KR20220129345A - Pharmaceutical composition for the prevention or treatment of COVID-19 comprising Venetoclax and Remdesivir as active ingredients - Google Patents

Abstract

The present invention analyzed a combined treatment effect of remdesivir, an FDA-approved drug, and venetoclax, a chronic lymphocytic leukemia treatment, in order to develop an effective antiviral treatment for SARS coronavirus 2. As a result, two drugs, remdesivir and venetoclas, are treated in combination to exhibit synergistic antiviral effects, which could be developed as a therapeutic agent for SARS coronavirus 2 causing serious problems.

Description

A pharmaceutical composition for preventing or treating COVID-19 comprising Venetoclax and Remdesivir as active ingredients {Pharmaceutical composition for the prevention or treatment of COVID-19 comprising Venetoclax and Remdesivir as active ingredients}

The present invention relates to a pharmaceutical composition comprising venetoclax and remdesivir as active ingredients for the treatment of COVID-19 (COVID-19) infection caused by novel SARS-CoV-2 infection.

Novel SARS-CoV-2 is a virus with a single-stranded positive RNA genome of about 30,000 base pairs, and the surface of the virus is surrounded by an envelope. After it was first reported in Wuhan, China at the end of December 2019, the number of infected people has increased rapidly worldwide (Non-Patent Document 1). Human-to-human transmission of the virus is so rapid that the World Health Organization has declared it a pandemic that can be spread around the world and is seeking countermeasures. According to the report as of February 13, 2020, there are more than 60,000 confirmed cases and the fatality rate is 2.2% (Non-Patent Document 2). In general, patients with COVID-19 infection show relatively mild symptoms such as body temperature rise, cough, and chest pain, but in some patients, respiratory difficulties and serious diseases such as pneumonia develop and lead to death (Non-Patent Document 3).

Remdesivir, an antibody treatment drug and direct-acting antiviral (DAA) that has been urgently approved to date, does not significantly reduce the symptoms of COVID-19 infection, so further development of therapeutic drugs is urgently needed situation is necessary. Drug repositioning is a method of developing new drugs with different indications 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 pharmacological properties and drugs that have undergone many clinical trials. Drugs that inhibit SARS-CoV-2 infection can be screened using FDA-approved drugs, and through this, it will be possible to quickly develop a therapeutic agent.

Various approaches are being studied to develop such a treatment for COVID-19 (COVID-19) infection, and most of them are studying a method of discovering a single active ingredient and discovering it as a therapeutic substance. However, in the case of the monotherapy method as described above, there are disadvantages in that a limitation of the therapeutic effect due to a single mechanism, the occurrence of a resistant reaction, and a toxic reaction due to high dose administration are involved.

The present invention is to solve various problems including the above problems, and to provide a combination therapy for the treatment of COVID-19 (COVID-19) infection.

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.

It is an object of the present invention to provide a combination pharmaceutical composition for the treatment of COVID-19 infection caused by SARS-CoV-2 infection, and to re-create a drug with a compound that inhibits SARS-CoV-2 proliferation by treating it in combination with remdesivir. As a result of screening, it was confirmed that when Venetoclax, approved as a treatment for chronic lymphocytic leukemia, was used in combination with remdesivir as an active ingredient, the SARS-CoV-2 proliferation inhibitory effect was improved. Accordingly, it is an object of the present invention to provide an antiviral composition for SARS-CoV-2 containing venetoclax and remdesivir as active ingredients, and a combined pharmaceutical composition for preventing or treating COVID-19 (COVID-19).

In order to achieve the above object, the present invention provides an antiviral composition for SARS coronavirus-2 comprising Venetoclax and Remdesivir as active ingredients.

As an example of the present invention, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 infection comprising Venetoclax and Remdesivir as active ingredients.

As another example of the present invention, the present invention provides a food composition for improving COVID-19 infection comprising Venetoclax and Remdesivir as active ingredients.

In the present invention, the COVID-19 infection may be flu, cold, sore throat, bronchitis or pneumonia.

Also, benetoclax and remdesivir of the present invention may be administered at 10-1000 mg per day, respectively, and benetoclax and remdesivir may be administered simultaneously or sequentially.

In another embodiment of the present invention, administering the screening target drug to the human lung cell line Calu-3 cells; infecting SARS-CoV-2; measuring the toxicity of SARS-coronavirus-2 to Calu-3 cells; And as a control, Venetoclax (Venetoclax) and the step of treating remdesivir (Remdesivir); provides a method of screening a drug for treating COVID-19 (COVID-19) infection, including.

The present invention shows an antiviral synergistic effect that inhibits the proliferation of SARS-CoV-2 by treating venetoclax and remdesivir in combination, and has high safety and clinical applicability, so it can be usefully used to develop a therapeutic agent for COVID-19 infection. have.

1 is a venetoclax chemical structural formula (A) according to the present invention and a result showing cytotoxicity after venetoclax alone or remdesivir combination treatment in Calu-3, a human lung cell line infected with SARS coronavirus 2 (B) it has been shown Figure 2 shows the results showing the anti-SARS-coronavirus 2 virus efficacy by treatment alone or in combination with venetoclax and remdesivir in Calu-3, a human lung cell line infected with SARS-CoV-2 according to the present invention. (A) is a result of analyzing the intracellular (intracellular) SARS coronavirus 2 mRNA level, (B) is a result of analyzing the SARS coronavirus 2 mRNA level released to the extracellular (extracellular). 3 shows the results showing the infectivity of SARS-CoV-2 released outside the cell after treatment with venetoclax and remdesivir alone or in combination in Calu-3, a human lung cell line infected with SARS-CoV-2 according to the present invention. . (A) It is the result of confirming the virus infectivity through the focus forming unit using the SARS coronavirus 2 nucelocapsid antibody. (B) is the result of re-analyzing the results of (A) based on the virus concentration used in the experiment.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, many specific details such as specific components are shown in the following description, which are provided to help a more general understanding of the present invention, and it is common 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 of And, 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 thereof will be omitted.

As used herein, the term “prevention” refers to inhibiting or delaying the occurrence of a disease from the cause.

As used herein, the term "treatment" means to stop the progression of damage by suppressing the progression and/or exacerbation of symptoms even if not completely cured, or to ameliorate some or all of the symptoms to guide them in the direction of healing.

In the present invention, the term “improvement” refers to suppressing or alleviating symptoms by being administered to a subject. Here, the “subject” may be a mammal other than a human (eg, a rat, a dog, a pig, etc.) or a human.

In order to achieve the object of the present invention, the present invention provides an antiviral composition for SARS coronavirus 2 comprising Venetoclax and Remdesivir as active ingredients.

In the present invention, Venetoclax (trade names Venclexta, Venclyxto) is an anticancer agent developed by AbbVie and Roche, and chronic lymphocytic leukemia (chronic). It is prescribed for the treatment of lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) or acute myeloid leukemia (AML). It was first approved by the FDA in 2016 for the treatment of chronic lymphocytic leukemia. The IUPAC name of Venetoclax is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(3- nitro-4-{[(oxan-4-yl)methyl]amino}benzenesulfonyl)-2-{1H-pyrrolo[2,3-b]pyridin-5-yloxy}benzamide.

It has been suggested that Venetoclax binds to the SARS-CoV-2 Spike protein (Maffucci and Maffucci, J. Proteome Res., 2020, 19:4637-4648), and Venetoclax is also a key protease of SARS-CoV-2. In combination with Mpro, the effect on inhibition of viral infection and therapeutic potential has been reported (Li X et al., biorxiv. https://doi.org/10.1101/2020.10.28.359042). In addition, it has been reported that venetoclax promotes autophagy by increasing the activity of beclin protein, an autophagy regulator, through the binding of Bcl-2 protein (Malik et al., Oncogene 2011 30(37):3918- 29). It has been reported that Venetoclax is related to the antiviral efficacy against MERS virus (Gassen et al., Nat. Commun., 2019, 10:1-16), and furthermore, the viral life cycle through the autophagy pathway. The potential for antiviral efficacy of venetoclax in regulated proliferation of SARS-CoV-2 has been raised (Bello-Perez et al., Cells, 2020, 9(7):1619).

Remdesivir (brand name Veklury) is an antiviral agent made from specific nucleotide analogues developed by Gilead Sciences. It was originally developed by Gilead Sciences as a drug for the treatment of Ebola hemorrhagic fever and Marburg virus. Subsequently, in various experiments, the therapeutic effect on various coronavirus-type viral diseases, including single-stranded RNA viruses, respiratory syncytial virus, hunin virus, Lassa fever virus, henipavirus, coronavirus (including MERS and SARS viruses), and SARS coronavirus 2. has been reported

When remdesivir is administered intracellularly, it is metabolized to its active form, GS-441524, which disrupts the RNA polymerase of SARS coronavirus 2 and acts as an adenosine nucleotide analog that interferes with the corrective function of the viral exonuclease. Interferes with RNA genome replication of 2. In October 2020, the US FDA officially approved remdesivir as a treatment for COVID-19. The IUPAC name of remdesivir is (2S)-2-{(2R,3S,4R,5R)-[5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5-cyano-3,4-dihydroxy-tetrahydro-furan-2-ylmethoxy]phenoxy-(S)-phosphorylamino}propionic acid 2-ethyl-butyl ester.

Drugs for combination therapy are being developed to prevent side effects of the drug by lowering the high-dose administration of remdesivir, and at the same time to increase the clinical effect of treating COVID-19 in preparation for treatment with remdesivir alone. It is being studied through the search for drugs.

As an example of the present invention, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 infection comprising Venetoclax and Remdesivir as active ingredients.

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. may include

The pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of the composition. The pharmaceutical composition of the present invention may be used in combination with venetoclax and remdesivir for the prevention and treatment of diseases, or additionally in combination with surgery, drug therapy, and methods using biological response modifiers.

The pharmaceutical composition of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods, and powders , tablets, capsules, injections and liquids are more preferred. Such formulation may be performed by a method conventionally performed in the pharmaceutical field, and may be preferably formulated according to each disease or component 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.

In the case of formulation, it may 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, and the like, and these solid preparations include at least one excipient, for example, 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 formulations for oral administration include suspensions, solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous agent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base of the suppository, witepsol, macrogol, tween, cacao butter, laurin, 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 severity of the disease, the drug form, 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, preferably 0.1 to 99% by weight per day. The daily dose may be about 0.1 to 1,000 mg/kg, preferably 100 to 300 mg/kg. More preferably, the venetoclax and remdesivir of the present invention may be administered at 10 to 1000 mg per day, respectively, but is not limited thereto.

In the present invention, Corona-19 (COVID-19) infection may include flu, cold, sore throat, bronchitis, or pneumonia, but is not limited thereto if it is a disease caused by SARS-CoV-2 infection.

As another example of the present invention, the present invention provides a food composition for improving COVID-19 infection comprising Venetoclax and Remdesivir as active ingredients.

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

In addition, the component may be added to food or beverage for the purpose of improving the effect of disease. At this time, the amount of the component in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, and the health drink 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 in other components other than containing the above components as an essential component in the indicated ratio, and may contain additional components such as various flavoring agents or natural carbohydrates like a conventional beverage. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents such as taumatin, stevia extract, such as rebaudioside A, glycyrrhizine, and the like; and synthetic flavoring agents such as saccharin, aspartame and the like may 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 flavorants, colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective properties colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the ingredients of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. At this time, the proportion of the additive is not very important, but is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the component of the present invention.

In another embodiment of the present invention, administering the screening target drug to the human lung cell line Calu-3 cells; infecting SARS-CoV-2; measuring the toxicity of SARS coronavirus 2 to Calu-3 cells; And as a control, Venetoclax (Venetoclax) and remdesivir (Remdesivir) treatment step; provides a method of screening a drug for treating COVID-19 (COVID-19) infection, including.

Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the 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, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

In a specific example of the present invention, for the development of a novel treatment regimen for COVID-19, the improved corona-19 treatment efficacy results by the combined treatment of venetoclax and remdesivir are shown.

<Example 1> Evaluation of cytotoxicity of Venetoclax in human lung cells infected with SARS-CoV-2

SARS-CoV-2 virus was provided from the National Pathogen Resource Bank of the Korea Centers for Disease Control and Prevention and used after propagation in Vero cells (ATCC). Virus titer was measured by conventional plaque assay. Venetoclas (5 uM) and remdesivir (50 nM, approximately 1/100 of the IC ₅₀ concentration confirmed in the results of anti-SARS-CoV-2 efficacy evaluation using Vero cells) in the human lung cell line Calu-3 Cell viability was confirmed by MTT assay to determine whether the drug was cytotoxic after 48 hours of drug treatment in the single treatment group and the Venetoclas + remdesivir combination treatment group. As shown in FIG. 1(B), no specific cytotoxicity by drug treatment was observed in both the group treated with Venetoclass and remdesivir alone and the group treated with the combination.

<Example 2> Evaluation of antiviral efficacy of venetoclax in human lung cells infected with SARS-CoV-2

To see the anti-SARS-CoV-2 efficacy in venetoclax alone and in combination with remdesivir, human lung cell line Calu-3 was infected with SARS-CoV-2 for 48 hours, and then intracellularly ( SARS-CoV-2 RNA levels released into intracellular and extracellular were analyzed by real-time qRT-PCR.

Primer Information: Centers for Disease Control and Prevention (CDC)

2019-nCoV_N2 Forward Primer : 5'-TTA CAA ACA TTG GCC GCA AA-3'

2019-nCoV_N2 Reverse Primer: 5'-GCG CGA CAT TCC GAA GAA-3'

2019-nCoV_N2 Probe : FAM-ACA ATT TGC CCC CAG CGC TTC AG-BHQ1

As shown in Figure 2, it was confirmed that the combined treatment of venetoclax and remdesivir significantly reduced the SARS-CoV-2 RNA level in both intracellular and extracellular released SARS-CoV-2 RNA levels. became

<Example 3> Efficacy of inhibiting SARS-CoV-2 infectivity by venetoclax

In order to select antiviral drugs against SARS-CoV-2 virus, we established an FFU assay antiviral efficacy evaluation method considering the SARS-CoV-2 characteristic that does not induce significant cell death, and using this, Venetoclax and remdesivir The inhibition of SARS-CoV-2 infectivity by the combination treatment was analyzed. After SARS-CoV-2 infected Calu-3 cells were treated with Venetoclas, remdesivir or Venetoclas+remdesivir for 48 hours, the supernatant of Calu-3 cells was collected and transferred to Vero E6 cells for 8 hours. while infected. After fixing the cells, Vero E6 cells infected with SARS-CoV-2 were identified using a SARS-CoV-2 nucleocapsid-specific antibody. Cells positively stained with SARS-CoV-2 nucleocapsid antibody were measured using ELISpot (unit: focus forming unit (FFU)). 3 as shown The number of FFUs was significantly reduced in the remdesivir and bedetoclas combination treatment group than in the benetoclas and remdesivir alone treatment group, confirming that the combined treatment had the effect of inhibiting SARS-CoV-2 infectivity.

Claims (12)

Composition for antiviral against SARS coronavirus 2 comprising Venetoclax and Remdesivir as active ingredients The composition for antiviral according to claim 1, wherein benetoclax and remdesivir are administered in an amount of 10 to 1000 mg per day, respectively. The composition for antiviral according to claim 1, wherein venetoclax and remdesivir are administered simultaneously or sequentially. Pharmaceutical composition for preventing or treating COVID-19 infection comprising Venetoclax and Remdesivir as active ingredients [Claim 5] The pharmaceutical composition for preventing or treating COVID-19 (COVID-19) infection according to claim 4, wherein the infection is flu, cold, sore throat, bronchitis or pneumonia. [Claim 5] The pharmaceutical composition for preventing or treating COVID-19 (COVID-19) infection according to claim 4, wherein Benetoclax and Remdesivir are administered in an amount of 10 to 1000 mg per day, respectively. The pharmaceutical composition for preventing or treating COVID-19 (COVID-19) infection according to claim 4, wherein the administration of venetoclax and remdesivir is simultaneous or sequential administration. Food composition for improving COVID-19 infection comprising Venetoclax and Remdesivir as active ingredients The food composition for improving COVID-19 infection according to claim 8, wherein the infection is flu, cold, sore throat, bronchitis or pneumonia. The food composition for improving corona-19 (COVID-19) infection according to claim 8, wherein benetoclax and remdesivir are administered in an amount of 10 to 1000 mg per day, respectively. The food composition for improving COVID-19 (COVID-19) infection according to claim 8, wherein the administration of venetoclax and remdesivir is simultaneous or sequential administration. administering the screening target drug to Calu-3 cells, a human lung cell line; infecting SARS-CoV-2; Measuring the toxicity of SARS-CoV-2 to Calu-3 cells; And Treatment of Venetoclax (Venetoclax) and Remdesivir (Remdesivir) as a control; Corona-19 (COVID-19) method of screening a drug for treating infection comprising a.

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