KR102641011B1 - Pharmaceutical composition for preventing or treating COVID-19 comprising of ST034307 as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating COVID-19 containing ST034307 as an active ingredient.

Description

Pharmaceutical composition for preventing or treating COVID-19 comprising ST034307 as an active ingredient {Pharmaceutical composition for preventing or treating COVID-19 comprising of ST034307 as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating COVID-19 containing ST034307 as an active ingredient.

Coronavirus disease-19 (COVID-19) first emerged in Wuhan, Hubei Province, China in December 2019 and began to spread. It is a pandemic infectious disease that has continued to spread around the world to this day, recording numerous confirmed cases and deaths, and is a zoonotic disease that infects both humans and animals. It is an infectious disease. The main symptoms are fever, fatigue, dry cough, and difficulty breathing, and if symptoms worsen, it can lead to death. Elderly people and people with underlying diseases (those with asthma, diabetes, heart disease) are more likely to develop serious symptoms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a beta coronavirus and is an enveloped virus with a single-stranded positive RNA genome of 25 to 32 kb. Beta coronaviruses that infect humans include SARS-CoV (the causative agent of severe acute respiratory syndrome), MERS-CoV (the causative agent of Middle east respiratory syndrome), and HCoV-229E (human coronavirus). etc. are included.

Currently, the injectable treatment Remdesivir and the oral treatments Paxrovid and Lagebrio have been approved for COVID-19 and are being used for mild and severe patients at high risk of developing severe disease. Remdesivir is a broad-spectrum antiviral nucleotide, an adenosine analog, and is an antiviral agent that inhibits RNA-dependent polymerase (RdRp) action by binding to RNA strands and preventing the addition of additional nucleotides. It is reported to be effective in shortening the time it takes to clinical recovery in some severely ill patients. Paxrovid is the first oral treatment introduced in Korea and consists of nirmatrelvir, a 3CL protease inhibitor essential for SARS-CoV-2 replication, and ritonavir, which inhibits the liver metabolism of nirmatrelvir by CYP3A. The effect of reducing the risk of death by 88% in moderately ill patients has been reported, but caution is required when dosing because it may affect drug metabolism by ritonavir. Lagebrio, another oral treatment, is an antiviral agent that is inserted into the virus replication process as a nucleotide analog and inhibits replication. It has lower antiviral efficacy than Paxrovid, and there have recently been reports that Lagebrio causes mutations in the coronavirus.

Currently, treatments are prescribed only to mild and moderate patients with a high risk of progressing to severe disease, and depending on the characteristics of the drug, there is a risk of mutation or development of resistant viruses. Accordingly, there is a need to continue to pay attention to the development of treatments with different mechanisms of action and that are easy to prescribe and administer.

Korean Patent Publication No. 2022-0105609

The purpose of the present invention is to solve the above problems by confirming the efficacy of the ST034307 compound selected through a cell-based compound library screening method to inhibit the proliferation of SARS-CoV-2 (COVID-19 virus), and using ST034307 as an active ingredient. The aim is to provide a pharmaceutical composition for preventing or treating COVID-19, comprising:

In order to solve the above problems, the present invention provides a composition for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a compound represented by the following formula (1) or a salt thereof as an active ingredient.

[Formula 1]

The compound represented by Formula 1 or its salt is administered in an amount of 0.001 to 10 g per day, and the composition may further include an ingredient that has an effect of inhibiting severe acute respiratory syndrome coronavirus 2.

As an example, the present invention provides a pharmaceutical composition for preventing or treating coronavirus disease 19 (COVID-19), comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The compound represented by 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 further include an ingredient effective against COVID-19.

As another example, the present invention provides a food composition for preventing or improving coronavirus disease 19 (COVID-19) containing a compound represented by the following formula (1) or a foodologically acceptable salt thereof as an active ingredient.

[Formula 1]

The compound represented by Formula 1 or a food-acceptable salt thereof is administered in an amount of 0.001 to 10 g per day, and the composition may further include an ingredient effective against COVID-19.

As another example, the present invention provides a method for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, comprising administering a compound represented by the following formula (1) or a salt thereof.

[Formula 1]

As another example, the present invention provides a method for preventing or treating coronavirus disease-19 (COVID-19) in non-human animals, comprising the step of administering a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof: do.

[Formula 1]

The pharmaceutical composition for preventing or treating COVID-19 containing ST034307 of the present invention as an active ingredient not only provides new information necessary for follow-up research and development of SARS-CoV-2 antiviral treatment, but also provides a new choice in the antiviral market. It can provide an opportunity.

Figure 1 shows the cytotoxicity results of ST034307 according to the present invention on Vero E6 cells.
Figure 2 shows the degree of virus growth inhibition of ST034307 according to the present invention based on genome copy.
Figure 3 shows the degree of virus growth inhibition of ST034307 according to the present invention based on plaque forming units (PFU).

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 shown, but this is provided to facilitate a more general understanding of the present invention, and it is known in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

In the present invention, the term “prevention” or “prevention” means suppressing 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 even if not completely cured, or improving some or all of the symptoms and leading toward healing.

In the present invention, the term “improvement” refers to any action that improves or beneficially changes symptoms.

In order to achieve the object of the present invention, the present invention provides a composition for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a compound represented by the following formula (1) or a salt thereof as an active ingredient.

[Formula 1]

ST034307, a compound represented by Formula 1, is a compound having the structure of Formula 1 and has Cas No. 133406-29-8 is a compound (IUPAC name: 6-chloro-2-(trichloromethyl)chromen-4-one). It is a selective inhibitor of adenylyl cyclase 1 and is known to be used as an anesthetic.

The compound of the present invention may include a salt form, which can be prepared by methods common in the art, for example, inorganic salts such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium bisulfate, phosphoric acid, and carbonic acid. Salts of acids or with organic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin). It means forming or reacting with alkali metal ions such as sodium and potassium to form metal salts thereof, or reacting with ammonium ions to form another type of salt.

The composition of the present invention can be manufactured in any form by further including appropriate carriers, excipients and diluents commonly used in the preparation of the composition, and is preferably manufactured in the form of a pharmaceutical composition or (health function) food composition. However, it is not limited to this.

As an example, the present invention provides a pharmaceutical composition for preventing or treating coronavirus disease 19 (COVID-19), comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[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, external preparations, suppositories, and sterile injectable solutions according to conventional methods. , tablets, capsules, injections and liquid formulations are more preferable. This formulation can be performed by a method commonly performed 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, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it can be prepared by additionally using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

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

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous agents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween, cacao, laurin, glycerogeratin, etc. can be used.

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

In addition, in the composition according to one embodiment of the present invention, the composition may include additional ingredients useful for suppressing, preventing, treating or improving severe acute respiratory syndrome coronavirus 2 or COVID-19, and the additional ingredients are compounds. , may contain all ingredients with known effects, including natural products.

As another example, the present invention provides a food composition for preventing or improving coronavirus disease 19 (COVID-19) containing a compound represented by the following formula (1) or a foodologically acceptable salt thereof as an active ingredient.

[Formula 1]

Food compositions to which the above composition can be added (health function) can be manufactured in the form of powders, 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 allitiamine, fursultiamine, benfotiamine, or salts thereof, as well as ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, and seasonings. Includes agents 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 common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (thaumatin, stevia extract (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 allitiamine, fursultiamine, benfotiamine or salts thereof, citric acid, high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic acid extract, jujube extract, and licorice. Extracts, etc. may be additionally included.

Additionally, the composition can be added to food or beverages for the purpose of preventing diseases. At this time, the amount of the active ingredient in the food or beverage can be added at 0.001 to 15% by weight of the total food weight, and the food composition can be added at a rate of 0.002 to 5 g, preferably 0.03 to 1 g, based on 100 g. and, if necessary, the active 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 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, comprising administering a compound represented by the following formula (1) or a salt thereof.

[Formula 1]

As another example, the present invention provides a method for preventing or treating coronavirus disease-19 (COVID-19) in non-human animals, comprising the step of administering a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof: do.

[Formula 1]

The 'administration' means introducing a predetermined substance into a cell or organism 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, intrapulmonaryly, or rectally, but is not limited thereto. However, when administered orally, proteins are digested, so it is desirable for oral compositions to be coated with the active agent or formulated to protect them from decomposition in the stomach. Additionally, the pharmaceutical composition can be administered by any device that allows the active agent to move to target cells.

The advantages and features of the present invention and methods 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 and will be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have the scope of the invention, and the invention is only defined by the scope of the claims.

<Example 1> Preparation of materials

Vero E6 cells were purchased from ATCC (Manassas, USA) and incubated with 10% inactivated FBS (Fetal bovine serum, Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) in an _incubator at 37°C with 5% CO 2 . Cultured in supplemented Dulbecco Modified Eagle Medium (DMEM, Gibco, USA). SARS-CoV-2 (passage 4, betaCoV/Korea/KCDC03/20) was isolated from a patient in Korea and was used under strict control at a Korea Disease Control and Prevention Agency biosafety level 3 (BSL-3) research facility. SARS-CoV-2 was propagated in Vero E6 cells, and titer was measured using the plaque test method. STO34307 used in the experiment was purchased in powder form from Chemscene (NJ, USA) and dissolved in DMSO solvent.

<Example 2> Measurement of cell viability using WST

24 hours before the experiment, 1.5 x 10 ⁵ Vero E6 cells were distributed and cultured in each well of a 24-well plate (Eppendorf, Germany). Medium containing 10 μM of the compound was added to each well and cultured at 37°C in a CO ₂ incubator for 48 hours. To remove cell debris, it was washed with 1ml of PBS. To this, medium containing WST substrate (EZ-cytox, Dogen, Korea) was added, cultured in an incubator for 30 minutes, and absorbance was measured at 450 nm.

<Example 3> Virus infection and compound treatment

24 hours before the experiment, 1.5 x 10 ⁵ Vero E6 cells were distributed and cultured in each well of a 24-well plate. After transferring the cells to a biosafety level 3 experimental area, the virus was inoculated at an MOI of 0.1 for 1 hour. After removing the inoculum, it was washed with 1ml of PBS to remove uninfected viruses, and medium containing 1ml of the compound was added. The final concentration of the compound was 10 μM. After 48 hours, the cell culture was harvested and stored in a -70°C freezer until the next experiment.

<Example 4> RNA extraction

Total RNA was extracted from the cell culture lysate obtained above in a volume of 100 μl using the MagNA Pure 96 DNA and Viral NA Small Volume kit (Roche, Switzerland) and the MagNA 96 system, an automated device.

<Example 5> qRT-PCR

The total RNA of the cell culture medium was subjected to qRT-PCR experiment to detect SARS-CoV-2 RNA and quantitatively analyze it. The amount of the E gene was measured in 5 μl of RNA using the PowerChek 2019-nCoV real-time PCR kit (Kogen biotech, Korea). To measure genome copy, the SARS-CoV-2 genome of a known concentration was diluted to a certain concentration and qRT-PCR was performed to determine a reference curve. Geonome copy was calculated using the reference curve formula.

<Example 6> Virus proliferation inhibition experiment by plaque test method

24 hours before the experiment ^, 2.0 After removing the medium, the diluted virus solution was inoculated into the cultured cell monolayer and adsorbed in a CO ₂ incubator at 37°C for 1 hour. 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 agarose overlay medium (DMEM with 2% FBS, 0.5% agarose, 100 U/ml Penicillin, and 100 μg/ml Streptomycin). . After culturing in a CO ₂ incubator at 37°C for 48 hours, 4% paraformaldehye was added and the cells were fixed for more than 1 hour. After removing the agarose overlay medium and washing with PBS, crystal violet solution was added and stained for 1 hour. After removing the crystal violet solution, it was washed with distilled water and dried. The plaque forming unit (PFU) was calculated by counting the number of plaques.

<Experimental Example> Antiviral efficacy of STO34307

STO34307 itself did not show toxicity to Vero E6 cells, and remdesivir (RDV), which was used as a positive control, was also used at a non-toxic concentration (Figure 1).

STO34307 shows that it inhibits SARS-CoV-2 virus replication (Figures 2 and 3). It was confirmed that it inhibits virus growth similar to remdesivir, which has known SARS-CoV-2 antiviral efficacy. In addition, the degree of virus growth inhibition and cell survival rate were indicated by observing genome copy and plaque formation in the ST034307 treated group compared to the untreated group.

Claims (11)

A composition for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a compound represented by the following formula (1) or a salt thereof as an active ingredient:
[Formula 1]
The composition for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to claim 1, wherein the compound represented by Formula 1 or a salt thereof is administered in an amount of 0.001 to 10 g per day. The composition for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to claim 1, wherein the composition further comprises an ingredient that has an effect of inhibiting severe acute respiratory syndrome coronavirus 2. A pharmaceutical composition for preventing or treating coronavirus disease 19 (COVID-19) containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
The pharmaceutical composition for preventing or treating coronavirus disease 19 (COVID-19) according to claim 4, wherein the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 0.001 to 10 g per day. The pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19) according to claim 4, wherein the composition further contains an ingredient effective against coronavirus infection-19. A food composition for preventing or improving coronavirus disease 19 (COVID-19) containing a compound represented by the following formula (1) or a food-acceptable salt thereof as an active ingredient:
[Formula 1]
The food composition for preventing or improving coronavirus disease 19 (COVID-19) according to claim 7, wherein the compound represented by Formula 1 or a food-acceptable salt thereof is administered in an amount of 0.001 to 10 g per day. The food composition for preventing or improving coronavirus infection-19 (COVID-19) according to claim 7, wherein the composition further comprises an ingredient effective against coronavirus infection-19. A method of inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro comprising administering a compound represented by the following formula (1) or a salt thereof:
[Formula 1]
A method for preventing or treating coronavirus disease 19 (COVID-19) in non-human animals comprising administering a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:
[Formula 1]