WO2025121761A1 - Pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome comprising ml-18 as active ingredient - Google Patents

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

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

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 a two-year 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. After that, cases of infection were reported in China, Korea, and Japan in 2012, and recently in Vietnam and Thailand. In severe cases, it can lead to death due to systemic inflammatory response syndrome and multiple organ failure, with a mortality rate of 6-30%.

The causative agent of SFTS is Dabie bandavirus , which belongs to the genus Banyangvirus in the family Phenuiviridae of the order Bunyavirales . It is 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 nucleoprotein (NP) and non-structural proteins (NSs).

In Korea, the first case of infection was reported in 2012, and the number of confirmed cases has been steadily increasing. In Korea, it was designated as a Class 4 notifiable infectious disease in 2013, 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 mainly transmitted through the bite of a tick infected with SFTSV, and it occurs frequently from February to November, mainly from spring to fall. The elderly aged 50 or older account for more than 90% of all confirmed cases, and the cumulative number of deaths from 2013 to 2021 is 279, showing a fatality rate of 18.5%.

SFTS is mainly transmitted by the small Haemaphysalis longicornis tick and is known to be mainly distributed in China, Korea, Japan, Russia, Australia, etc., and has recently been discovered in the United States. The SFTS virus can infect not only humans, but also livestock such as cows, 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 body fluids, blood, etc. of infected patients, or close contact through aerosols. Although the risk of SFTS virus infection and the number of infected patients are increasing, there is currently no effective vaccine or treatment, so conservative treatment is mainly performed, and methods such as administration of ribavirin (an antiviral treatment), steroids, immune globulin, and plasma exchange are being attempted.

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. Its antiviral efficacy has been confirmed in an SFTSV susceptible animal model, interferon receptor deficient mice (C57BL/6 IFNAR-/-), and a phase 3 clinical trial is in progress. Monoclonal antibody Ab10 targets glycoprotein Gn and its antiviral efficacy against SFTSV has been confirmed in interferon receptor deficient mice (A129). However, the animal model that verified the in vivo efficacy is an immunosuppressed mouse that does not reflect the complete human infection response, and favipiravir is not approved in Korea, making it difficult to use as a treatment for SFTS.

The purpose of the present invention is to solve the above problems by screening the ML-18 compound that inhibits the growth of SFTS virus 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.

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

[Chemical Formula 1]

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.

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.

[Chemical Formula 1]

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 a component effective for severe fever with thrombocytopenia syndrome.

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-wise acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

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.

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

[Chemical Formula 1]

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.

[Chemical Formula 1]

The pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome, comprising ML-18 of the present invention as an active ingredient, can provide new information necessary for follow-up research and development of anti-SFTS virus therapeutic agents, as well as provide a new opportunity for selection in the antiviral market.

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

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

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

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are illustrated, but these are provided only to help a more general understanding of the present invention, and it will be obvious to those skilled in the art that the present invention can be practiced without these specific details. In addition, when describing the present invention, if it is determined that a specific description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted.

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

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.

The term “improvement” in the present invention means any act by which symptoms are improved or beneficially changed.

In order to achieve the purpose of the present invention, the present invention provides a composition for inhibiting severe fever with thrombocytopenia syndrome, comprising a compound represented by the following chemical formula 1 or a salt thereof as an effective ingredient.

[Chemical Formula 1]

ML-18, a compound represented by the above chemical formula 1, is a compound having a structure of chemical formula 1 and is a Cas No. 1422269-30-4 compound (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 as a selective inhibitor of bombesin receptor subtype-3 for the treatment of lung cancer, etc.

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 bisulfate, 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.

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

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.

[Chemical Formula 1]

The pharmaceutical composition of the present invention can be formulated in the form of 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 liquids are more preferable. 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 method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

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.

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.

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

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

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, 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 administration route, and the period, 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. Depending on necessity, the active ingredient can be administered in an amount of 0.001 to 10 g per day, but is not limited thereto.

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

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-wise acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

The (health functional) food composition to which the above composition can be added can be manufactured in the form of powder, granules, tablets, capsules or beverages. For example, there are various general foods, beverages, gum, tea, vitamin complexes or health supplements.

The food composition of the present invention may include not only allithiamine, fursultiamine, benfotiamine or a salt thereof, but also components commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. 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.

In addition, the composition may be added to food or beverage for the purpose of disease prevention. At this time, the amount of the effective ingredient in the food or beverage may be added in an amount of 0.001 to 15 wt% of the total food weight, and the food composition may be added in an amount of 0.002 to 5 g, preferably 0.03 to 1 g, based on 100 g, and the effective ingredient may be administered in an amount of 0.001 to 10 g per day, but is not limited thereto.

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

[Chemical Formula 1]

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.

[Chemical Formula 1]

The above 'administration' means introducing a given substance into a cell or an individual by an appropriate method, and in the present invention, the route of administration of the composition to an animal may be through any general route as long as it can reach the target tissue. For example, it may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonary, or rectally, but is not limited thereto. However, when administered orally, since proteins are digested, it is preferable that the oral composition be formulated to coat the active agent or protect it from decomposition in the stomach. In addition, the pharmaceutical composition may be administered by any device through which the active agent can move to the target cell.

The advantages and features of the present invention, and the method for achieving them, will become clear 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, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person having ordinary skill in the art to which the present invention belongs of the scope of the invention, and the present invention is defined only by the scope of the claims.

<Example 1> Preparation of materials

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% CO 2 , 37℃ 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 used after being dissolved in DMSO. Compounds used in animal experiments were dissolved in saline solution containing 10% DMSO, 40% polyethylene glycol 200 (PEG300, Chemscene, USA), and 5% Tween 80.

<Example 2> Measurement of cell viability using WST

Vero E6 cells were seeded at 0.2 × 10 ⁵ cells per well of a 96-well plate (Corning, USA) 24 hours before the experiment and cultured. Medium containing diluted compounds was added to each well and cultured in a CO ₂ incubator at 37°C for 72 hours. Compounds were serially diluted 4-fold from a maximum concentration of 40 μM to a minimum concentration of 0.0097 μM and treated with 7 concentrations. Cell debris was removed by washing with 0.2 ml of PBS. Medium containing WST substrate (EZ-cytox, Dogen, Korea) was added to the well and cultured in an incubator for 30 minutes and the absorbance was measured at 420 nm. CC ₅₀ (concentration showing 50% cytotoxicity) was calculated by nonlinear regression analysis using the GraphPad Prism9 program (GraphPad Prism Software Inc., USA).

<Example 3> Virus infection and compound treatment

Vero E6 cells were seeded at 0.2 × 10 ⁵ cells per well of a 96-well plate 24 hours before the experiment and cultured. After the cells were transferred to the biosafety level 3 experimental area, the viruses were inoculated at an MOI (multiplicity of infection) 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 compounds was added. The compounds were serially diluted 4-fold from a maximum concentration of 40 μM to a minimum concentration of 0.0097 μM and treated with seven concentrations. After 72 hours, 0.2 ml of the cell culture was harvested and stored in a -70℃ ultra-low temperature freezer until performing the immunostaining experiment.

<Example 4> Immunostaining experiment (Infectious center assay)

Vero E6 cells were seeded at 1.5 × 10 ⁵ cells per well of a 24-well plate 24 hours before the experiment, 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 CO ₂ 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 viruses produced after inoculation to the surroundings, 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 37°C, CO ₂ incubator, the methylcellulose overlay medium was removed. After washing each well with PBS, 4-paraformaldehyde was added and the cells were fixed for more than 5 minutes. After detection with a primary antibody against the self-made SFTS virus nucleoprotein (NP), infection was observed by attaching a horseradish peroxidase (HRP)-conjugated secondary antibody (Abfrontier, Korea). The IC ₅₀ (concentration of the compound that inhibits the virus concentration by 50%) value was calculated from the experimental results obtained by repeating the experiment three times using the GraphPad Prism 9 program (GraphPad Prism Software Inc., USA), and the selectivity index (SI) was obtained by dividing the CC ₅₀ value by the IC ₅₀ value.

<Example 5> Analysis of antiviral efficacy in animal models

Interferon receptor deficient mice (IFNAR-/-) were used for animal experiments, and were performed under strict management 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 virus infection, the compound was administered orally for 5 days, and body weight changes and survival were observed for 12 days.

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

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

<Experimental Example 2> Antiviral efficacy of ML-18 in animal models

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

Claims (11)

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]

In claim 1, a composition for suppressing severe fever with thrombocytopenia syndrome characterized in that the compound represented by the chemical formula 1 or a salt thereof is administered in an amount of 0.001 to 10 g per day. In claim 1, the composition further comprises a component having a severe fever with thrombocytopenia syndrome virus inhibitory effect, characterized in that the composition inhibits severe fever with thrombocytopenia syndrome. 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]

In claim 4, 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 is administered in an amount of 0.001 to 10 g per day. In claim 4, 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. 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-based salt thereof as an effective ingredient [Chemical Formula 1]

In claim 7, 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 acceptable salt thereof is administered in an amount of 0.001 to 10 g per day. In claim 7, 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. 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]

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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