Pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome comprising ML-18 as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, comprising ML-18 as an active ingredient.

Description

Pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome containing ML-18 as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, comprising ML-18 as an active ingredient.

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a febrile hemorrhagic disease caused by the SFTS virus (SFTSV), a tick-borne, emerging zoonotic infectious disease. In 2009, a large number of patients with fever of unknown etiology, accompanied by thrombocytopenia, gastrointestinal symptoms, and leukopenia, occurred in central and northeastern China. After two years of epidemiological investigation and pathogen research, a single-stranded RNA virus was isolated from the blood of patients and was first reported as SFTSV in 2011. Following reports in China in 2012, cases were subsequently reported in Korea and Japan, and more recently in Vietnam and Thailand. In severe cases, systemic inflammatory response syndrome and multiple organ failure can lead to death, with a mortality rate of 6-30%.

[0003] The causative agent of SFTS is severe fever with thrombocytopenia syndrome virus (SFTS virus, SFTSV). The official name is Dabie bandavirus, which belongs to the genus Banyangvirus, family Phenuiviridae, order Bunyavirales, and commonly called Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV). It is a spherical, lipid-encapsulated virus with a genome composed of three negative-sense single-stranded RNA segments. The L segment encodes RNA-dependent RNA polymerase (RdRp), and the M segment encodes two glycoproteins (Gn / Gc). The S segment expresses a nucleoprotein (NP) and a non-structural protein (NSs).

[0004] In South Korea, the first case of infection was reported in 2012, and the number of confirmed cases has been steadily increasing. In 2013, it was designated as a Class 4 notifiable infectious disease, and surveillance for patient occurrence began. Since 2020, it has been classified as a Class 3 notifiable infectious disease, and outbreak and epidemic surveillance are being conducted. Infection is primarily transmitted through the bite of infected ticks, with a high incidence seasonally from February to November, with a majority occurring from spring to fall. People aged 50 and older account for more than 90% of confirmed cases, and the cumulative number of deaths from 2013 to 2021 has reached 279, representing an 18.5% fatality rate.

[0005] SFTS is mainly transmitted by the small tick (Haemaphysalis longicornis) and is known to be mainly distributed in China, Korea, Japan, Russia, Australia, etc., and was recently discovered in the United States. The SFTS virus can infect not only humans but also livestock animals such as cattle, pigs, and horses, and pets such as cats and dogs, and human infection can occur through these intermediate hosts. It can also be transmitted from person to person through contact with the body fluids or blood of an infected person, or through close contact through aerosols. As such, the risk of SFTS virus infection and the number of infected patients are increasing, but there is currently no effective vaccine or treatment, so conservative treatment is mainly performed, and methods such as ribavirin (an antiviral treatment), steroids, immune globulin, and plasma exchange are being tried.

[0006] Several research teams have confirmed the antiviral efficacy against SFTSV in vitro or in vivo using existing FDA-approved compounds, nucleoside analogs, monoclonal antibodies, and antisera from recovered SFTS patients. Favipiravir (T-705) is a guanosine analog that targets the RNA-dependent RNA polymerase of RNA viruses and inhibits viral replication. It has confirmed antiviral efficacy in interferon receptor-deficient mice (C57BL / 6 IFNAR- / -), a susceptible animal model for SFTSV, and is currently in phase 3 clinical trials. The monoclonal antibody Ab10 targets the glycoprotein Gn and has confirmed antiviral efficacy against SFTSV in interferon receptor-deficient mice (A129). However, the animal model that verified the in vivo efficacy is an immunosuppressed mouse that does not fully reflect the human infection response, and favipiravir is not approved in Korea, making it difficult to use as a treatment for SFTS.

[0007] The purpose of the present invention is to solve the above problems by selecting a compound ML-18 that inhibits SFTS virus growth through a cell-based compound library screening method, and to partially confirm the antiviral efficacy against SFTSV in a susceptible animal model, thereby providing a pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome containing ML-18 as an active ingredient.

[0008] In order to solve the above problem, the present invention provides a composition for suppressing severe fever with thrombocytopenia syndrome, which comprises a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient.

[0009] [Chemical Formula 1]

[0010]

[0011] The compound represented by the above chemical formula 1 or a salt thereof is administered in an amount of 0.001 to 10 g per day, and the composition may additionally contain a component having a severe fever with thrombocytopenia syndrome virus inhibitory effect.

[0012] As an example, the present invention provides a pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] [Chemical Formula 1]

[0014]

[0015] The compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 0.001 to 10 g per day, and the composition may additionally contain an ingredient effective for severe fever with thrombocytopenia syndrome.

[0016] As another example, the present invention provides a food composition for preventing or improving severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a food-related acceptable salt thereof as an active ingredient.

[0017] [Chemical Formula 1]

[0018]

[0019] The compound represented by the above chemical formula 1 or a food-based salt thereof is administered in an amount of 0.001 to 10 g per day, and the composition may additionally contain an ingredient effective for severe fever with thrombocytopenia syndrome.

[0020] As another example, the present invention provides a method for inhibiting severe fever with thrombocytopenia syndrome virus in vitro, comprising administering a compound represented by the following chemical formula 1 or a salt thereof.

[0021] [Chemical Formula 1]

[0022]

[0023] As another example, the present invention provides a method for preventing or treating severe fever with thrombocytopenia syndrome in a non-human animal, comprising administering a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0024] [Chemical Formula 1]

[0025]

[0026] The pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, which comprises ML-18 of the present invention as an active ingredient, not only provides new information necessary for the subsequent research and development of anti-SFTS virus therapeutic agents, but also provides a new opportunity for selection in the antiviral market.

[0027] Figure 1 shows the results of plaque reduction rate (green) and cell viability (purple) of ML-18 according to the present invention.

[0028] Figure 2 shows the survival rate of interferon receptor deficient mice administered ML-18 according to the present invention.

[0029] Figure 3 shows the change in body weight of interferon receptor deficient mice administered ML-18 according to the present invention.

[0030] Hereinafter, preferred embodiments of the present invention will be described in detail. Furthermore, the following description includes numerous specific details, such as specific components, but these are provided to facilitate a more comprehensive understanding of the present invention. It will be apparent to those skilled in the art that the present invention can be practiced without these specific details. Furthermore, in describing the present invention, detailed descriptions of known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention.

[0031]

[0032] In the present invention, the term “prevention” or “prevention” means inhibiting or delaying the occurrence of a disease from its cause.

[0033] In this specification, “treatment” means stopping the progression of damage by suppressing the progression and / or worsening of symptoms without completely curing, or leading toward healing by improving some or all of the symptoms.

[0034] In the present invention, the term “improvement” means any act that improves or beneficially changes symptoms.

[0035] In order to achieve the purpose of the present invention, the present invention provides a composition for suppressing severe fever with thrombocytopenia syndrome, which comprises a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient.

[0036] [Chemical Formula 1]

[0037]

[0038] ML-18, a compound represented by the above chemical formula 1, is a compound having the structure of chemical formula 1 and is a compound with Cas No. 1422269-30-4 (IUPAC name: (2S)-3-(1H-indol-3-yl)-N-[[1-(4-methoxyphenyl)cyclohexyl]methyl]-2-[(4-nitrophenyl)carbamoylamino] propanamide). It is known to be used for the treatment of lung cancer, etc. as a selective inhibitor of bombesin receptor subtype-3.

[0039] The compound of the present invention may include a salt form, which can be prepared by a method conventional in the art, for example, a salt with an inorganic acid such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, or carbonic acid, or an acid salt with an organic acid such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin), or a reaction with an alkali metal ion such as sodium or potassium to form a metal salt thereof, or a reaction with an ammonium ion to form another form of a salt.

[0040] The composition of the present invention can be prepared in any form of composition by further including appropriate carriers, excipients and diluents commonly used in the preparation of compositions, and can preferably be prepared in the form of a pharmaceutical composition or a (health functional) food composition, but is not limited thereto.

[0041] As an example, the present invention provides a pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0042] [Chemical Formula 1]

[0043]

[0044] The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and powders, tablets, capsules, injections, and liquid preparations are more preferred. Such formulation can be performed by methods conventionally practiced in the pharmaceutical field, and can be preferably formulated according to each disease or ingredient using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

[0045] Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0046] When formulating, it can be prepared by additionally using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0047] Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0048] Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous and suspending agents can be used such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be witepsol, macrogol, tween, cacao butter, laurin butter, and glycerogelatin.

[0049] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the preferred dosage varies depending on the patient's condition and weight, the degree of the disease, the drug form, the route and period of administration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition of the present invention can be included in an amount of 0.001 to 99.9 wt%, preferably 0.01 to 99 wt%, per day. The daily dosage can be about 0.01 to 1,000 mg / kg, preferably 100 to 300 mg / kg. If necessary, the active ingredient can be administered in an amount of 0.001 to 10 g per day, but is not limited thereto.

[0050] In addition, in a composition according to one embodiment of the present invention, the composition may include an additional ingredient useful for suppressing, preventing, treating or improving severe fever with thrombocytopenia syndrome virus or severe fever with thrombocytopenia syndrome, and the additional ingredient may include any ingredient known to have an effect including a compound or natural product.

[0051] As another example, the present invention provides a food composition for preventing or improving severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a food-related acceptable salt thereof as an active ingredient.

[0052] [Chemical Formula 1]

[0053]

[0054] The (health functional) food composition to which the above composition can be added may be manufactured in the form of powder, granules, tablets, capsules, or beverages. Examples include various general foods, beverages, gum, tea, vitamin complexes, or health supplements.

[0055] The food composition of the present invention may include not only allithiamine, fursultiamine, benfotiamine or salts thereof, but also ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to allithiamine, fursultiamine, benfotiamine or a salt thereof, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, Eucommia extract, jujube extract, licorice extract, etc. may be additionally included.

[0056] In addition, the composition may be added to food or beverages for the purpose of disease prevention. At this time, the amount of the effective ingredient in the food or beverage may be added at 0.001 to 15 wt% of the total food weight, and the food composition may be added at a ratio of 0.002 to 5 g, preferably 0.03 to 1 g, based on 100 g. If necessary, the effective ingredient may be administered at a daily dose of 0.001 to 10 g, but is not limited thereto.

[0057] As another example, the present invention provides a method for inhibiting severe fever with thrombocytopenia syndrome virus in vitro, comprising administering a compound represented by the following chemical formula 1 or a salt thereof.

[0058] [Chemical Formula 1]

[0059]

[0060] As another example, the present invention provides a method for preventing or treating severe fever with thrombocytopenia syndrome in a non-human animal, comprising administering a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0061] [Chemical Formula 1]

[0062]

[0063] The above "administration" refers to introducing a given substance into a cell or organism through an appropriate method. In the present invention, the route of administration of the composition to an animal may be through any common route as long as it can reach the target tissue. For example, it may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, but is not limited thereto. However, since proteins are digested during oral administration, it is preferable that the oral composition be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device that allows the active agent to travel to the target cell.

[0064]

[0065] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0066]

[0067] <Example 1> Preparation of materials

[0068] Vero E6 cells were purchased from ATCC (Manassas, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% inactivated FBS (Fetal bovine serum, Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA) in a 5% CO2, 37°C incubator. SFTS virus (passage 7, KADGH strain) was isolated from a domestic patient and used under strict management in a biosafety level 3 (BSL-3) research facility. The virus was propagated in Vero E6 cells, and the virus titer was measured using an immunostaining experiment (Infectious center assay). Compound ML-18 used in cell experiments was purchased in powder form from Chemscene (NJ, USA) and dissolved in DMSO before use. Compounds used in animal experiments were dissolved in saline solution containing 10% DMSO, 40% Polyethylene glycol 200 (PEG300, Chemscene, USA), and 5% Tween80.

[0069]

[0070] <Example 2> Measurement of cell viability using WST

[0071] 24 hours before the experiment, 0.2 x 10 Vero E6 cells were seeded into each well of a 96-well plate (Corning, USA). 5Each well was divided into individual wells and cultured. Medium containing diluted compounds was added to each well and cultured for 72 h in a CO2 incubator at 37°C. Compounds were serially diluted 4-fold from a maximum concentration of 40 μM to a minimum concentration of 0.0097 μM, resulting in seven concentrations. Cells were washed with 0.2 ml of PBS to remove cell debris. Medium containing WST substrate (EZ-cytox, Dogen, Korea) was added to the wells and cultured for 30 min in an incubator, and the absorbance was measured at 420 nm. CC 50 (The concentration showing 50% cytotoxicity) was calculated by nonlinear regression analysis using the GraphPad Prism9 program (GraphPad Prism Software Inc., USA).

[0072]

[0073] <Example 3> Virus infection and compound treatment

[0074] 24 hours before the experiment, 0.2 x 10 Vero E6 cells were seeded into each well of a 96-well plate. 5 Cells were cultured by distributing them individually. After transferring the cells to a biosafety level 3 experimental area, the virus was inoculated at a multiplicity of infection (MOI) of 0.1 for 1 hour. After removing the inoculum, the cells were washed with 0.2 ml of PBS to remove uninfected viruses, and 0.2 ml of medium containing the compound was added. The compound was serially diluted 4-fold from a maximum concentration of 40 μM to a minimum concentration of 0.0097 μM, and treated with 7 concentrations. After 72 hours, 0.2 ml of cell culture was harvested and stored in a -70°C ultra-low temperature freezer until performing immunostaining experiments.

[0075]

[0076] <Example 4> Immunostaining experiment (Infectious center assay)

[0077] Vero E6 cells were seeded at 1.5 x 10 in each well of a 24-well plate 24 hours before the experiment. 5 Each well was divided into 10 wells and cultured, and the cell culture medium containing the virus was serially diluted 10-fold in DMEM medium containing 2% FBS to prepare an inoculum. After removing the medium, the diluted virus inoculum was inoculated onto the cultured cell monolayer and allowed to adsorb for 1 hour in a CO2 incubator at 37°C. The inoculum was removed and washed with 1 ml of PBS to remove uninfected virus. To minimize the spread of progeny virus produced after inoculation, the cell layer was covered with methylcellulose overlay medium (DMEM with 10% FBS, 0.5% methylcellulose, 100 U / ml Penicillin, and 100 μg / ml Streptomycin). After culturing for 24 hours in a CO2 incubator at 37°C, the methylcellulose overlay medium was removed. Each well was washed with PBS, 4-paraformaldehyde was added, and the cells were fixed for more than 5 minutes. After detection with a self-produced primary antibody against SFTS virus nucleoprotein (NP), infection was observed by attaching a horseradish peroxidase (HRP)-conjugated secondary antibody (Abfrontier, Korea). The results of the experiment obtained by repeating the experiment three times were analyzed using the IC program (GraphPad Prism 9 program) (GraphPad Prism Software Inc., USA). 50 (The concentration of the compound that inhibits the virus concentration by 50%) was calculated, and CC 50 IC value 50 The selectivity index (SI) was calculated by dividing by the value.

[0078]

[0079] <Example 5> Analysis of antiviral efficacy in animal models

[0080] Animal experiments used interferon receptor-deficient (IFNAR- / -) mice and were conducted under strict supervision in a Biosafety Level 3 (BSL-3) research facility. Mice were infected with 100 μl of 3 infectious units (IFU) via intramuscular injection into the thigh. After viral infection, the compound was administered orally for 5 days, and body weight changes and survival were observed for 12 days.

[0081]

[0082] <Experimental Example 1> Antiviral efficacy of ML-18 at the cellular level

[0083] ML-18 inhibits SFTS virus replication in a concentration-dependent manner (Fig. 1). The plaque reduction rate (green) and cell viability (purple) of the treated group are shown compared to the untreated group.

[0084]

[0085] <Experimental Example 2> Antiviral efficacy of ML-18 in an animal model

[0086] ML-18 increases the survival rate of interferon receptor-deficient mice, a susceptible animal model for SFTSV. The survival rate (Fig. 2) and body weight changes (Fig. 3) of mice in the untreated and compound-treated groups are shown.

Claims

1. A composition for suppressing severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a salt thereof as an active ingredient. [Chemical Formula 1] 2. A composition for suppressing severe fever with thrombocytopenia syndrome, characterized in that the compound represented by the chemical formula 1 or a salt thereof in the first paragraph is administered in an amount of 0.001 to 10 g per day.

3. In the first paragraph, the composition is characterized in that it additionally contains a component having a severe fever with thrombocytopenia syndrome virus inhibitory effect.

4. A pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Chemical Formula 1] 5. A pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, characterized in that the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof in paragraph 4 is administered in an amount of 0.001 to 10 g per day.

6. In the fourth paragraph, a pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, characterized in that the composition additionally contains a component effective for severe fever with thrombocytopenia syndrome.

7. Food composition for preventing or improving severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a food-based salt thereof as an effective ingredient [Chemical Formula 1] 8. A food composition for preventing or improving severe fever with thrombocytopenia syndrome, characterized in that the compound represented by the chemical formula 1 or a food-based salt thereof in clause 7 is administered in an amount of 0.001 to 10 g per day.

9. In the 7th paragraph, the composition is characterized in that it additionally contains a component effective for severe fever with thrombocytopenia syndrome, a food composition for preventing or improving severe fever with thrombocytopenia syndrome.

10. A method for inhibiting severe fever with thrombocytopenia syndrome virus in vitro, comprising administering a compound represented by the following chemical formula 1 or a salt thereof. [Chemical Formula 1] 11. A method for preventing or treating severe fever with thrombocytopenia syndrome in a non-human animal, comprising administering a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof. [Chemical Formula 1]

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