIzol reagent, and then stored at -70 °C for further analysis. The same procedure was applied to the whole samples 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) Relative viral RNA expression level analysis

[0092] To study the changes in viral RNA expression in the collected samples, RNA was isolated from cell-based and animal-based samples treated with TRIzol reagent, and then cDNA synthesis was performed using MLVRT TM reverse transcriptase.

[0093] For target gene amplification, each primer was designed taking into account the overlap between two exons of the target gene with a GC content of less than 50%, and the expected amplicon size was approximately 100 bp. As shown in Table 2, the primer sequences were identified. To quantify viral RNA, quantitative real-time PCR (qRT-PCR) was performed. Each reaction was carried out 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 viral titer (FFU / mL) and the corresponding threshold cycle (CT) value.

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

[0095] [Table 2]

[0096]

[0097] (10) Pathological analysis

[0098] A series of procedures such as paraffin embedding, sectioning, and hematoxylin and eosin staining were performed on the organ samples fixed with 4% paraformaldehyde.

[0099] (11) Statistical analysis

[0100] The data graphs using concentration-dependent analysis were analyzed using OriginPro 8. Briefly, each data set used a log S curve. Error bars represent ±SEM unless otherwise indicated. The statistical significance of in vitro and in vivo antiviral efficacy assays was evaluated by two-tailed t-tests. The statistical significance of survival rates was analyzed by the log-rank test using R (R Foundation for Statistical Computing, Austria).

[0101] 2. Results

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

[0103] The degree of inhibition of dengue virus RdRp enzyme activity was confirmed to depend on the concentration of 14 compounds (IC 50 values, Figures 1 to 3 ).

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

[0105] (2) Confirmation of inhibition of helicase activity

[0106] The degree of inhibition of dengue virus helicase activity was confirmed to depend on the concentration of 6 compounds (IC 50 values, Figure 4 ).

[0107] Refer to Figure 4 , it can be seen that 6 compounds efficiently inhibited dengue virus helicase.

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

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

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

[0111] (4) Confirmation of the effect of reducing dengue virus

[0112] Cell-based evaluations were performed to evaluate the anti-dengue virus activity of each compound. In vitro virus focus formation assays were performed using Vero E6 cells to study the concentration-dependent antiviral effects of each compound. Virus focus images are shown in Figure 6。

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

[0114] (5) Confirmation of virus growth inhibition and increased survival rate

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

[0116] Referring to Figure 7 , it can be seen that the montelukast-administered group had a lower degree of body weight loss, a higher survival rate, and lower congestion.

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

[0118] For each major organ, the tissue-specific antiviral effect of montelukast has been determined.

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

[0120] The relative dengue virus RNA expression was measured from each tissue sample, and the results are shown in Figure 8 .

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

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

[0123] Montelukast-treated dengue virus-infected mice were used to obtain histological data for the treated and untreated groups. After excision of the spleen and liver from each group, H&E staining was performed. The results are shown in Figure 9 .

[0124] Referring to Figure 9 , it can be seen that montelukast treatment reduced virus replication.

Claims

1. A pharmaceutical composition for preventing or treating flavivirus infection, the composition comprising: At least one of cephalosporin or a pharmaceutically acceptable salt of the compound.

2. The composition according to claim 1, wherein the flavivirus infection is an infection caused by the following viruses: Apoi virus, Aroa virus, Bagaza virus, Banj virus, Boboí virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadget Valley virus, Ilheus virus, Israel Turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Maban 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 Farm virus, Saboya virus, Saint Louis encephalitis virus, Sarbejo virus, San Palita virus, Somaliland reef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundé virus, Yellow fever virus, Yokose virus, or Zika virus.

3. The composition according to 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, remisostat, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, micafungin sodium, efavirenz, miltefosine, netilmicin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, and micafungin; or at least one of a pharmaceutically acceptable salt of the compound.

5. A composition for inhibiting the growth of flavivirus or inactivating it, the composition comprising: At least one of cephalosporin or a pharmaceutically acceptable salt of the compound.

6. The composition according to claim 5, wherein the flavivirus is Apoi virus, Aroa virus, Bagaza virus, Bhanja virus, Boboí virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadget Valley virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Mbandza 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 Farm virus, Saboya virus, Saint Louis encephalitis virus, Sarbeho virus, San Perlita virus, Somaliland reef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundé virus, Yellow fever virus, Yokose virus, or Zika virus.

7. The composition according to 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, remisiran, montelukast, norfloxacin, nedaplatin, bleomycin, nystatin, micafungin sodium, efavirenz, miltefosine, netilmicin, paromomycin, amikacin, capreomycin, trifluoperazine, dihydrostreptomycin, hydroxychloroquine, and micafungin; or at least one of the pharmaceutically acceptable salts of the compounds.

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