OA21043A - Pharmaceutical composition for preventing or treating epidemic RNA viral infectious disease. - Google Patents

Abstract

The present invention relates to a use of pyronaridine or a pharmaceutically acceptable salt thereof, and/or artemisinin or a derivative thereof for preventing or treating an epidemic RNA viral infectious disease, and more specifically, to a pharmaceutical composition for preventing or treating an epidemic RNA viral infectious disease, in particular, Coronavirus Disease 2019 (COVID19), the composition comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof, and/or artemisinin or a derivative thereof, together with a pharmaceutically acceptable carrier.

Description

DESCRIPTION

TITLE OF INVENTION

PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING EPIDEMIC RNA VIRAL INFECTIOUS DISEASE

TECHNICAL FIELD

The present invention relates to use of pyronaridine or a pharmaceutically acceptable sait thereof, and/or artemisinin or a dérivative thereof for the prévention or treatment of épidémie RNA virus infections. More specifically, the present invention relates to a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections—specifically, coronavirus disease 2019 (COVID-19), which comprises a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable sait thereof, and/or artemisinin or a dérivative thereof, together with a pharmaceutically acceptable carrier.

BACKGROUND ART

RNA viruses having RNA genomes hâve a higher mutation rate than DNA viruses and easily generate mutants adapted to changes in the host and environment. Due to this property, it is difficult to control RNA viruses through antiviral agents or prophylactîc vaccines. In addition, RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that synthesizes RNA using RNA as a template in the viral genome, and RNA polymerases of host cells—-which synthesize RNA using DNA as a template—cannot act on réplication of RNA viruses. RNA viruses are divided into posîtive-sense single-stranded, negative-sense sîngle-stranded and double-stranded dsRNA viruses according to the polarity of the genome and whether or not the genomic RNA is of an identical polarity to mRNA.

Acute viral infections—which hâve recently spread around the world and caused a global public health crisis—are rapidly spreading through transportation and trade from tire country where virus is originated to other countries, and there is a great global demand for the development of therapeutic agents. In particular, épidémies by influenza H INI flu in 2009, Ebola in West Africa in 2014 as well as in the Démocratie Republic of the Congo in 2019, and Zika in 2016 are ail RNA viruse infections.

Coronavîruses are viruses belonging to a positive-stranded RNA virus famîly like Zîka virus, hâve a positive-sense single-stranded RNA genome with a size of 25-32 kb, and are zoonotic viruses capable of infecting both human and animal cells, such as avian and niammalîan cells.

Coronavîruses hâve a structure in which characteristîc club-shaped spike proteins protrude from their outer envelopes. Coronavîruses are a family of viruses with various members including SARS-CoV, MERS-CoV, or SARS-CoV-2 (2019-nCoV) which causes Severe Acute Respiratory Syndrome (SARS) emerged in 2003, Middle East Respiratory Syndrome (MERS) newly emerged în Saudi Arabia in 2012, or Coronavirus Disease 2019 (COVID-19, 2019-nCoV infection) recently declared as a public health emergency of international concem (PHEIC) by the World Health Organization (WHO), respectively.

SARS-CoV causes Severe Acute Respiratory Syndrome, which originated în China in 2002 and spread worldwide, recording a mortality rate of about 10% in 8,096 patients. It is usually accompanied by high fever and myalgia, and after 2-7 days, a dry cough without sputum appears, causing respiratory failure in 10-20% of patients. Since an appropriate treatment has notyet been established, antibacterial agents for atypical pneumonia may be administered, in combination with antiviral agents such as oseltamivir or ribavîrin, or steroids.

MERS-CoV is assumed to be a virus transmitted from animai hosts such as camels to humans, causing severe acute respiratory syndrome and rénal failure, causing around 2,000 infections in 26 countries including the Middle East, with a mortality rate of 35.6% (WHO, 2016). The incubation period is about 5 days, and it is accompanied by fever, cough, shortness of breath, and pneumonia. It was prévalent in a mariner of lîmited transmission among family members or members within medical institutions, and commonly progressed to severe disease in people with underlying diseases such as diabètes.

SARS-CoV-2 (2019-nCoV) is a virus that causes COVID-19, and the first case was identified in Wuhan, China in 2019. After an incubation period of 1 -14 days, various respiratory symptoms, ranging from mîld to severe, such as cough, fever, malaise, shortness of breath, pneumonia, or acute respiratory distress syndrome, appear, and rarely sputum, sore throat and diarrhea appear. Because there is no sélective antiviral agent for it, symptomatic management alone or in combination with treatment with antiviral agents previously indîcated for other viral diseases is being used.

Specifically, COVID-19, the most recent outbreak among them, is spreading very quickly without any treatment or vaccine currently available (Li et al., 2020), and an appropriate cell or animal assay system for the disease has not yet been established. Currently, médications that hâve been reported to be clinically used or that are suggested in expert recommendations in China and Korea, includes chloroquine, remdesivir (Wang et ah, 2020), lopinavir, favipîravir, ribavîrin, interferon, etc., and more than 80 clinical trials are in progress (Maxmen et al., 2020). In particular, since SARS-CoV-2 belongs to the coronavirus family, reagents—previously known to hâve antiviral effects against MERS-CoV or SARS-CoV, which has approximately 79.5% homology in the nucléotide sequence to SARS-CoV-2—are attracting attention (Zhou et al., 2020), includîng reagents such as niclosamîde (Xu et al., 2020).

In view of SARS or COVID-19 cases, when having close contact to a patient, the risk of infection is very hîgh: viruses hâve a fairly high transmissîbility to many people in a densely populated environment by aerosolized respîratory droplets. In addition, acute viral infections caused by these coronaviruses are rapidly spreadîng through transportation and trade from the country where virus îs originated to other countries, thereby causing a global public health crisis.

Nevertheless, the development of an appropriate regimens for effectively inhibiting, treating or preventing such respîratory virus pathogens causing épidémies has been însufficient to date. As such, there îs an urgent need to develop a drug for counteracting theses diseases for the health and welfare of human beîngs around the world.

In addition, although the clinical symptoms of theîr respîratory infections are somewhat simîlar and they belong to the same RNA virus family, there are différences according to genetic and structural levels among viruses, and it is reported that these différences affect the sensitîvity and efficacy of antiviral drugs. Furthermore, these molecular-genetic différences in viruses cause différences in transmission routes, host receptors for virus binding, transmission rates, incubation periods and/or infection sites, leading to différences in clinical symptoms and therapeutic efficacy, and thus the development and application of appropriate reagent against the target virus are very important.

Also, given tire rapid transmission, high mortality, and global health and économie risks caused by the respîratory infections disease, in addition to the development of new drugs and vaccines that takes at least one to several years, it can be a very effective and cost-effective strategy to explore the possibility of preventing, improving or treating RNA virus infections, specifically coronavirus-induced respîratory diseases, based on drug repositioning of drugs whose safety has been guaranteed by previous clinical trials and practîcal use expérience.

DISCLOSURE OF INVENTION

TECHNICAL PROBLEM

An object of the présent invention is to provide use of pyronaridîne or a pharmaceutically acceptable sait thereof, and/or artemisinin or a dérivative thereof in the prévention or treatment of épidémie RNA virus infections.

Another object of the présent invention is to provide a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections comprising a therapeutically effective amount of pyronaridîne or a pharmaceutically acceptable sait thereof, and/or artemisinin or a derivatîve thereof, together with a pharmaceutically acceptable carrier.

Still another object of the present invention is to provide a method for the prévention or treatment of épidémie RNA virus infections by using pyronaridîne or a pharmaceutically acceptable sait thereof, and/or artemisînin or a dérivative thereof.

SOLUTION TO PROBLEM

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections comprising a therapeutically effective amount of pyronaridîne or a pharmaceutically acceptable sait thereof, and/or artemisînin or a dérivative thereof, together with a pharmaceutically acceptable carrier.

In addition, the present invention provides use of pyronaridîne or a phannaceutically acceptable sait thereof, and/or artemisînin or a dérivative thereof in the prévention or treatment of épidémie RNA virus infections.

Furthermore, the present invention provides a method for the prévention or treatment of épidémie RNA virus infections which comprises administering a therapeutically effective amount of pyronaridîne or a phannaceutically acceptable sait thereof, and/or artemisînin or a dérivative thereof to a subject in need thereof.

The present invention is described in detail hereinafter.

Accordîng to one aspect of the present invention, there is provided a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections comprising a therapeutically effective amount of pyronaridîne of the following Formula 1 or a pharmaceutically acceptable sait thereof, together with a pharmaceutically acceptable carrier:

[Formula 1]

Accordîng to another aspect of the present invention, there is provided a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections comprising a therapeutically effective amount of artemisînin of the following Formula 2 or a dérivative thereof, together with a pharmaceutically acceptable carrier:

According to another aspect of the présent invention, there is provîded a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections comprising a therapeutically effective amount of pyronarîdine of the above Formula l or a pharmaceutically acceptable sait thereof, and artemisinîn of the above Fonnula 2 or a derîvative thereof, together with a pharmaceutically acceptable carrier.

In one embodiment according to the présent invention, examples of the pharmaceutically acceptable sait of pyronarîdine may include an acid-addition sait which is formed from phosphoric acid, sulfuric acid, hydrochloric acid, acetîc aeîd, methanesulfonic acid, benzenesulfonîc acid, toluenesulfonic acid, maleic acid or fumaric acid, but are not limited thereto. In another embodiment according to the présent invention, the pharmaceutically acceptable sait of pyronarîdine may be pyronarîdine tetraphosphate.

In another embodiment according to the présent invention, examples of the artemisinîn derîvative may include dîhydroartemisinin, artesunate, artemether and arteether, but are not limited thereto. In another embodiment according to the présent invention, the artemisinîn derîvative may be artesunate.

According to another embodiment of the présent invention, in the pharmaceutical composition comprising a therapeutically effective amount of pyronarîdine or a pharmaceutically acceptable sait thereof, and artemisinîn or a derîvative thereof, together with a pharmaceutically acceptable carrier, a weight ratio of the pyronarîdine or a pharmaceutically acceptable sait thereof to the artemisinîn or a derîvative thereof may be 10:1 to l : 10. In another embodiment according to the présent invention, the weight ratio of the pyronarîdine or a pharmaceutically acceptable sait thereof to the artemisinîn or a derîvative thereof may be 1:1 to 6:1. In another embodiment according to the présent invention, the weight ratio of the pyronaridine or a pharmaceutically acceptable sait thereof to the artemîsinin or a dérivative thereof may be 1:1 to 4:1. In another embodiment according to the présent invention, the weight ratio of the pyronaridine or a pharmaceutically acceptable sait thereof to the artemisinin or a dérivative thereof may be 3:1.

As used herein, the term “pharmaceutically acceptable” refers to intoxicity that îs physiologically acceptable and does not inhibit the action of an active ingrédient when administered to humans, and does not usually cause gastrointestinal disorders, allergie réactions such as dizziness or simîlar reactions. The pharmaceutical composition of the présent invention may be formulated in various ways according to the route of administration by methods known in the art together with a pharmaceutically acceptable carrier. The route of administration is not limited thereto, but may be administered orally or parenterally. Parentéral routes of administration include, for example, various routes such as transdermal, nasal, intraperitoneal, întramuscular, subeutaneous, întravenous and the like.

When the pharmaceutical composition of the présent invention is orally administered, the pharmaceutical composition of the présent invention may be formulated in the form of powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer, injection, suppository and the like according to a method known in the art together with a suitable oral administration carrier. Examples of suitable carriers may include sugars including lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; starches including corn starch, wheat starch, rice starch and potato starch; celluloses including cellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose and low-substîtuted hydroxymethyl cellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, if necessary, crospovidone, sodium starch glycolate, croscarmellose sodium, sodium carboxymethyl cellulose, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may addîtionally include a glidant, an anti-aggregating agent, a plastîcizer, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative and the like.

Also, when administered parenterally, the pharmaceutical composition of the présent invention may be formulated in the form of injection, suppository, transdermal administration and nasal inhalant according to methods known in the art together with a suitable parentéral carrier. In the case of the injection, it must be sterilized and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injection include, but are not limited to, a solvent or dispersion medium containing water, éthanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), a mixture thereof and/or vegetable oil. More preferably, suitable carriers include Hanks’ solution, Ringer’s solution, phosphate buffered saline (PBS) or stérile water for injection containing triethanolamine, 10% éthanol, 40% propylene glycol, and isotonie solutions such as 5% glucose. In order to protecting the injection from microbial contamination, it may further comprise varions antibacterial and antifungal agents such as parabens, chlorobutanol, phénol, sorbic acid, thimerosal and the like. In addition, in most cases, the injection may further comprise an isotonie agent such as sugar or sodium chloride.

These formulations are described in the document (Remîngton’s Pharmaceutical Science, 15^th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania) commonly known prescription in pharmaceutical chemistry.

In the case of administration by inhalation, the compound for use according to the présent invention may be conveniently delivered in the form of an aérosol spray from a pressurized pack or a nebulizer by using a suitable propellant—for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aérosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, capsules and cartridges for use in inhalers or insufflators may be formulated to contain a powder mixture based on a suitable powder base.

As other pharmaceutically acceptable carriers, reference may be made to those described in the foilowing document (Remîngton’s Pharmaceutical Sciences, 19^th Edition, 1995 Mack Publishing Company, Easton, Pennsylvania).

In another embodiment of the présent invention, pyronarîdine or a phannaceutically acceptable sait thereof was formulated together with pharmaceutically acceptable carriers.

As described above, the “pharmaceutically acceptable carrier” that can be used in the présent invention may be any one conventionally used in the pharmaceutical field. Représentative examples may include lactose, dextrin, starch, pregelatinized starch, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, lowsubstituted hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, polyethylene glycol, Silicon dioxide, hydrotalcite, aluminum magnésium silicate, aluminum hydroxide, aluminum silicate, magnésium aluminometasilicate, bentonite and a mixture thereof. In addition to the carrier, the pharmaceutical composition of the présent invention may further comprise a disintegrant for raptd disintegration and dissolution in contact with an aqueous medium when adminîstered in vivo, a solubilizer or surfactant to increase dissolution or absorption, and a glidant or lubricant to increase fluidity or lubrîcation. Examples of the disintegrant may include crospovidone, sodium starch glycolate, croscarmellose sodium, sodium carboxymethylcellulose, agar, alginîc acid or sodium alginate. Examples of the glidant or lubricant may include colloïdal

Silicon dioxide, Silicon dioxide, talc, magnésium stéarate, calcium stéarate, zinc stéarate, sodium stéarate fumarate, stearic acid or Silicon dioxide. However, it is not limited to the recited examples. According to another embodiment of the présent invention, there is provided a pharmaceutical composition comprising 40 to 80% by weight of pyronaridine or a pharmaceutically acceptable sait thereof, l to 30% by weight of microcrystalline cellulose, 0.1 to 5% by weight of Silicon dioxide, 1 to 10% by weight of hydroxypropyl cellulose, 1 to 10% by weight of low-substituted hydroxypropyl cellulose, 2 to 20% by weight of sodium starch glycolate and 1 to 10% by weight of magnésium stéarate.

In another embodiment of the présent invention, artemîsinin or a dérivative thereof was formulated together with pharmaceutically acceptable carriers. In addition to the carrier, the pharmaceutical composition of the présent invention may further comprise a disintegrant for rapid disintegration and dissolution in contact with an aqueous medium when administered in vivo, a solubîlizer or surfactant to increase dissolution or absorption, and a glidant or lubricant to increase fluidity or lubrication. Examples of the carrier may include microcrystalline cellulose, lactose hydrate, mannitol, starch, pregelatînized starch, low-substituted hydroxycellulose, hydroxycellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose or hydroxypropyl methylcellulose. Examples of the disintegrant may include crospovidone, sodium starch glycolate, croscarmellose sodium, sodium carboxymethyl cellulose, agar, alginic acid or sodium alginate. Examples of the glidant or lubricant may include colloïdal Silicon dioxide, Silicon dioxide, talc, magnésium stéarate, calcium stéarate, zinc stéarate, sodium steaiyl fumarate, stearic acid or Silicon dioxide. Représentative examples of the surfactant may include sodium lauryl sulfate and a dérivative thereof, poloxamer and a dérivative thereof, saturated polyglycolized glyceride (aka gelucire), labrasol, various of polysorbate (for example, polyoxyethylene sorbitan monolaurate (hereinafter, Tween 20), polyoxyethylene sorbitan monopalmitate (hereinafter, Tween 40), polyoxyethylene sorbitan monostearate (hereinafter, Tween 60), polyoxyethylene sorbitan monooleate (hereinafter, Tween 80)), sorbitan esters (for example, sorbitan monolaurate (hereinafter, Span 20), sorbitan monopalmitate (hereinafter, Span 40), sorbitan monostearate (hereinafter, Span 60), sorbitan monooleate (hereinafter, Span 80), sorbitan trilaurate (hereinafter, Span 25), sorbitan trioleate (hereinafter, Span 85), sorbitan tristearate (hereinafter, Span 65)), cremophor, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil, sodium lauryl glutamate or disodium cocoamphodîaçetate. However, it is not limited to the recited examples. According to another embodiment of the présent invention, there is provided a pharmaceutical composition comprising 10 to 50% by weight of artemîsinin or a dérivative thereof, 30 to 70% by weight of microcrystallîne cellulose, 2 to 20% by weight of low-substituted hydroxypropyl cellulose, 2 to 20% by weight of sodium starch glycolate, 0.1 to 5% by weight of Silicon dioxide, 0.5 to 15% by weight of sodium lauryl sulfate and 0.1 to 5% by weight of magnésium stéarate.

In another embodiment of the present invention, pyronarîdine or a pharmaceutically acceptable sait thereof and artemisinin or a dérivative thereof were formulated together with pharmaceutically acceptable carriers. In addition to the carrier, the pharmaceutical composition of the present invention may further comprise a disintegrant for rapîd désintégration and dissolution în contact with an aqueous medium when adminîstered in vivo, a solubilizer or surfactant to increase dissolution or absorption, and a glidant or lubricant to increase fluidity or lubri cation. Examples of the carrier may include lactose, dextrin, starch, pregelatînized starch, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, lowsubstituted hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, polyethylene glycol, Silicon dioxide, hydrotalcite, aluminum magnésium silicate, aluminum hydroxide, aluminum silicate, magnésium aluminum metasilîcate, bentonite, butylhydroxytoluene and a mixture thereof. Représentative examples of the surfactant may include sodium lauryl sulfate and a dérivative thereof, poloxamer and a dérivative thereof, saturated polyglycolized glyceride (aka gelucire), labrasol, varions of polysorbate (for example, polyoxyethylene sorbitan monolaurate (hereinafter, Tween 20), polyoxyethylene sorbitan monopalmitate (hereinafter, Tween 40), polyoxyethylene sorbitan monostearate (hereinafter, Tween 60), polyoxyethylene sorbitan monooleate (hereinafter, Tween 80)), sorbitan esters (for example, sorbitan monolaurate (hereinafter, Span 20), sorbitan monopalmitate (hereinafter, Span 40), sorbitan monostearate (hereinafter, Span 60), sorbitan monooleate (hereinafter, Span 80), sorbitan trilaurate (hereinafter, Span 25), sorbitan trioleate (hereinafter, Span 85), sorbitan tristearate (hereinafter, Span 65)), cremophor, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil ), sodium lauryl glutamate or disodium cocoamphodiacetate, but are not limited thereto. Examples of the disintegrant may include crospovidone, sodium starch glycolate, croscarmeilose sodium, sodium carboxymethylcellulose, agar, alginic acid or sodium alginate. Examples of the glidant or lubricant may include colloïdal Silicon dioxide, Silicon dioxide, talc, magnésium stéarate, calcium stéarate, zinc stéarate, sodium stéarate fumarate, stearic acid or Silicon dioxide. However, it is not limited to the recited exampies. According to another embodiment of the présent invention, there is provided a pharmaceutical composition comprising 15 to 60% by weight of pyronarîdine or a pharmaceutically acceptable sait thereof, 5 to 20% by weight of artemisinin or a dérivative thereof, 5 to 30% by weight of microcrystalline cellulose, 10 to 40% by weight of crospovidone, 2 to 15% by weight of low-substituted hydroxypropyl cellulose, 1 to 10% by weight of sodium lauryl sulfate, 5 to 30% by weight of polyethylene glycol, 0.1 to 5% by weight of hydroxypropyl cellulose, 0.001 to 1% by weight of butylhydroxytoluene, 0.1 to 5% by weight of Silicon dîoxide and 0.5 to 10% by weight of magnésium stéarate.

The pharmaceutical composition of the présent invention may be formulated in powder, granule, tablet, capsule, dry syrup, coating préparation, injection, suppository, transdermal administration, inhalation administration and the like.

In another embodiment of the présent invention, the pharmaceutical composition of the présent invention may be administered in combination with one or more additional drugs having antiviral efficacy to prevent and treat épidémie RNA virus infections.

In another embodiment of the présent invention, examples of the other antiviral agents may include viral réplication inhibitors, helîcase inhibitors, viral protease inhibitors and viral cell entry inhibitors, but are not limited thereto. In another embodiment of the présent invention, the other antiviral agent may be, for example, ribavîrin, interferon, nîclosamide or a combination thereof, but is not limited thereto.

In another embodiment of the présent invention, examples of the épidémie RNA virus infections disease may include, but are not limited to, Zika virus infection, Ebola virus infection, and respiratory diseases caused by novel influenza virus and coronavirus infections. In another embodiment of the présent invention, examples of the respiratory diseases caused by the coronavirus infections may include, but are not limited to, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) or Coronavirus Disease 2019 (COVID-19). In another embodiment of the présent invention, the respiratory disease caused by the coronavirus infection may be Coronavirus Disease 2019 (COVID-19).

In the présent invention, the term “prévention” refers to any action that inhibîts or delays the occurrence, spread and récurrence of épidémie RNA virus infections by administering the phamaceutical composition of the présent invention, and the term “treatment” refers to any action in which the symptoms of the disease are improved or beneftcially changed by administering the pharmaceutical composition of the présent invention.

In addition, as used herein, the term “therapeutically effective amount” refers to an amount that exhibîts a higher response than a négative control, and preferably refers to an amount suffîcient to prevent or treat épidémie RNA virus infections. The therapeutic dose for a patient îs generally 50 to 2,000 mg/day, and more preferably 100 to 1,000 mg/day, depending on the severity of the condition and whether administered alone or in combination, or in combination with other drugs. It may be administered once a day or in divided doses via the oral or parentéral route. However, the therapeutically effective amount may be appropriatefy changed depending on varions factors such as the type and severity of disease, the âge, body weight, health status and gender of a patient, administration route, and treatment period.

Although the present invention describes the prévention and treatment of épidémie RNA virus infections in humans, preferably respiratory diseases caused by coronavirus infections in humans, and more preferably COVID-19 in humans, the present invention may be useful for the treatment of infectious RNA viruses, specifically viruses in Coronaviridae causing respiratory diseases, and more specifically the virus causing COVID-19, in animais and humans.

The problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned would be clearly understood by a person skilled in the art from the following description. In addition, the above description does not limit the claimed invention in any manner, furthermore, the combination of discussed features is not absolutely necessary for the inventive solution.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides a pharmaceutical composition for the prévention or treatment of épidémie RNA virus infections comprising a therapeutically effective amount of pyronaridîne or a phannaceutically acceptable sait thereof, and/or artemisînin or a dérivative thereof as active ingredient(s). The pharmaceutical composition accordîng to the present invention can be effectively used for the prévention or treatment by effectîvely înhibitîng épidémie RNA virus infections—for example, respiratory diseases such as COVID-19 caused by coronaviruses.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a concentration-response curve regarding the inhibitory effects against SARSCoV-2 and cytotoxicity by the pretreatment with pyronaridîne tetraphosphate—which is one of the active ingrédients of the present invention— when measured at 24 hours post-infection.

Figure 2 is a concentration-response curve regarding the inhibitory effects against SARSCoV-2 and cytotoxicity by the co-treatment with pyronaridîne tetraphosphate when measured at 24 hours post-infection.

Figure 3 shows the results in which the inhibitory effects against SARS-CoV-2 by cotreatment of artesunate—one of the active ingrédients of the present invention—were measured at hours and 48 hours post-infection and compared with those by chloroquine as a control.

Figure 4 shows the results comparing the inhibitory effects by the combination in various ratios of pyronarîdine tetraphosphate and artesunate in SARS-CoV-2-infected cells by the combinations, and at the optimal ratio, the inhibitory effects against viruses at 24 hours and 48 hours post-infection were compared.

Figure 5 is concentration-response curves regarding the inhibitory effects against SARSCoV-2 and cytotoxicity measured 24 hours and 48 hours post-infection in the human lung cell line, Calu-3 cells, in comparison with those by hydroxychloroquine as a control, when pyronarîdine tetraphosphate or artesunate was treated simultaneously with virus infection.

Figure 6 is a concentration-response curve regarding the inhibitory effects against SARSCoV-2 by post-infection treatment with pyronarîdine tetraphosphate or artesunate in the human lung cell line, Calu-3 cells, when measured 48 hours after drug treatment.

Figure 7 shows the results in which hamsters were infected with SARS-CoV-2 and orally administered with low or high doses of pyronarîdine tetraphosphate and artesunate in combination at 3:1 ratio at 1 hour post-infection once a day for 3 days, or with high dose of pyronarîdine tetraphosphate alone at 25 hours post-infection, and then the viral titers in lhe lungs were analyzed at day 4 post-infection and compared the virus-inoculated control group in which the drugs were not administered.

MODE FOR THE INVENTION

Hereinafter, the constitutions and effects of the présent invention will be described in more detail through examples. However, these examples are only illustrative, and the scope of the présent invention is not limited thereto.

Example 1: Préparation of pyronarîdine tetraphosphate monotablet

Hydroxypropyl cellulose was dissolved in éthanol to préparé a binding solution. After wet granulation of pyronarîdine tetraphosphate using the prepared binding solution, the obtained product was dried and granulated. Low-substituted hydroxypropyl cellulose, sodium starch glycolate, microcrystalline cellulose and Silicon dioxide were mixed. After lubrication by adding magnésium stéarate, tablets were prepared by tableting.

[Table 1]

Ingrédient content (mg/preparatîon)

Pyronaridine tetraphosphate	360
Microcrystalline cellulose	48
Silicon dioxide	6
Hydroxypropyl cellulose	12
Low-substituted hydroxypropyl cellulose	18
Sodium starch glycolate	24
Magnésium stéarate	12

Example 2: Préparation of artesunate monotablet

Silicon dioxide and sodium lauryl sulfate were sieved using a sieve. The sieved Silicon 5 dioxide and sodium lauryl sulfate were mixed with artesunate, microcrystalline cellulose, lowsubstituted hydroxypropyl cellulose and sodium starch glycolate, lubricated by adding magnésium stéarate, and then tabîetted to préparé tablets. The obtained product was coated with a filmcoating agent.

[Table 2]

Ingrédient content (mg/preparation)
Artesunate	100
Microcrystalline cellulose	228
Low-substituted hydroxypropyl cellulose	30
Sodium starch glycolate	20
Silicon dioxide	5
Sodium lauryl sulfate	12
Magnésium stéarate	5
Film-coating agent (Opadry)	12

Example 3: Préparation of pyronaridine tetraphosphate/artesunate combination tablet

Polyethylene glycol as a melting dispersîng carrier, butylhydroxytoluene and artesunate as an active ingrédient were mixed, melted by heating, and then rapidly cooled and finely pulverized. Then, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, 15 crospovidone and magnésium stéarate were mixed thereto to obtain Mixture 1. After dissolving hydroxypropyl cellulose în éthanol, pyronaridine tetraphosphate was wet-granulated, dried and granulated to obtain Mixture 2. Mixture 1, Mixture 2, sodium lauryl sulfate, Silicon dioxide and crospovidone were mixed, and then magnésium stéarate was added thereto to lubricate the mixture.

followed by tableting to préparé tablets. The obtained product was coated with a film-coating agent.

[Table 3]

Ingrédient content (mg/preparation)
Artesunate	60
Pyronaridîne tetraphosphate	180
Microcrystalline cellulose	93
Crospovidone	120
Low-substituted hydroxypropyl cellulose	38
Sodium lauryl sulfate	23
Polyethylene glycol	90
Hydroxypropyl cellulose	6
Butylhydroxytoluene	0.12
Silicon dioxide	4.5
Magnésium stéarate	16.5
Film-coating agent (Opadry)	20

In order to détermine whether pyronaridîne or a sait thereof, and artemisinin or a dérivative thereof of the présent invention hâve antiviral activity against coronavirus, the reagents were treated alone and in combination as in the following Experimental Examples, and the inhibitory rates against viral infection were evaluated.

Experimental Example 1: Evaluation of antiviral effects of pyronaridîne tetraphosphate (pretreatment)

In Experimental Example 1, before infecting cells with SARS-CoV-2 (a Korean isolate), pyronaridîne tetraphosphate was pretreated for 1 hour, and the inhibitory efficacy against virus infection was evaluated.

1) Préparation of viruses and host cells

Vero cells were purchased from the American Type Culture Collection (ATCC) and incubated at 37°C with 5% COs in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% heat-inactivated fêtai bovine sérum (FBS) and an antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prévention (KCDC). After virus amplification, the viral titers were determined by a plaque assay by counting viral plaques formed in the cells used for virus amplification upon infection with the virus.

2) Détermination of antiviral efficacy using immunofluorescence staining îmagings

Vero cells were seeded at 1.2 χ 10⁴ cells per well in pClear plates, and 24 hours prior to the experiment cells were pre-treated for 1 hour with a sériés of 10 dilutions of drugs în culture media in the range of 0.05-50 μΜ, and then SARS-CoV-2 was inoculated to the cells at a multiplicity of infection (MOI) of 0.0125. Twenty-four hours after infection, the cells were fixed with 4% formaldéhyde, and the infected cells were analyzed by immunofluorescence stainîng using an antibody against N protein of SARS-CoV-2. The infection rate was calculated as the ratio of the number of infected cells to the total number of cells compared to the positive and négative Controls through the imaging analysis program. The antîviral effect of the drug is represented as a concentration-response curve, and using the Graph Prism (Ver. 8) analysis program, 50% effective concentration (ECso, concentration that inhibits virus infection-induced cytotoxicity by 50%) and 50% cytotoxic concentration (CC50, the concentration of the compound that causes damage in 50% of cells in coniparîson with normal cells) was calculated as shown în Equation 1.

<Equation 1>

Sigmoidal model, Y= Bottom + (Top - Bottom)/(1 + (IC5o/X)^Hllls!ope)

As a resuit, as shown in Figure 1, in the case of Vero cells infected with SARS-CoV-2, 70% virus inhibition rate was observed at a concentration of 50 μΜ of pyronaridine, but cytotoxicity was also increased by 17% by drug pretreatment. The Vero cells were isolated from the kidney épithélial cells of African green monkeys (Chlorocebus sp.) and hâve been known as type-1 IFN-deficient cells. In the previous study, when measuring the in vitro antiviral efficacy of pyronaridine against Ebola virus in Vero cells, no anti viral activity was observed at a concentration below CC50 (CC50 = 1.3 μΜ), but when inoculated into human-derived Hela cells, ît was reported that the CC50 is higher and the antiviral activity showed at a non-toxic concentration (EC50 = 0.42-1.12 μΜ, CCso = 3.1 μΜ). In addition, pyronaridine significantly inhibited mortality and viral infection rate in mouse models challenged with Ebola virus (Lane et al., 2015). It is well known that the efficacy of antiviral agents may dîffer in in vitro or in vivo assay Systems dependîng on the différences in characterîstics of the host cells tested and their intracellnlar immune signaling pathways (Lane et al., 2015), and thus human-derived host cell-based assays were additionally established, and the antiviral efficacy of pyronaridine was further confirmed in various cell fines and animal studies.

Experimental Example 2: Inhibitory effects of pyronaridine tetraphospliate against SARSCoV-2 virus (co-treatment)

In Experimental Example 2, the inhibitory effects of pyronaridine against viral infection were evaluated when co-treated cells at the time of SARS-CoV-2 (a Korean isolate) infection. Chloroquine îs known to exhîbit antiviral efficacy by increasing endosomal pH leading to inhibition of viral binding to the cells and glycosylation of host receptors to SARS-CoV (Vincent et al._s 2005). Since pyronarîdine—which has a structure similar to chloroquine—was also expected to act via a similar mechanism, pyronarîdine was simultaneously treated at the time of viral infection and its antiviral efficacy was measured. By optimizing some experimental conditions used in Experimental Example 1, the experiment was carried out under test conditions with relatively low cytotoxicity.

1) Préparation of viruses and host cells

Vero cells were incubated at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented wîth 10% heat-inactivated fêtai bovine sérum (FBS) and an antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prévention (KCDC). After viral amplification, the viral titers were determined through qRT-PCR measuring RM A copy numbers.

2) Measurement of antiviral efficacy using RNA copy numbers

After dissolving pyronarîdine tetraphosphate in DMSO, it was diluted to a concentration of 0.033-100 μΜ using culture media. Twenty-four hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2^X 10⁴ cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. Twenty-four hours after infection, the cell supematant was collected, RNA was extracted, and qRT-PCR was performed against the RdRp gene. The antiviral efficacy of the drug was analyzed by comparing the copy number of viral RNA with the drug with that with the control. A drug concentrationresponse curve was drawn with the viral infection inhibition rate (% inhibition) from the virus titer inversely calculated from the RNA copy number, and 50% effective concentration (EC50, the concentration that înhîbits virus titer by 50%) was calculated using the Graph Prism (Ver. 8) analysis program, as in Experimental Example 1.

3) Measurement of cytotoxicity (%cytotoxicity)

Cytotoxicity was measured using a tétrazolium salts-based assay (WST-1). WST-1 is converted into a chromogenic substance called fonnazan by mitochondrial dehydrogenases, which are présent only in livîng cells. After adding 10 μΕ of WST-1 premix to each well, celles were incubated for 1 additional hour, and the amount of fonnazan produced was calculated with its absorbance measured by ELISA. The 50% cytotoxic concentration (CC₅o, the concentration of compound that causes damage in 50% of cells compared with normal cells) was calculated.

As a resuit, as shown in Figure 2, pyronarîdine exhibîted the antiviral effect in a concentration-dependent manner when co-treated, and cytotoxicity was observed at some high concentrations, but antî viral activîty against SARS-CoV-2 virus, more than 90% inhibition, at noncytotoxîc concentrations (EC50 = 8.27 μΜ, CCso ~ II .54 μΜ; selectivity index, SI>1.40).

Experimental Example 3: Inhibitory effects of artesunate against SARS-CoV-2 (cotreatment)

In Experimental Example 3, the antiviral efficacy of artesunate was measured under the same experimental conditions as in Experimental Example 2.

1) Préparation of virus and host cell

Vero cells and viruses were prepared in the same manner as shown in Experimental Example 2.

2) Measurement of antiviral efficacy using RNA copy numbers

Artesunate was dissolved in DMSO and then diluted to concentrations of 3.13, 12.5, and 50 μΜ using media. Twenty-four hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2*10⁴ cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. At 24 hours and 48 hours after infection, the cell supematant was collected, and qRT-PCR was performed against the RdRp gene to calculate the virus titer and to calculate the virus infection inhibition rate (% inhibition) as shown in Experimental Example 2. As the control, chloroquine was used.

3) Measurement of cytotoxicity (%cytotoxîcîty)

Cytotoxicity was measured in the same manner as shown in Experimental Example 2.

As a resuit, as shown in Figure 3, artesunate exhibîted the antiviral activîty in a concentratîon-dependent manner and showed 83% inhibition rate against virus at a concentration of 50 μΜ. At 12.5 μΜ, the inhibition rates were 40% and 51% at 24 hours post-infection (24 hpi) and 48 hours post-infection (48 hpi), respectively. In addition, at 3.13 μΜ, the inhibition rate was 39% at 48 hours post-infection. There was no signifîcant cytotoxicity in ail conditions tested. Artesunate had a lower ECso and slower onset time for SARS-CoV-2 inhibition compared to pyronarîdine or chloroquine used as the control, but had a long-lastîng antiviral effect, showing a pattern in which the virus inhibition rate increased slowly over time.

Experimental Exampk 4: Inhibitory effects of pyronaridine tetraphosphate/artesunate combination against SARS-CoV-2

Unlike chloroquine, which did not show antiviral effect in the guinea pig models infected with Ebola virus, pyronaridine significantly improved the virus titer and survival rate in Ebola vîrus-challenged mouse models. As such, it was assumed that chloroquine may hâve different mechanisms of action în addition, among which immunomodulatory mechanisms such as type 1 IFN-1 pathway were suggested (Lane et al., 2019). Artesunate also showed an antiviral efficacy against Ebola virus in in vitro assays but weaker than pyronaridine (Gîgnox et al., 2016). Therefore, in Experimental Example 4, the changes in antiviral efficacy according to treatment in combination at different combination ratio of the two drugs were evaluated.

1) Préparation of virus and host cells

Vero cells and virus were prepared in the same manner as shown in Experimental Example 2.

2) Measurement of antiviral efficacy using RNA copy numbers

Pyronaridine tetraphosphate and artesunate were dissolved in DMSO, diluted to various concentrations using media at various combination ratios such as 1:1,3:1,10:1, etc. Twenty-four hours before the experiments, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2*10⁴ cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. At 24 hours after infection, the cell supematant was collected, and qRT-PCR was performed against the RdRp gene to calculate the virus titer and the virus infection inhibition rate (% inhibition) as shown in Experimental Example 2. When 10 μΜ of pyronaridine tetraphosphate and 3.3 μΜ of artesunate were treated in combination, virus titers were measured at 24 and 48 hours after infection each, and compared with chloroquine and lopinavir used as the Controls.

3) Measurement of cytotoxicity (%cytotoxicity)

Cytotoxicity was measured in the same manner as shown in Experimental Example 2.

As a resuit, as shown in Figure 4, the antiviral effect of artesunate treated in combination with pyronaridine was higher than that of artesunate treated alone, and the antiviral effect increased as the ratio of pyronaridine in the combination was higher. Specifically, when 10 μΜ of pyronaridine tetraphosphate and 3.3 μΜ of artesunate were combined (ratio 3:1), it exhibited the inhibition rate of 90-100% against virus infection, which is a higher antiviral effect than that of chloroquine or lopinavir used as the Controls. In this case, the inhibition rate against infection was maintained up to 48 hours. No significant cytotoxicity was observed in ail combination ratios shown in Figure 4, and when 10 μΜ of pyronarîdine tetraphosphate and 3.3 μΜ of artesunate were treated in combination, Iower cytotoxicity was observed in comparison to the treatment with 10 μΜ of pyronarîdine tetraphosphate alone (49.5% decrease).

Experimental Example 5: Inhibitory effects of pyronarîdine tetraphosphate or artesunate against SARS-CoV-2 in human lung cell lines (co-treatment)

It has been reported that there may be species différence among the antiviral actions in humans and other animais, such as receptor structures. Therefore, in order to confirm the efficacy in human lung cell lines, in Experimental Example 5, when SARS-CoV-2 (a Korean isolate) was inoculated into Calu-3 cells (human lung cell line), pyronarîdine phosphate or artesunate was treated and evaluated for efficacy in inhibiting viral infection, the inhibitory effects of pyronarîdine tetraphosphate or artesunate against viral infection were evaluated in human lung cell lines, Calu3 cells, when co-treated cells at the time of SARS-CoV-2 (a Korean isolaie) infection.

) Préparation of viruses and host cells

Calu-3 cells were incubated at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% heat-inactivated fêtai bovine sérum (FBS) and an antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prévention (KCDC).

2) Measurement of antiviral efficacy using RNA copy numbers

After dissolving in DMSO, pyronarîdine tetraphosphate was diluted to a concentration of 0.033-100 μΜ using media. Twenty-four hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2xl0⁴ cells/well (MOl=0.01), and the culture media containing various dilutions of drug were added to each well. At 24 hours and 48 hours after infection, qRT-PCR was performed against the RdRp gene as shown in Experimental Example 2. A drug concentration-response curve was drawn with the viral infection inhibition rate (% inhibition) from the virus titer inversely calculated from the RNA copy number, and 50% effective concentration (EC50, the concentration that inhibits virus titer by 50%) was calculated using the Graph Prism (Ver. 8) analysis program, as shown in Experimental Example 1.

3) Measurement of cytotoxicity (%cytotoxicity)

Cytotoxicity was measured in the same manner as in Experimental Example 2.

As a resuit, as shown in Figure 5, both pyronarîdine and artesunate exhibited antiviral effects in human lung cell lines in a concentration-dependent manner when co-treated at the time of infections. In addition, at both 24 hours post-infection (24 hpi) and 48 hours post-infection (48 hpî), they exhibited antiviral activities against SARS-CoV-2, more than 90% inhibition, at noncytotoxîc concentrations (pyronaridine 48 hours post-infection IC50 = 8.58 μΜ, CC50 > 100 μΜ; selectivity index, SI>11.66; artesunate 48 hours post-infection IC50 = 0.45 μΜ, CC50 >100 μΜ;

selectivity index, SI >220.8). Specificaliy, in the case of artesunate, the effect was significantly increased in the human lung cell lines compared to that in the monkey kidney cell lines, Vero cells. Contrary to those, hydroxychloroquine showed no anti viral effect at less than 50 μΜ in the human lung cell lines, whereas hydroxychloroquine showed antiviral efficacy in the monkey cell lines.

Experimental Example 6: Inhibitory effects of post-infection treatment with pyronaridine tetraphosphate or artesunate against SARS-CoV-2 in human lung cell lines

In Experimental Example 6, pyronaridine tetraphosphate or artesunate was treated each in Calu-3 cells at 0, 2, 4, 6, 8, 10, 12, 24 and 36 hours after SARS-CoV-2 (a Korean isolate) inoculation, and evaluated how long hours the inhibitory effect of each drug were retained against 15 virus infections.

1) Préparation of viruses and host cells

Calu-3 cells and viruses were prepared in the same manner as shown in Experimental Example 5.

2) Détermination of antiviral efficacy using viral plaque assay

After dissolving pyronaridine tetraphosphate and artesunate in DMSO each, it was diluted to 12.5 μΜ using media. At 1 hour after the inoculation with SARS-CoV-2 (MOI=0.1), the supematant was removed, and Calu-3 cells were washed, followed by the addition of DMEM culture media containing 2% bovine sérum. The culture media containing drugs were added at

0, 2, 4, 6, 8,10,12, 24 and 36 hours each. At 48 hours after each drug treatment, cell supernatants were harvested and a plaque assay—in which the plaques generated by infections virus infection were counted in Vero cells, cells used for virus amplification—was performed. The DMEM-F12 medium layer containing 2% agarose was laid on the layer of infected Vero cells, and the number of plaques was counted by using counter-staining with crystal violet, after incubation for 72 hours.

The antiviral efficacy of the drug was analyzed with the viral infection inhibition rate (% inhibition) from the virus titer inversely calculated from the number of plaques formed and compared with the control.

As a resuit, as seen in Figure 6, the maximum antiviral efficacy was shown when 12.5 μΜ 35 pyronaridine was treated (>99% inhibition when added at up to 6 hours post-infection, >94% inhibition when added at up to 12 hours post-infection, and 90% inhibition when added at up to 24 hours post-infection). On the other hand, 12.5 pM artesunate treatment showed 92-96% inhibition when added at up to 6 hours post-infection, >90% inhibition when added at up to 12 hours post-infection, and 48% inhibition when added at up to 24 hours post-infection.

Experimental Example 7: Inhibitory effects of pyronarîdine tetraphosphate/artesunate combination against SARS-CoV-2 in COVID-19 animal models

In Experimental Example 7, pyronarîdine tetraphosphate and artesunate (combination in a 3:1 ratio) were orally administered to hamsters infected with SARS-CoV-2 (a Korean isolate) to evaluate in vivo antiviral efficacy in animais.

1) Préparation of viruses and hamsters for SARS-CoV-2 inoculation

SARS-CoV-2 virus was provîded by the Korea Centers for Disease Control and Prévention (KCDC). As experimental animal models, Syrian hamsters—which showed high susceptibility to SARS-CoV-2 and had low restriction in supply—were used, and SARS-CoV-2 (1 x 10⁶ PFU/100 pL) was inoculated to each of both nasal passages of the hamster with an amount of 50 pL.

2) In vivo antiviral efficacy measurement using plaque assay

At 1 hour after nasal inoculation with SARS-CoV-2, pyronarîdine tetraphosphate (180 mg/kg or 360 mg/kg) and artesunate (60 mg/kg or 120 mg/kg) were orally administered as the combination of 3:1 ratio once a day for 3 days, and in vivo antiviral efficacy of the combination of two drugs against SARS-CoV-2 was evaluated. As the comparative experimental group, pyronarîdine 360 mg/kg alone was orally administered once at 25 hours after infection to evaluate the duration of post-infection efficacy of pyronarîdine alone. Both pyronarîdine tetraphosphate and artesunate were prepared just before use, completely dissolved in 5% sodium bicarbonate, and orally administered. As the control groups, a normal control group (Mock) în which virus was not inoculated and a vehicle control group in which only a solvent was administered at the same time were used. At 4-day post-infection, both the left and rîght lobes of the lungs were excised. the virus was extracted, and the virus titers în the lung tissues were analyzed by a plaque assay as described în Experimental Example 6. The viral tîter in the lungs quantified by a plaque assay was normalized to the total weight (g) of the lung tissues, and then converted to log value to calculate the final tîter (Logw plaque forming unit/g, LogioPFU/g).

As a result, as shown in Figure 7, at 4-day post-infection, the réduction in tire titer of the infectious virus in the lung tissues was statistically significant in both the pyronarîdine tetraphosphate (P)-artesunate (A) 180/60 mg/kg and 360/120 mg/kg co-administration groups [médian LogioPFU: 8.30 for virus-challenged vehicle control group vs. 7.22 for PA 180/60 mg/kg Co-administration group (p<0.001); vs. 7.61 for PA 360/120 mg/kg co-administration group (p=0.046)]. On the other hand, when pyronaridine tetraphosphate alone was orally administered, 5 a significant decrease in the infectious virus titer was observed in the lung tissue when high dose of 360 mg/kg was administered, and a significant inhibitory effect was observed even if it was administered once at 25 hours post-infection [médian LogioPFU 8.30 for virus-challenged vehicle control group vs. single high-dose 25 hpi administration group 7.22 (p<0.001)].

Claims (16)

1. Use of a therapeutically effective amount of pyronaridîne or a pharmaceutically acceptabl e sait thereof in the manufacture of a médicament for the prévention or treatment of épidémie RNA virus infections.

2. Use of a therapeutically effective amount of artemisînin or a dérivative thereof in the manufacture of a médicament for the prévention or treatment of épidémie RNA virus infections.

3. Use of a therapeuticalfy effective amount of pyronaridîne or a pharmaceutically acceptable sait thereof, and artemisînin or a dérivative thereof in the manufacture of a médicament for the prévention or treatment of épidémie RNA virus infections.

4. The use accordîng to Claim 1 or 3, wherein the pharmaceutically acceptable sait of pyronaridîne is selected from the group consisting of phosphate, sulfate, hydrochlorîde, acetate, methanesulfonate, benzenesulfonate, toluenesulfonate, maleate and fumarate.

5. The use accordîng to Claim 4, wherein the phannaceutically acceptable sait of pyronaridîne is pyronaridîne tetraphosphate.

6. The use accordîng fo Claim 3, wherein a weight ratio of the pyronaridîne or a pharmaceutically acceptable sait thereof to the artemisînin or a derivatîve thereof is 10:1 to 1:10.

7. The use accordîng to Claim 6, wherein the weight ratio of the pyronaridîne or a pharmaceutically acceptable sait thereof to the artemisînin or a derivatîve thereof is 1:1 to 6:1.

8. The use accordîng to Claim 7, wherein the weight ratio of the pyronaridîne or a pharmaceutically acceptable sait thereof to the artemisînin or a derivatîve thereof is 3:1.

9. The use accordîng to Claim 2 or 3, wherein the artemisînin derivatîve is selected from the group consisting of dihydroartemisinin, artesunate, artemether and arteether.

10. The use accordîng to Claim 9, wherein the artemisînin derivatîve is artesunate.

11. The use accordîng to any one of Claims 1 to 3, wherein the médicament further comprises at least one other antiviral agent.

12. The use according to Claim 11, wherein the other antiviral agent is selected from the group consisting of a viral réplication inhibitor, a helicase inhibitor, a viral protease inhibitor and a viral cell entry inhibitor.

13. The use according to Claim 12, wherein the other antiviral agent is selected from the group consisting of rîbavirin, interferon, nîclosamide and a combination thereof.

14. The use according to any one of Claims 1 to 3, wherein the épidémie RNA virus infectious disease is selected from the group consisting of Zika virus infection, Ebola virus infection, respiratory diseases caused by novel înfluenza virus or coronavirus infections.

15. The use according to Claim 14, wherein the respiratory disease caused by coronavirus infection is selected from the group consisting of Severe Acute Respiratory syndrome (SARS), Mîddle East Respiratory Syndrome (MERS) and Coronavirus Disease 2019 (COVID-19).

16. The use according to Claim 15, wherein the respiratory disease caused by coronavirus infection is Coronavirus Disease 2019 (COVID-19).