KR102185945B1 - Antiviral composition - Google Patents

Abstract

In the present invention, an antiviral drug effective on novel SARS-coronavirus-2 is screened by using an FDA-approved drug library. Thirty-one kinds of drugs are selected through screening for two times, of which two drugs are simultaneously screened and a total of 29 drugs exhibit efficacy. By using drugs showing excellent antiviral efficacy, drugs can be developed as a treatment for novel SARS-coronavirus-2 causing serious problems.

Description

Antiviral composition {Antiviral composition}

The present invention relates to an antiviral composition.

The new SARS-coronavirus-2 is a virus with a single-stranded, positive RNA genome of about 30,000 base pairs. It is a virus whose surface is surrounded by an envelope. Since it was first reported in Wuhan, China at the end of December 2019, the number of infected people has exploded around the world (Non-Patent Document 1). The virus spreads very efficiently from person to person, and the World Health Organization is also proclaiming it as a pendemic that can spread worldwide and seeking response. As of February 13, 2020, it is reported that there are more than 60,000 confirmed cases and the mortality rate is 2.2% (Non-Patent Document 2). In the case of a typical Covid-19 patient, relatively mild symptoms such as an increase in body temperature, cough, and chest pain are shown, but in some patients, it develops into serious diseases such as dyspnea and pneumonia, leading to death (non-patent document 3). Treatments or vaccines that have been approved to date are treating patients with symptomatic therapy that relieves symptoms in an unreported situation. Therefore, there is an urgent need to develop therapeutic drugs. Drug repositioning is a method of developing a new drug with a different application status using FDA-approved drugs with high safety and clinical applicability (Non-Patent Document 4). It has the advantage of rapidly developing drugs using well-known drug properties and drugs that have undergone many clinical trials. Drugs that inhibit the new SARS-coronavirus-2 infection can be screened using FDA-approved drugs, and through this, it will be possible to quickly develop a therapeutic agent.

1. Emerg Microbes Infect. (2020) 9: 542-544. 2. Trop Med Int Health. (2020) 25: 278-280 3. Clin Med (Lond). (2020) 20: 124-127. 4. Nature Reviews Drug Discovery (2004) 3: 673-683.

The present invention was derived from the above requirements, and the present inventors completed the present invention by confirming the antiviral effect against the new SARS-coronavirus-2 for about 1,500 FDA-approved drug libraries.

In order to solve the above problems, the present invention flunarizine (Flunarizine), romerizine (Lomerizine), losartan (Losartan), selexipag (Selexipag), quinidine gluconate (Quinidine gluconate), hydroquinidine (Hydroquinidine) ), reserpine (Reserpine) and colporsine daproate (NKH477, Colforsin-daproate) provides an antiviral composition comprising at least one compound selected from the group consisting of.

In addition, the present invention is flunarizine (Flunarizine), romerizine (Lomerizine), losartan (Losartan), selexipag (Selexipag), quinidine gluconate (Quinidine gluconate), hydroquinidine (Hydroquinidine), reserpine (Reserpine) and Colforsin daproate (NKH477, Colforsin-daproate) provides a pharmaceutical composition for preventing or treating viral infections comprising at least one compound selected from the group consisting of.

In another example, the present invention is flunarizine, lomerizine, losartan, selexipag, quinidine gluconate, hydroquinidine, le It provides a food composition for preventing or improving viral infections comprising at least one compound selected from the group consisting of Serpine and Colforsin Daproate (NKH477, Colforsin-daproate).

In another embodiment, the present invention provides a step of administering a drug to be screened to Vero cells;

Infecting SARS-coronavirus-2;

Measuring cell death by microscopic or MTS method;

Reacting with an antibody specific for double-stranded RNA; And

As controls, flunarizine, lomerizine, losartan, selexipag, quinidine gluconate, hydroquinidine, reserpine And treating at least one compound selected from the group consisting of colforsine daproate (NKH477, Colforsin-daproate); it provides a method for screening a SARS-coronavirus-2 inhibitory drug comprising.

The drugs selected according to the present invention exhibit excellent antiviral effects against the new SARS-coronavirus-2, and have high safety and clinical applicability, so they can be usefully used to develop a therapeutic agent for the new SARS-coronavirus-2 infection. .

1 is a result of establishing an antiviral activity screening method for confirming the efficacy of a drug that inhibits cytopathic effect (CPE) induced by a novel SARS-coronavirus-2 virus according to an embodiment of the present invention. Is shown.
2 shows the results of establishing an assay method for evaluating the efficacy of a drug by staining cells infected with a novel SARS-coronavirus-2 virus according to an embodiment of the present invention with an antibody.
Figure 3 is a result of screening a drug that inhibits cellular degeneration induced by a new SARS-coronavirus-2 virus using an FDA-approved drug library according to an embodiment of the present invention by microscopic observation, and evaluating their efficacy Is shown.
FIG. 4 is a result of screening drugs that inhibit cellular degeneration induced by the new SARS-coronavirus-2 virus using an FDA-approved drug library according to an embodiment of the present invention by the MTS method, and evaluating their efficacy Is shown.

Hereinafter, a preferred embodiment of the present invention will be described in detail. In addition, in the following description, there are many specific matters such as specific elements, etc., which are provided to help a more general understanding of the present invention, and the present invention can be practiced without these specific matters. It is self-evident to those who have the knowledge of Further, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

In the present invention, the term "prevention" means inhibiting or delaying the occurrence of the disease.

In the present specification, the term "treatment" refers to stopping the progression of damage by inhibiting the progression and/or worsening of symptoms even without complete cure, or improving some or all of the symptoms and inducing them in the direction of healing.

In order to achieve the object of the present invention, the present invention is flunarizine (Flunarizine), romerizine (Lomerizine), losartan (Losartan), selexipag (Selexipag), quinidine gluconate (Quinidine gluconate), hydroquinidine (Hydroquinidine), reserpine (Reserpine) and colporsine daproate (NKH477, Colforsin-daproate) provides an antiviral composition comprising at least one compound selected from the group consisting of.

In addition, in order to achieve the object of the present invention, the present invention is flunarizine (Flunarizine), romerizine (Lomerizine), losartan (Losartan), selexipag (Selexipag), quinidine gluconate (Quinidine gluconate), hydro Quinidine (Hydroquinidine), reserpine (Reserpine) and colforsine daproate (NKH477, Colforsin-daproate) provides a pharmaceutical composition for the prevention or treatment of viral infections comprising at least one compound selected from the group consisting of.

Flunarizine according to an embodiment of the present invention is a drug classified as a calcium antagonist used for various indications, and Flunarizine sold under the brand Sibelium. Not available over the counter in the US or Japan, the drug was discovered in 1968 in Janssen Pharmaceutica (R14950). It is used as a treatment for various cardiovascular systems including hypertension.

Lomerizine (also known as KB-2796) is a diphenyl piperazine class L-type and T-type calcium channel blocker. This drug is currently used clinically in the treatment of migraine headaches, and is also used experimentally in the treatment of glaucoma and optic nerve damage, and its effect on treating hypertension is known.

Losartan is a drug commonly used to treat high blood pressure, marketed under the trade name Cozaar. It is also used in diabetic kidney disease, heart failure and left ventricular dilatation.

Selexipag is a drug developed by Actelion for the treatment of pulmonary arterial hypertension (PAH). Selexipag and the active metabolite ACT-333679 (or free carboxylic acid MRE-269) are agonists of the prostacycline receptor, which cause vasodilation in the pulmonary circulation.

Quinidine gluconate and hydroquinidine are drugs that act as first-class antiarrhythmic drugs (Ia) in the heart.

Reserpine is a drug commonly used in the treatment of hypertension in combination with thiazide diuretics or vasodilators.

Colforsin daproate (NKH477, Colforsin-daproate) is a water-soluble derivative of forskolin and is used as a treatment for hypertension.

In addition, in the pharmaceutical composition according to an embodiment of the present invention, the virus is selected from coronavirus, and is preferably a SARS-coronavirus-2, SARS or MERS, but is not limited thereto.

In addition, in the pharmaceutical composition according to an embodiment of the present invention, the viral infection may include flu, cold, sore throat, bronchitis, or pneumonia, but is not limited thereto if it is a disease caused by viral infection.

In addition, in the pharmaceutical composition according to an embodiment of the present invention, the composition may include an additional component effective against viral infection, and the additional component includes all components known to be effective against viral infection including compounds and natural products. Can include.

The composition of the present invention may further include suitable carriers, excipients, and diluents conventionally used in the preparation of the composition.

The pharmaceutical composition of the present invention may be used alone or in combination with surgery, drug treatment, and methods using biological response modifiers for the prevention and treatment of diseases.

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 injectable solutions according to a conventional method. , Tablets, capsules, injections and liquids are more preferable. Such formulation may be carried out by a method commonly performed in the pharmaceutical field, and may be preferably formulated according to each disease or component using a 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, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In the case of formulation, it may be prepared by additionally using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( 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, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous agent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition of the present invention may be included in an amount of 0.01 to 99.9% by weight per day, preferably 0.1 to 99% by weight. The daily dosage may be about 0.1 to 1,000 mg/kg, and preferably 100 to 300 mg/kg.

In addition, in order to achieve the object of the present invention, the present invention is flunarizine (Flunarizine), romerizine (Lomerizine), losartan (Losartan), selexipag (Selexipag), quinidine gluconate (Quinidine gluconate), hydro It provides a food composition for preventing or improving viral infections comprising at least one compound selected from the group consisting of quinidine (Hydroquinidine), reserpine, and colforsine daproate (NKH477, Colforsin-daproate).

The food composition includes, for example, various general foods, beverages, gum, tea, and vitamin complexes.

In addition, the ingredients may be added to food or beverages for the purpose of preventing diseases. At this time, the amount of the ingredients in the food or beverage may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 g. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients other than containing the above ingredients as essential ingredients in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates as in ordinary beverages. have. Examples of the natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, taumatin, which is a natural flavoring agent, stevia extract, such as rebaudioside A, glysirhizin, etc.; And synthetic flavoring agents, such as saccharin, aspartame, and the like can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention. In addition to the above, the ingredients of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and heavy agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective properties. Colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonates used in carbonated beverages, and the like may be contained. In addition, the ingredients of the present invention may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. At this time, the ratio of the additive is not very important, but it is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the component of the present invention.

In addition, in order to achieve the object of the present invention,

The present invention comprises the steps of administering a drug to be screened to Vero cells;

Infecting SARS-coronavirus-2;

Measuring cell death by microscopic or MTS method;

Reacting with an antibody specific for double-stranded RNA; And

As controls, flunarizine, lomerizine, losartan, selexipag, quinidine gluconate, hydroquinidine, reserpine And treating at least one compound selected from the group consisting of colforsine daproate (NKH477, Colforsin-daproate); it provides a method for screening a SARS-coronavirus-2 inhibitory drug comprising.

In this case, in the case of cells not infected with the virus, the cell viability is regarded as 100%, and the viability of the infected cells is measured, but is not limited thereto.

In addition, in order to measure the dose-response of the drug, a method of measuring double-stranded RNA generated in a host when a virus is infected is used. In this case, the double-stranded RNA is detected using an antibody, but is limited thereto. no.

In the screening of the drug, the effect of the drug is compared using the drug Remdesivir as a control, but all drugs known as SARS-coronavirus-2 inhibitory drugs in this field can be used, but are not limited thereto. .

The cells are treated with a drug diluted 5 times from 100 μM, and the virus is simultaneously infected, and after 24 hours after infection, the cells are fixed, stained with antibodies, and an image of the cells can be obtained with an operetta (PerkinElmer, Operetta) equipment. At this point, the infection rate can be calculated with Harmony software. If only virus is infected, it is regarded as 100%, and if not infected as 0%, the infection rate when the drug is treated is calculated. Through Prism software (GraphPad, Prism), the concentration (IC ₅₀ ) that inhibits virus infection by 50% can be calculated. The concentration that reduces the viability of 50% cells (CC ₅₀ ) is calculated as the cytotoxic concentration, and the Selectivity Index (SI) is an index indicating how good the compound is compared to toxicity, and can be calculated by dividing the CC ₅₀ value by the IC ₅₀ value. A variety of methods can be used to evaluate the efficacy of a drug.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims.

[ Example 1] Establishment of cell-based antiviral efficacy evaluation method

An antiviral efficacy evaluation method was established in order to select a drug that has a therapeutic effect against the new SARS-coronavirus-2. The new SARS-coronavirus-2 was provided by the Centers for Disease Control and Prevention, and was used by proliferating in Vero (ATCC, CCL-81) cells. Virus titer was measured using a plaque assay. In order to establish an antiviral efficacy evaluation method, a method was used to determine how much a drug inhibits CPE (Cytopathic effect) induced by a virus. When a virus infects, it deforms the cells, which can be checked under a microscope and eventually kills the cells. If the drug is effective, it prevents cell death in this way, thereby increasing the survival rate of cells, and this can be measured using MTS (Promega, G3582). In the case of cells not infected with the virus, the cell viability was regarded as 100%, and the viability of the infected cells was measured (FIG. 1). When infected with a virus, conditions for consistently lowering cell viability were established and used to screen FDA-approved drug libraries.

In order to measure the dose-response of the drug selected through screening, an assay method was established to evaluate the efficacy of the drug by staining the virus-infected cells with an antibody. When a virus is infected, double-stranded RNA that is difficult to observe in host cells is formed, and the virus-infected cells can be stained using an antibody that reacts specifically to it (English and Scientific Consulting Kft. J2). Cells infected with the virus can be observed in green by using an antibody that exhibits green fluorescence as a secondary antibody. Cell nuclei were stained in blue through DAPI staining (FIG. 2). The effect of inhibiting SARS-coronavirus-2 infection was confirmed by using the drug Remdesivir. The cells were treated with drugs diluted 5 times from 100 μM, and virus was simultaneously infected. Cells were fixed 24 hours after infection and stained with antibodies. Images of the cells were obtained with an operetta (PerkinElmer, Operetta) device, and the infection rate was calculated with Harmony software. When only virus was infected, it was considered as 100% and non-infected as 0%, and the infection rate when the drug was treated was calculated. The concentration (IC ₅₀ ) that inhibits virus infection by 50% was calculated through Prism software (GraphPad, Prism) (Fig. 2). For cytotoxicity, only the drug was treated at the same concentration and time in cells without virus infection, and the cell viability was measured by the MTS method. At this time, the concentration to reduce the survival rate of 50% cells (CC ₅₀ ) was calculated as the cytotoxic concentration. The Selectivity Index (SI) is an index indicating how excellent a compound is compared to toxicity, and was calculated by dividing the CC ₅₀ value by the IC ₅₀ value.

[ Example 2] Screening for antiviral efficacy of 1469 FDA approved drug libraries

In order to screen for drugs that can inhibit the new SARS-coronavirus-2, 1469 FDA-approved drug libraries were received from the Korea Research Institute of Chemical Technology Compound Bank. These were treated with 20 μM to Vero (ATCC, CCL-81) cells grown in 96-well plates and infected with viruses. Before the assay method for quantitatively measuring cellular lesions (CPE) caused by viruses was established, cellular lesions were confirmed under a microscope. As a result of checking all wells treated with 1469 kinds of drugs, only when 10 kinds of drugs were treated, cell lesions were suppressed and healthy cells could be observed. The drug efficacy (IC ₅₀ ) by measuring the dose-response of these 10 drugs through the image-based antiviral efficacy evaluation established in Example 1 Was confirmed, and at the same time, cytotoxicity (CC ₅₀ ) was obtained. Using these two values, the selectivity index value could also be calculated (Fig. 3). Ten drugs showing excellent efficacy were found.

[ Example 3] Screening for antiviral efficacy of 1473 FDA approved drug libraries

After establishing an assay method for quantitatively measuring cellular lesions (CPE), the antiviral efficacy of 1473 FDA-approved drug libraries was further screened. In the same manner as in Example 2, drugs at a concentration of 20 μM were treated on Vero (ATCC, CCL-81) cells grown in 96-well plates, and virus was infected. 72 hours after infection, cell viability was measured using MTS, and 26 drugs showing a cell viability of 60% or more were selected. Their efficacy (IC ₅₀ ) was confirmed by an image-based assay, and at the same time, cytotoxicity (CC ₅₀ ) values were obtained. Based on these results, 21 drugs showing an effect on the new SARS-coronavirus-2 were additionally selected (FIG. 4).

Claims (12)

A composition for antiviral against SARS-coronavirus-2, including flunarizine. delete delete A pharmaceutical composition for preventing or treating viral infections against SARS-coronavirus-2, including flunarizine. delete delete The pharmaceutical composition for preventing or treating viral infections according to claim 4, wherein the viral infection is flu, cold, sore throat, bronchitis or pneumonia. Food composition for preventing or improving viral infections against SARS-coronavirus-2, including flunarizine. delete delete The food composition for preventing or improving viral infection according to claim 8, wherein the viral infection is flu, cold, sore throat, bronchitis or pneumonia. delete

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