KR20220138835A - A composition for anti-coronavirus combination therapy comprising a coronavirus entry inhibitor and a viral protease inhibitor - Google Patents

Abstract

One aspect relates to an anti-coronavirus composition comprising a coronavirus cell entry inhibitor and a coronavirus protease activity inhibitor or a pharmaceutical composition for preventing or treating a coronavirus infection. According to one aspect, when a coronavirus cell entry inhibitor and a coronavirus protease activity inhibitor are administered in combination, entry of the coronavirus into cells is inhibited, and even protease activity of the coronavirus is inhibited, thereby exhibiting a more remarkable synergistic effect in the anti-coronavirus effect and the prevention, alleviation, or treatment effect of a coronavirus infection.

Description

Anti-coronavirus combination therapy comprising a coronavirus cell entry inhibitor and a coronavirus protease activity inhibitor {A composition for anti-coronavirus combination therapy comprising a coronavirus entry inhibitor and a viral protease inhibitor}

It relates to an anti-coronavirus co-administration therapeutic composition comprising a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus.

In December 2019, a novel coronavirus occurred in Wuhan, Hubei Province, China. This virus, which started spreading from the seafood market in Wuhan City, was initially called the Wuhan seafood market pneumonia virus because it causes pneumonia, but later officially named COVID-19 by the WHO, and it is a beta-coronavirus. Because the gene sequence is very similar to the SARS-CoV to which it belongs, the International Commission on Virus Classification named it Severe Acute Respiratory Syndrome Coronavirus2 (SARS-CoV-2). After the outbreak, it rapidly spread to about 20 countries by February 2020, and the WHO declared a pandemic on March 11, 2020. SARS-CoV, which appeared in 2003, caused about 8,000 infections and about 700 deaths (about 9.6%) during the outbreak period (November 2002 to July 2003), while SARS-CoV-2 spread The rate is exceptionally fast, and the mortality rate is lower than that of SARS-CoV so far, but the number of deaths worldwide is increasing. SARS-CoV-2 is still ongoing and is being continuously monitored by all countries.

SARS coronavirus 2 is a positive single-stranded RNA virus belonging to the coronavirus genus of the Coronaviridae family in terms of its genetic structure, and has a gene of about 30 kb in size. This viral gene has a cap structure at the 5' end and a 3'-poly(A) sequence at the 3' end. After the virus enters the cell, the RNA gene is translated and a nonstructural protein is made from two ORFs (ORF1a and ORFb), which is translated into polypeptide 1a (pp1a, 440 - 500 kDa), which is then translated to the viral protease. 11 nonstructural proteins are produced. Specifically, in the translation of ORF1b, a -1 ribosome frameshift occurs upstream of the ORF1a stop codon, causing no stop, and as ORF1b translation continues, a long polypeptide (pp1ab, 740 - 810 kDa) is produced. . This protein is cleaved by virus-derived proteases, nonstructural protein 3 (nsp3) and nonstructural protein 5 (nsp5) to produce 16 nonstructural proteins. Among the 16 nsp, a negative-stranded RNA intermediate is generated using genomic RNA (gRNA) as a template by the RNA-dependent RNA polymerase activity of nonstructural protein 12 (nsp12), which is known to be important for replication. Subgenomic RNA (sgRNA) is generated from this negative-stranded RNA intermediate. gRNAs are assembled by structural proteins to make infectious virus particles, and sgRNAs of various lengths include 4 structural proteins (spike, envelope, membrane and nucleocapsid proteins) and 6 accessory proteins (ORF3a, 6, 7a, 7b, 8 and 10).

Virus-targeting drugs are being developed in two major strategies. The first is a strategy to inhibit the entry of viruses into cells, and the second is a strategy to inhibit intracellular proliferation. The method to block the entry of the virus is the most efficient and effective method to block the penetration of the virus into the cell. In addition, if administered together with a drug for inhibiting intracellular proliferation, it is possible to more effectively inhibit the proliferation of viruses. A typical drug development process takes a long time and a lot of development cost because the development stages such as drug action point selection, screening, preclinical, and clinical are complex. In order to quickly respond to a new virus such as SARS-CoV-2, the use of a drug whose safety has been verified through the re-creation of a new drug can contribute to early suppression of the spread of the virus. In addition, it may be possible to discover substances with improved drug efficacy and delivery effects by finding the site of action of the discovered drug and changing the structure of the drug based on this information.

Therefore, in the present invention, pyronaridine and ebselen were discovered by testing the FDA-approved drug group in order to control SARS coronavirus 2, which started in December 2019 and has recorded a high infection rate and mortality rate so far. . Pyronaridine was developed as a treatment for malaria in 1970 and is currently undergoing phase 2/3 clinical trials as a treatment for SARS coronavirus 2 in combination with artesunate. Another drug, Epselen, is in clinical trials for its anti-inflammatory, antioxidant and antidepressant, deafness treatment and SARS-CoV-2 treatment. nsp5, a viral protease, is an enzyme required for virus propagation involved in SARS-CoV-2 viral polyprotein processing. When the activity of this enzyme is inhibited, the individual non-structural proteins cannot be produced, so that replication does not occur normally, thereby inhibiting virus proliferation. Therefore, the substance having an activity-inhibiting function discovered through the establishment of an nsp5-specific inhibitor screening assay can be used as a virus-specific therapeutic agent. Abselen used in the present invention has an activity of inhibiting the activity of nsp5 protein, which is a SARS coronavirus 2 protease, and is consistent with the results of Examples of the present invention.

The present inventors found that when pyronaridine, an inhibitor of coronavirus entry into cells, and epselen, an inhibitor of protease activity of coronavirus, are administered in combination, they simultaneously inhibit virus entry and intracellular proliferation, thereby showing a synergistic effect and showing a synergistic effect on SARS coronavirus 2 By revealing that it is possible to control the present invention was completed.

Korean Patent Publication No. 10-2021-0116008

One aspect is to provide an anti-coronavirus composition comprising a cell entry inhibitor of a coronavirus and an inhibitor of protease activity of a coronavirus.

Another aspect is to provide a pharmaceutical composition for the prevention or treatment of a coronavirus infection comprising a cell entry inhibitor of the coronavirus and an inhibitor of protease activity of the coronavirus.

Another aspect is to provide a method for preventing or treating a coronavirus infection, comprising administering a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus to a subject in need thereof.

Another aspect is to provide the use of a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus for the manufacture of a medicament for the prevention or treatment of coronavirus infection.

One aspect provides an anti-coronavirus composition comprising a cell entry inhibitor of a coronavirus and an inhibitor of protease activity of a coronavirus.

In one aspect, the cell entry inhibitor of the coronavirus may be one or more selected from the group consisting of chloroquine, hydroxychloroquine, tilorone and pyronaridine, specifically may be pyronaridine.

The chloroquine ((RS)-N'-(7-chloroquinolin-4-yl)-N, N-diethyl-pentane-1,4-diamine) is a compound of Formula 1 below, and has a molecular weight of 319.872 and C ₁₈ It consists of a molecular formula of H ₂₆ ClN ₃ , and may be derived from nature, synthesized using a known organic synthesis method, non-protein compounds, peptides, extracts of plant-derived tissues or cells, microorganisms (eg, For example, it may be a product obtained by culturing bacteria or fungi, and in particular yeasts):

[Formula 1]

.

.

The hydroxychloroquine (hydroxychloroquine, 2-[4-[(7-chloroquinolin-4-yl)amino]pentyl-ethylamino]ethanol) is a compound of Formula 2 below, and has a molecular weight of 335.872 and C ₁₈ H ₂₆ ClN ₃ O It consists of a molecular formula, may be derived from nature, may be synthesized using a known organic synthesis method, and may include non-protein compounds, peptides, extracts of plant-derived tissues or cells, microorganisms (eg, bacteria or fungi, and especially products obtained by culturing yeast:

[Formula 2]

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.

The tilorone (2,7-Bis[2-(diethylamino)ethoxy]-9-fluorenone dihydrochloride) is a compound of Formula 3 below, and has a molecular weight of 410.558 and a molecular formula of C ₂₅ H ₃₄ N ₂ O ₃ , , may be derived from nature, may be synthesized using known organic synthesis methods, non-protein compounds, peptides, extracts of plant-derived tissues or cells, microorganisms (eg, bacteria or fungi, and especially yeast) It may be a product obtained by culturing of:

[Formula 3]

.

.

The pyronaridine (pyronaridine, 4-[(7-chloro-2-methoxybenzo[b][1,5]naphthyridin-10-yl)amino]-2,6-bis(pyrrolidin-1-ylmethyl)phenol) is It is a compound of Formula 4 below, has a molecular weight of 518.06 and a molecular formula of C ₂₉ H ₃₂ ClN ₅ O ₂ , may be derived from nature, may be synthesized using a known organic synthesis method, and may be a non-protein compound , peptides, extracts of tissues or cells of plant origin, or products obtained by culturing microorganisms (eg bacteria or fungi, and in particular yeasts):

[Formula 4]

.

.

In one embodiment, in order to confirm the anti-coronavirus efficacy of pyronaridine (inhibitor of cell entry of coronavirus) on SARS-CoV-2, after infecting SARS-CoV-2 with a multiplicity of infection, pyronaridine was administered by concentration (0.5 μM, 1 μM, 2 μM, 4 μM, 10 μM, 20 μM, and 40 μM), cell viability was maintained over 80% from the 4 μM concentration of pyronaridine, and the 2 μM concentration showed no cytotoxicity. It was confirmed that the intracellular viral gene copy number was reduced (see Example 1).

In another embodiment, chloroquine, hydroxychloroquine, which is a cell entry inhibitor of coronavirus, in a cell line expressing hACE2, a SARS-coronavirus 2 receptor, in order to confirm the virus entry inhibitory effect of pyronaridine on SARS-CoV-2 As a result of treatment with (hydroxychloroquine) and pyronaridine at different concentrations (1 μM, 2 μM and 10 μM), the inhibitory effect of pyronaridine was statistically Significance was shown, and it was confirmed that it exhibited the most excellent effect compared to other compounds (see Example 2).

In one aspect, the inhibitor of protease activity of the coronavirus may be ebselen (ebselen).

The ebselen (2-Phenyl-1,2-benzoselenazol-3(2H)-one) is a compound of the following Chemical Formula 5, has a molecular weight of 274.18 and a molecular formula of C ₁₃ H ₉ NOSe, and is derived from nature. Non-protein compounds, peptides, extracts of plant-derived tissues or cells, and products obtained by culturing microorganisms (eg, bacteria or fungi, and especially yeast) It can be:

[Formula 5]

.

.

In one embodiment, in order to analyze the anti-coronavirus efficacy of a non-structural protein target drug, Huh7 cells (human liver cancer cells) were transfected with the SARS coronavirus subgenomic replicon plasmid, followed by remdesi, known as a viral RNA polymerase inhibitor. Cells were treated with 2 μM of remdesivir and 10 μM of epselen (an inhibitor of protease activity of coronavirus), which is known to inhibit protease activity, and the virus replication inhibitory ability was confirmed through luciferase activity. By confirming that both remdesivir and epselen lowered the luciferase activity, it was confirmed that these drugs acted on their respective targets to exhibit the effect of inhibiting virus replication.

In addition, using a FRET-based assay, Apselen's ability to inhibit SARS coronavirus 2 nsp5 3CL protease activity was confirmed. As a result, it was confirmed that in the group treated with epselen, protease activity was inhibited by epselen, and the fluorescence intensity was significantly suppressed (see Example 3).

In one aspect, the protease of the coronavirus may be a 3C-like protease (3CL protease: 3C-like protease) family of enzymes, specifically, nsp5 protease.

The term “protease” refers to an enzyme that hydrolyzes proteins, and is an enzyme that makes a mixture of amino acids or peptides.

The term "3C-like protease (3CL protease: 3C-like protease)" refers to a major protease found in coronaviruses, and cleaves coronavirus polyproteins at 11 conserved positions.

The "nsp5 protease" is a protease and is an enzyme necessary for virus propagation involved in viral polyprotein processing. When the activity of the enzyme is inhibited, the individual non-structural proteins cannot be produced, so that replication does not occur normally, thereby inhibiting virus proliferation.

In one aspect, the coronavirus may be SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus2.

The term "Corona virus" belongs to the Coronaviridae and is a single-stranded positive RNA virus with a size of about 100 to 220 nm having a spherical outer membrane.

The term "SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus2)" is a positive single-stranded RNA virus belonging to the coronavirus genus of the Coronaviridae family in its genetic structure.

The term "anti-coronavirus" refers to inhibiting the proliferation of viruses in a way that prevents the first corona virus from adsorbing/entering the cell or by preventing the synthesis of genetic material such as RNA of the virus that has entered the cell do.

The term "cell" means a cell that can be infected with a virus, in particular a coronavirus, and may be a plant or animal cell, and may be an isolated cell or a cell not isolated from a living organism, organ or tissue. . Specifically, the cell may be a mammalian cell, and more specifically, a human cell.

The term "cell entry" means that the genetic information of a virus is introduced into a host cell, and the term "cell entry inhibition" means preventing the virus from entering the cell in various ways, such as blocking a receptor. .

According to one aspect, when the composition comprises the inhibitor of protease activity of the coronavirus and the inhibitor of protease activity of the coronavirus, the composition inhibits the entry into cells of the coronavirus and inhibits the activity of the protease of the coronavirus By doing so, in the anti-coronavirus, a more pronounced synergistic effect can be exhibited.

In one aspect, the concentration ratio of the coronavirus cell entry inhibitor and the coronavirus protease activity inhibitor in the composition is 1:0.1 to 1:10, 1:0.1 to 1:5, 1:0.1 to 1: 4, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:4, 1:2 to 1:10, 1:2 to 1:5 or 1:2 to 1:4 days can

In one aspect, when the concentration ratio of the coronavirus cell entry inhibitor and the coronavirus protease activity inhibitor in the composition is 1:0.1 to 1:10, the number of viral gene copies in the cell is significantly reduced, more It may exhibit an excellent anti-coronavirus effect.

In one embodiment, by using pyronaridine and epselen in Vero E6 cells in parallel, epselen or pylorine prior to analyzing whether the anti-coronavirus efficacy is increased by simultaneously inhibiting SARS-coronavirus 2 entry inhibition and protease activity The effect on cell activity when naridine and epselen were used in combination was analyzed. As a result, epselen was not toxic to cells at the 40 μM treatment concentration, and the concentrations of epselen (2.5 μM, 5.0 μM, 7.5 μM and 10 μM) and pyronaridine (0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM and 2.5 μM), it was confirmed that cell activity was maintained at 80% or more even when treated with a combination of concentrations (see Example 4).

In addition, as a result of analyzing the anti-coronavirus efficacy using pyronaridine and epselen in parallel in Vero E6 cells, each concentration interval (pyronaridine concentration interval: 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM and 2.5 μM) It was confirmed that the viral gene copy number in cells was significantly decreased in the concentration interval of μM, epselen: 2.5 μM, 5.0 μM, 7.5 μM, and 10 μM), and the maximum in the combined treatment condition of pyronaridine 2.5 μM and epselen 7.5 μM It was confirmed that an anti-coronavirus synergistic effect appeared (see Example 4).

In addition, the combination treatment of pyronaridine 2.5 μM and epselen 7.5 μM showed high anti-coronavirus activity that reduced plaque formation to a level below the detection level (lower detection limit: 10 PFU/ml) than when used alone. was confirmed (see Example 4).

In another example, the use of pyronaridine/artesunate, which is currently undergoing clinical trials by changing its use from a treatment for malaria to a treatment for SARS-CoV-2, shows a synergistic effect and shows an anti-coronavirus effect when treated in combination. evaluated. As a result, it can be seen that artesunate reduced the cell activity to less than 20% at the concentration of 40 μM, and even when co-treated with pyronaridine 2.5 μM and artesunate at the concentrations of 2.5 μM, 7.5 μM and 12.5 μM, the cells It was confirmed that there was no significant toxicity (reduction in cell activity of 20% or less). The antiviral effect of pyronaridine in this co-administration combination was not augmented by artesunate. Through this, it was confirmed that the synergistic effect of the combined administration of pyronaridine and epselen was a combination-specific effect thereof (see Example 5).

In another example, the effect of concurrent administration of pyronaridine/epselen was compared with that of remdesivir, an RNA replication enzyme inhibitor, which is an injection. As a result, it was confirmed that remdesivir did not show any decrease in cell viability at concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM and 20 μM (see Example 6).

In addition, a comparison of the anti-coronavirus titers of remdesivir (0.5 μM, 1 μM, 5 μM and 10 μM) and pyronaridine/epselen (pyronaridine 2.5 μM and epselen 7.5, 10 μM) co-administration was performed. Experiments were performed on Vero E6 cell line infected with SARS-CoV-2. As a result of analyzing the residual viral RNA copy number in cells, it was confirmed that the combined administration of oral pyronaridine/epselen exhibited an anti-coronavirus effect to the extent that the US FDA-approved remdesivir injection drug (Example) 6).

Another aspect provides a pharmaceutical composition for preventing or treating a coronavirus infection, comprising a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus.

The "coronavirus cell entry inhibitor", "coronavirus protease activity inhibitor", "coronavirus", etc. may be within the above-described range.

The term "coronavirus infection (COVID-19: coronavirus disease 2019)" is a severe respiratory syndrome caused by a coronavirus, in one aspect, the coronavirus may be SARS coronavirus 2.

The term "prevention" refers to any action of suppressing the infection of the individual coronavirus or delaying the onset of the coronavirus infection by administration of the pharmaceutical composition according to an aspect.

The term "treatment" refers to any action in which the symptoms of an individual's coronavirus infection are improved or beneficially changed by administration of the pharmaceutical composition according to an aspect.

The term "administration" means introducing a given substance into a subject by an appropriate method, and "subject" means any living organism, including humans, rats, mice, livestock, etc., capable of carrying the coronavirus. As a specific example, it may be a mammal including a human.

In addition, the pharmaceutical composition may be provided as a pharmaceutical composition including an active ingredient alone, or one or more pharmaceutically acceptable carriers, excipients or diluents.

The term “pharmaceutically acceptable” means exhibiting properties that are non-toxic to cells or humans exposed to the composition.

Specifically, the carrier may be, for example, a colloidal suspension, powder, saline, lipid, liposome, microspheres or nanospherical particles. They may form complexes with, or be associated with, vehicles and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

When the pharmaceutical composition is formulated, commonly used lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. can Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycero geratin, etc. may be used, and well-known diluents or excipients may be used when preparing in the form of eye drops. have.

In one aspect, the pharmaceutical composition may be administered orally. The term "oral administration" means administering a drug through the mouth.

Specifically, solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, water It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. .

In one aspect, the entry inhibitor of the coronavirus and the inhibitor of protease activity of the coronavirus may be administered in combination, in addition to the case where they are provided simultaneously incorporated into the pharmaceutical composition.

The term "concomitant administration" refers to introducing two or more drugs that are used at the same time or almost simultaneously (one after another, administered on the same day, etc.) into a subject by an appropriate method.

In one aspect, when the corona virus entry inhibitor and the coronavirus protease activity inhibitor are co-administered, by inhibiting the protease activity of the coronavirus as well as inhibiting the entry into the cell of the coronavirus, In the prevention or treatment of coronavirus infection, a more pronounced synergistic effect may be exhibited. In addition, it is possible to obtain the effect of suppressing the appearance of drug-resistant virus than single treatment.

In one aspect, the concentration ratio when administering the corona virus entry inhibitor and the coronavirus protease activity inhibitor in combination is 1:0.1 to 1:10, 1:0.1 to 1:5, 1:0.1 to 1:4 , 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:4, 1:2 to 1:10, 1:2 to 1:5 or 1:2 to 1:4 have.

In one aspect, when the concentration ratio of the corona virus entry inhibitor and the coronavirus proteolytic activity inhibitor is 1:0.1 to 1:10, when the concentration ratio is 1:0.1 to 1:10, the number of viral gene copies in the cell is significantly reduced, a better corona It can show the effect of prevention or treatment of viral infection.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, activity of the drug, and the drug in the patient. Sensitivity, administration time, administration route and excretion rate, treatment duration, factors including concurrent drugs, and other factors well known in the medical field may be determined.

In one aspect, the administration of the pharmaceutical composition may be administered once a day, may be administered in divided doses. For example, it may be administered every other day, it may be administered one day a week. Specifically, the pharmaceutical composition may be administered at 0.001 to 1000 mg/kg/day, more specifically, at 0.1 to 100 mg/kg/day. The administration may be administered once a day or may be administered in several divided doses.

Another aspect provides a method for preventing or treating a coronavirus infection, comprising administering a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus to a subject in need thereof.

The "coronavirus cell entry inhibitor", "coronavirus protease activity inhibitor", "subject", "administration", "coronavirus infection", "prevention", "treatment", etc. may be within the above-described range. .

The method may be administered in parallel with a known composition or other pharmaceutical composition having a preventive or therapeutic effect on coronavirus infection, may be administered simultaneously, separately, or sequentially, may be administered single or multiple have. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

According to one aspect, when the cell entry inhibitor of the coronavirus and the protease activity inhibitor of the coronavirus are administered in combination, by inhibiting the entry of the coronavirus into the cell and inhibiting the activity of the protease of the coronavirus, In the prevention or treatment of coronavirus infection, a more pronounced synergistic effect may be exhibited. Furthermore, it is possible to obtain the effect of suppressing the emergence of drug-resistant viruses compared to the use of extensive poisoning of each drug.

Another aspect provides the use of a cell entry inhibitor of a coronavirus and an inhibitor of protease activity of a coronavirus for the manufacture of a medicament for the prevention or treatment of a coronavirus infection.

The "coronavirus infection", "prevention", "treatment", "coronavirus cell entry inhibitor", "coronavirus protease activity inhibitor", etc. may be within the above-described range.

According to an aspect, when the cell entry inhibitor of the coronavirus and the protease activity inhibitor of the coronavirus are administered in combination, the anti- It may exhibit a more pronounced synergistic effect in the coronavirus effect and the prevention, improvement or treatment effect of coronavirus infection.

1 is a diagram showing the results of analysis of the antiviral activity of pyronaridine against SARS coronavirus 2 (* is P <0.05).
2 is a SARS-CoV-2 spike pseudotyped virus in HEK293T-hACE2 [HEK293T cell line expressing human ACE2 (hACE2), a SARS-CoV-2 receptor] cells; The result of analyzing the inhibitory ability of chloroquine, hydroxychloroquine and pyronaridine to enter SARS-CoV-2 using a recombinant virus expressing the SARS-CoV-2 spike protein on the surface of murine leukemia retrovirus (MLV)] (** is P < 0.01, ns is not statistically significant).
3 is a diagram showing the results of analyzing the effect of remdesivir and epselen in inhibiting viral replication using the SARS coronavirus subgenomic replicon, and confirming the effect of epselen as an inhibitor of protease activity ( ***P < 0.001).
4 is a diagram showing the results of analysis of cytotoxicity, virus suppression ability and synergistic effect in Vero E6 cells during the combined treatment of pyronaridine and ebselen (** is P <0.01).
Figure 5 is a diagram showing the results of confirming the efficacy and cytotoxicity of the combined administration of pyronaridine and artesunate (artesunate) in Vero E6 cells in clinical trials (ns is not statistically significant).
6 is a diagram showing the results of analyzing the antiviral efficacy and cytotoxicity of the RNA polymerase inhibitor remdesivir, which has received FDA approval as a SARS coronavirus 2 treatment, and pyronaridine and epselen in combination (* is P < 0.05, ns is not statistically significant).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example

1. Analysis of antiviral activity of pyronaridine against SARS-CoV-2

Before examining the antiviral activity of pyronaridine against SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus2), Vero E6 cells (green monkey kidney cells; 2.2 x 10 ⁴ cells/96) -well-plate well) to measure the cytotoxicity of the drug.

After treating Vero E6 cells with pyronaridine by concentration (0.5 µM, 1 µM, 2 µM, 4 µM, 10 µM, 20 µM and 40 µM), 24 hours later, MTS (tetrazolium compound [3-(4,5) -dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) analysis showed that cell viability was not improved up to a concentration of 4 μM. It was confirmed that 80% or more was maintained (FIG. 1a).

In addition, after infecting SARS coronavirus 2 with a multiplicity of infection (MOI) of 0.01, it was treated at a concentration of 2 μM that did not show cytotoxicity, and the antiviral efficacy was confirmed after 24 hours. It was confirmed that 1log ₁₀ decreased (FIG. 1b).

2. Analysis of the mechanism of SARS-CoV-2 inhibition of pyronaridine

Transduction (transduction) of the SARS-CoV-2 spike-pseudotyped retrovirus into the HEK293T-hACE2 cell line (2 x 10 ⁵ cells/12-well-plate well) expressing the SARS-CoV-2 receptor hACE2 Chloroquine (chloroquine), hydroxychloroquine (hydroxychloroquine) and pyronaridine (pyronaridine) were treated by concentration (1 μM, 2 μM and 10 μM) before the hour (Fig. 2a). Then, a virus entry assay was performed to quantify the luciferase reporter activity expressed by the viral gene that entered the cell. Specifically, cells were washed 12 hours after transduction with a pseudotyped virus, and reporter activity was measured after an additional 36 hours incubation in complete medium.

As a result, it was confirmed that all three drugs had the effect of inhibiting entry, and the inhibitory effect of pyronaridine compared to chloroquine and hydroxychloroquine was statistically significant at a concentration of 2 μM, which did not significantly affect the cell viability of the drug, and excellent It was confirmed that the effect was shown (Fig. 2b).

3. Analysis of Antiviral Efficacy of Non-Structural Protein Targeted Drugs Using SARS Coronavirus Subgenomic Replicon

The antiviral efficacy of the non-structural protein target drug using the SARS-CoV subgenomic replicon was analyzed using the SARS-CoV subgenomic replicon plasmid ( FIG. 3A ). The replicon lacks a viral structural protein-coding gene, so it cannot form infectious virus particles, but a replica complex protein-coding gene required for replication, that is, a non-structural protein including nsp5 3CL protease (protease). Because it carries all of the coding genes, the antiviral efficacy of replication inhibitors and nsp5 protease inhibitors can be evaluated. Specifically, Huh7 cells (human liver cancer cells; 1 x 10 ⁵ cells/12-well-plate well) were transfected with the SARS coronavirus subgenomic replicon plasmid (transfection: transfection) and then a viral RNA polymerase inhibitor Cells were treated with 2 μM of remdesivir, known as remdesivir, and 10 μM of ebselen, which is known to inhibit nsp5 protease activity. evaluated.

As a result, it was confirmed that both remdesivir and epselen acted on the target to inhibit virus replication (FIG. 3b).

In addition, protease activity analysis was performed to confirm whether epselen actually inhibits the SARS-CoV-2 protease. Purified nsp5 protease and FRET substrate peptide (DABCYL-KTSAVLQSGFRKME-EDANS) were mixed and reacted at 25°C, and then the intensity of the fluorescence signal was measured using a fluorometer (excitation wavelength: 340 nm, emission wavelength: 535 nm). .

As a result, it was confirmed that the fluorescence intensity increased with time in the control group not treated with epselen, but it was confirmed that the fluorescence intensity was significantly suppressed by the protease activity was inhibited by epselen in the group treated with epselen ( Fig. 3c).

4. Analysis of the effect of combined treatment with pyronaridine and epselen

Since a single virus-targeting direct-acting antiviral agent (DAA) has a problem that can cause the emergence of a resistant virus rapidly, in order to effectively control SARS coronavirus 2, a combination of multiple direct-acting antiviral agents (DAAs) is used. may be desirable. In the present invention, by using pyronaridine and epselen, which have been approved by FDA for safety in Vero E6 cells (1 x 10 ⁵ cells/12-well-plate well), in parallel with SARS coronavirus 2 entry inhibition and proteolysis It was analyzed whether the antiviral efficacy was increased by simultaneously inhibiting the enzyme activity. Prior to the experiment, the cytotoxicity of the drugs alone and in combination treatment was evaluated. As a result, epselen showed no toxicity to cells at the 40 μM treatment concentration, and epselen (2.5 μM, 5.0 μM, 7.5 μM and 10 μM). ) and pyronaridine (0.5 µM, 1.0 µM, 1.5 µM, 2.0 µM and 2.5 µM), it was confirmed that cell activity was maintained at 80% or more (Figs. 4a and 4b). .

As a result of analyzing the antiviral efficacy under the same combined treatment conditions, each concentration interval (pyronaridine concentration interval: 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM and 2.5 μM, epselen concentration interval: 2.5 μM, 5.0 μM, 7.5 μM and 10 μM), it was confirmed that the viral gene copy number in the cell was significantly reduced (Fig. 4c). As a result of analyzing the synergistic effect using the Compusyn program based on these results, it was confirmed that the maximum antiviral synergistic effect (combination index, CI < 1) was shown in the combined treatment conditions of pyronaridine 2.5 μM and epselen 7.5 μM. (Fig. 4d).

In addition, the infectious virus titer was analyzed by a plaque formation assay using the supernatant recovered under the above conditions. As a result, it was found that the combination treatment of pyronaridine 2.5 μM and epselen 7.5 μM exhibited high antiviral activity that reduced plaque formation to the level below the detection level (lower detection limit: 10 PFU/ml), in contrast to treatment alone. was confirmed (Fig. 4e). In addition, as a result of Western blot analysis, it was confirmed that the expression of intracellular viral capsid protein (N) was significantly reduced ( FIG. 4f ).

The degree of decrease in the expression of the viral capsid protein is similar to the antiviral titer obtained when the RNA replication enzyme inhibitor, remdesivir, is treated at a concentration of 5 μM, and the above results show the possibility of using these combinations as oral therapeutics.

5. Pyronaridin/Artesunate Combination Tour Efficacy Analysis

A clinical trial is currently underway for the combined administration of pyronaridine/artesunate to evaluate the therapeutic effect of SARS-CoV-2. The present inventors co-administered pyronaridine/artesunate to Vero E6 cells (1 x 10 ⁵ cells/12-well-plate well), such as pyronaridine/ebselen The synergistic effect was evaluated.

As a result, in Vero E6 cells, artesunate did not show antiviral activity when administered alone at the concentrations of 2.5 μM, 7.5 μM, and 12.5 μM, and the antiviral titer of pyronaridine was decreased when administered in combination with pyronaridine 2.5 μM. It was confirmed that there is no enhancing effect (FIG. 5A).

In addition, MTS (tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) for the drug concentration Analysis was carried out. As a result, it was confirmed that there was no change in cell viability seen when pyronaridine was treated with 2.5 μM, and viability of 80% or more was maintained ( FIGS. 5B and 5C ).

The combined administration of pyronaridine and epselen exhibits synergy by inhibiting viral entry and nsp5 protease activity, thereby showing that it can exhibit higher antiviral efficacy than pyronaridine/artesunate. It shows that the co-administration of epselen is a drug-specific efficacy.

6. Comparative Analysis of Efficacy of Remdesivir and Pyronaridin/Epselen Combination Administration

Remdesivir, an injectable drug approved as a SARS-CoV-2 RNA replication enzyme-targeting treatment, shows high antiviral efficacy when analyzed in SARS-CoV-2 infected cells. After remdesivir treatment on Vero E6 cells (1 x 10 ⁵ cells/12-well-plate well), the pyronaridine/epselen combination that can be administered orally shows high antiviral efficacy found in the present invention under these conditions was treated to compare antiviral titers.

Prior to co-administration of drugs, cell viability of remdesivir alone was measured. As a result, it was confirmed that, when remdesivir was administered alone, it did not induce a decrease in cell activity (FIG. 6a).

As a result of comparing the antiviral efficacy of the pyronaridin/epselen combination and remdesivir alone, the two ratios of pyronaridine and epselen were administered (pyronaridine 2.5 μM and epselen 7.5 μM, pyronaridin 2.5). It was confirmed by analyzing the number of residual viral RNA copies in the cell that the combined administration of 10 µM and epselen (10 µM) could obtain a more significant inhibitory effect than the antiviral effect of 5 µM of remdesivir (Fig. 6b).

Claims (8)

A composition for an anti-coronavirus comprising a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus.
The composition of claim 1, wherein the protease of the coronavirus is a 3C-like protease (3CL protease: 3C-like protease) family of enzymes.
The composition of claim 1, wherein the protease of the coronavirus is nsp5 protease.
The method according to claim 1, wherein the coronavirus is SARS coronavirus 2 (SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus2), the composition.
The method according to claim 1, wherein the concentration ratio of the cell entry inhibitor of the coronavirus and the protease activity inhibitor of the coronavirus in the composition is 1:0.1 to 1:10, the composition.
A pharmaceutical composition for preventing or treating a coronavirus infection, comprising a cell entry inhibitor of coronavirus and an inhibitor of protease activity of coronavirus.
The pharmaceutical composition according to claim 6, wherein the concentration ratio of the coronavirus entry inhibitor and the coronavirus protease activity inhibitor in the pharmaceutical composition is 1:0.1 to 1:10.
The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is administered orally.

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