Pharmaceutical composition for preventing or treating epidemic RNA virus infectious diseases

Abstract

The present invention relates to the use of pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof for preventing or treating an epidemic RNA virus infectious disease, and more specifically, to a pharmaceutical composition for preventing or treating an epidemic RNA virus infectious disease, especially COVID-19, which composition comprises a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof, and a pharmaceutically acceptable carrier.

Description

Technical Field

[0001] The present invention relates to the use of pyronaridine or a pharmaceutically acceptable salt thereof, and / or artemisinin or a derivative thereof for preventing or treating epidemic RNA virus infection. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating epidemic RNA virus infection, especially COVID-19, which comprises a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof, and / or artemisinin or a derivative thereof, and a pharmaceutically acceptable carrier. Background Art

[0002] RNA viruses with RNA genomes have a higher mutation rate than DNA viruses and are prone to produce mutants that adapt to host and environmental changes. Due to this characteristic, it is difficult to control RNA viruses through antiviral agents or preventive vaccines. In addition, RNA viruses encode an RNA-dependent RNA polymerase (RdRp), which synthesizes RNA using the RNA in the viral genome as a template, while the host cell's RNA polymerase synthesizes RNA using DNA as a template and cannot act on the replication of RNA viruses. RNA viruses are divided into positive-stranded single-stranded RNA viruses, negative-stranded single-stranded RNA viruses, and double-stranded RNA (dsRNA) viruses based on the polarity of the genome and whether the polarity of the genomic RNA is the same as that of the mRNA.

[0003] Acute viral infections that have recently spread around the world and caused global public health crises are spreading rapidly from the country of origin of the virus to other countries through transportation and trade, and there is a huge global demand for the development of therapeutic drugs. In particular, the epidemics caused by H1N1 influenza in 2009, Ebola virus in West Africa in 2014 and the Democratic Republic of Congo in 2019, and Zika virus in 2016 are all RNA viral infections.

[0004] Coronaviruses, like Zika virus, are viruses belonging to the positive-strand RNA virus family, have a positive-stranded single-stranded RNA genome of 25-32 kb in size, and are zoonotic viruses capable of infecting cells of humans and animals such as birds and mammals. Coronaviruses have a structure in which a characteristic rod-shaped spike protein protrudes from its outer membrane. Coronaviruses are a family of viruses with multiple members, including SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV), which respectively cause severe acute respiratory syndrome (SARS) in 2003, Middle East respiratory syndrome (MERS) newly emerged in Saudi Arabia in 2012, and COVID-19 (2019-nCoV infection), which has recently been declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO).

[0005] SARS-CoV causes severe acute respiratory syndrome, which has been documented to have a mortality rate of approximately 10% in 8,096 patients. It is usually accompanied by high fever and myalgias, followed by a dry cough without sputum after 2-7 days, and respiratory failure in 10-20% of patients. As appropriate treatment has not yet been established, antibacterial drugs for atypical pneumonia can be used in combination with antiviral agents such as oseltamivir or ribavirin or steroids.

[0006] MERS-CoV is believed to be a virus transmitted to humans from animal hosts such as camels, which can cause severe acute respiratory syndrome and renal failure, causing about 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 spreads in a limited manner among family members or members of medical institutions, and often develops into severe disease in people with underlying diseases such as diabetes.

[0007] SARS-CoV-2 (2019-nCoV) is the virus that causes COVID-19. After an incubation period of 1-14 days, respiratory symptoms ranging from mild to severe appear, such as cough, fever, fatigue, shortness of breath, pneumonia or acute respiratory distress syndrome, and rarely sputum, sore throat and diarrhea. Since there are no selective antiviral agents against it, symptomatic treatment alone or in combination with treatment with antiviral agents previously used for other viral diseases is being used.

[0008] Specifically, the most recent outbreak of COVID-19 has spread rapidly without any treatment or vaccine currently available (Li et al., 2020), and suitable cell or animal detection systems for the disease have not yet been established. Currently, drugs reported for clinical use or recommended by experts in China and South Korea include chloroquine, remdesivir (Wang et al., 2020), lopinavir, favipiravir, ribavirin, interferon, etc., and more than 80 clinical trials are underway (Maxmen et al., 2020). In particular, since SARS-CoV-2 belongs to the coronavirus family, agents previously known to have antiviral effects against MERS-CoV or SARS-CoV (the homology of SARS-CoV nucleotide sequence to SARS-CoV-2 is about 79.5%) have attracted attention (Zhou et al., 2020), including agents such as niclosamide (Xu et al., 2020).

[0009] As in the case of SARS or COVID-19, the risk of infection is very high when in close contact with patients: the virus has a fairly high ability to spread to many people in densely populated environments through aerosolized respiratory droplets. In addition, the acute viral infections caused by these coronaviruses are spreading rapidly from the country of origin of the virus to other countries through transport and trade, thus causing a global public health crisis.

[0010] However, to date, the development of appropriate regimens for effectively inhibiting, treating or preventing such epidemic-causing respiratory viral pathogens has been inadequate. Therefore, for the health and well-being of people worldwide, there is an urgent need to develop a drug to combat these diseases.

[0011] In addition, although their clinical symptoms of respiratory infection are somewhat similar and they belong to the same RNA virus family, there are differences between the viruses at the genetic and structural levels, which have been reported to affect the sensitivity and efficacy of antiviral drugs. In addition, these molecular genetic differences in the viruses lead to differences in transmission routes, host receptors to which the virus binds, transmission rates, incubation periods, and / or infection sites, which in turn lead to differences in clinical symptoms and treatment effects, so it is very important to develop and apply reagents suitable for the target virus.

[0012] In addition, given the rapid spread of respiratory infectious diseases, high mortality rates, and global health and economic risks, in addition to spending at least one to several years to develop new drugs and vaccines, drug repositioning based on drugs whose safety has been ensured through clinical trials and actual use experience to explore the possibility of preventing, ameliorating, or treating RNA virus infections, especially respiratory diseases caused by coronaviruses, may be a very effective and cost-effective strategy. Summary of the invention

[0013] Technical issues

[0014] One object of the present invention is to provide the use of pyronaridine or a pharmaceutically acceptable salt thereof, and / or artemisinin or a derivative thereof in preventing or treating epidemic RNA virus infection.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating epidemic RNA virus infection, which composition comprises a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof, and / or artemisinin or a derivative thereof, and a pharmaceutically acceptable carrier.

[0016] Another object of the present invention is to provide a method for preventing or treating epidemic RNA virus infection by using pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof.

[0017] Technical solutions to technical problems

[0018] In order to achieve the above objectives, the present invention provides a pharmaceutical composition for preventing or treating epidemic RNA virus infection, the composition comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof, and a pharmaceutically acceptable carrier.

[0019] In addition, the present invention provides the use of pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof in preventing or treating epidemic RNA virus infection.

[0020] In addition, the present invention provides a method for preventing or treating epidemic RNA virus infection, which comprises administering a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof to an individual in need thereof.

[0021] The present invention is described in detail below.

[0022] According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating epidemic RNA virus infection, the composition comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof of the following formula 1, and a pharmaceutically acceptable carrier:

[0023] [Formula 1]

[0024]

[0025] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating epidemic RNA virus infection, comprising a therapeutically effective amount of artemisinin or a derivative thereof of the following formula 2, and a pharmaceutically acceptable carrier:

[0026] [Formula 2]

[0027]

[0028] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating epidemic RNA virus infection is provided, which comprises a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof of the above formula 1, and artemisinin or a derivative thereof of the above formula 2, and a pharmaceutically acceptable carrier.

[0029] In one embodiment according to the present invention, examples of the pharmaceutically acceptable salt of pyronaridine can include acid addition salts formed by phosphoric acid, sulfuric acid, hydrochloric acid, acetic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, maleic acid or fumaric acid, but are not limited thereto. In another embodiment according to the present invention, the pharmaceutically acceptable salt of pyronaridine can be pyronaridine tetraphosphate.

[0030] In another embodiment according to the present invention, examples of artemisinin derivatives may include dihydroartemisinin, artesunate, artemether and arteether, but are not limited thereto. In another embodiment according to the present invention, the artemisinin derivative may be artesunate.

[0031] According to another embodiment of the present invention, in a pharmaceutical composition comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof, artemisinin or a derivative thereof, and a pharmaceutically acceptable carrier, the weight ratio of pyronaridine or a pharmaceutically acceptable salt thereof to artemisinin or a derivative thereof may be 10:1 to 1:10. In another embodiment of the present invention, the weight ratio of pyronaridine or a pharmaceutically acceptable salt thereof to artemisinin or a derivative thereof may be 1:1 to 6:1. In another embodiment of the present invention, the weight ratio of pyronaridine or a pharmaceutically acceptable salt thereof to artemisinin or a derivative thereof may be 1:1 to 4:1. In another embodiment of the present invention, the weight ratio of pyronaridine or a pharmaceutically acceptable salt thereof to artemisinin or a derivative thereof may be 3:1.

[0032] As used herein, the term "pharmaceutically acceptable" refers to non-toxic as described below: physiologically acceptable, and does not inhibit the effect of the active ingredient when administered to humans, and does not usually cause gastrointestinal diseases, allergic reactions such as dizziness or similar reactions. The pharmaceutical composition of the present invention can be formulated in various ways together with a pharmaceutically acceptable carrier according to the route of administration by methods known in the art. The route of administration is not limited thereto, but can be administered orally or parenterally. Parenteral administration routes include, for example, various routes, such as transdermal, intranasal, intraperitoneal, intramuscular, subcutaneous, intravenous, etc.

[0033] When the pharmaceutical composition of the present invention is orally administered, the pharmaceutical composition of the present invention can be formulated into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, implant films, injections, suppositories, etc. together with suitable oral administration carriers according to methods known in the art. Examples of suitable carriers may include sugars, starches, celluloses, and fillers, 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 carboxymethylcellulose, hydroxypropyl methylcellulose, and low-substituted hydroxymethylcellulose; and fillers such as gelatin and polyvinyl pyrrolidone. In addition, if necessary, cross-linked polyvinylpyrrolidone, sodium starch glycolate, cross-linked sodium carboxymethylcellulose, sodium carboxymethylcellulose, agar, alginic acid, or sodium alginate may be added as a disintegrant. In addition, the pharmaceutical composition may also include glidants, anticoagulants, plasticizers, lubricants, wetting agents, flavor enhancers, emulsifiers, preservatives, and the like.

[0034] In addition, when administered parenterally, according to methods known in the art, the pharmaceutical composition of the present invention can be formulated into the form of injection, suppository, percutaneous administration and nasal inhalation together with suitable parenteral carriers. In the case of injection, it must be disinfected and protected from microbial contamination such as bacteria and fungi. Examples of suitable carriers for injection include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), their mixtures and / or vegetable oils. More preferably, suitable carriers include Hanks solution, Ringer solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and isotonic solutions (such as 5% glucose). In order to protect the injection from microbial contamination, various antibacterial and antifungal agents may be further contained, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In addition, in most cases, the injection may further contain isotonic agents, such as sugar or sodium chloride.

[0035] These formulations are described in the well-known formulation literature in pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th edition, 1975, Mack Publishing Company, Easton, Pennsylvania).

[0036] In the case of administration by inhalation, the compounds for use according to the invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix based on a suitable powder base.

[0037] As other pharmaceutically acceptable carriers, reference can be made to the acceptable carriers described in the following document (Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, PA, 1995).

[0038] In another embodiment of the present invention, pyronaridine or a pharmaceutically acceptable salt thereof is formulated together with a pharmaceutically acceptable carrier.

[0039] As mentioned above, the "pharmaceutically acceptable carrier" that can be used in the present invention can be any one of the conventionally used in the pharmaceutical field. Representative examples can include lactose, dextrin, starch, pregelatinized starch, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, polyethylene glycol, silicon dioxide, hydrotalcite, magnesium aluminum silicate, aluminum hydroxide, aluminum silicate, magnesium aluminum metasilicate, bentonite and mixtures thereof. In addition to the carrier, the pharmaceutical composition of the present invention can further include a disintegrant for rapid disintegration and dissolution when in contact with an aqueous medium during in vivo administration, a solubilizer or surfactant for increasing dissolution or absorption, and a glidant or lubricant for increasing fluidity or lubricity. The example of a disintegrant can include cross-linked polyvinylpyrrolidone, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, agar, alginic acid or sodium alginate. The example of glidant or lubricant can include colloidal silicon dioxide, silicon dioxide, talc, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, stearic acid or silicon dioxide. However, it is not limited to the examples listed. According to another embodiment of the present invention, a pharmaceutical composition is provided, which comprises 40% to 80% by weight of pyronaridine or a pharmaceutically acceptable salt thereof, 1% 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 magnesium stearate.

[0040] In another embodiment of the present invention, artemisinin or its derivatives are formulated with a pharmaceutically acceptable carrier. In addition to the carrier, the pharmaceutical composition of the present invention may further include a disintegrant for rapid disintegration and dissolution when in contact with an aqueous medium during in vivo administration, a solubilizer or surfactant for increasing dissolution or absorption, and a glidant or lubricant for increasing fluidity or lubricity. Examples of carriers may include microcrystalline cellulose, lactose hydrate, mannitol, starch, pregelatinized starch, low-substituted hydroxycellulose, hydroxycellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose or hydroxypropyl methylcellulose. Examples of disintegrants may include cross-linked polyvinylpyrrolidone, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, agar, alginic acid or sodium alginate. Examples of glidants or lubricants may include colloidal silicon dioxide, silicon dioxide, talc, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, stearic acid or silicon dioxide. Representative examples of surfactants may include sodium lauryl sulfate and its derivatives, poloxamer and its derivatives, saturated polyglycolized glyceride (also known as gelucire), labrasol, various polysorbates (e.g., polyoxyethylene sorbitan monolaurate (hereinafter referred to as Tween 20), polyoxyethylene sorbitan monopalmitate (hereinafter referred to as Tween 40), polyoxyethylene sorbitan monostearate (hereinafter referred to as Tween 60), polyoxyethylene sorbitan monooleate (hereinafter referred to as Tween 80)), sorbitan esters (e.g., sorbitan monolaurate (hereinafter referred to as Span 20), sorbitan monopalmitate (hereinafter referred to as Span 40), sorbitan monostearate (hereinafter referred to as Span 60), sorbitan monooleate (hereinafter referred to as Span 80)), 80), sorbitan trilaurate (hereinafter referred to as Span 25), sorbitan trioleate (hereinafter referred to as Span 85), sorbitan tristearate (hereinafter referred to as Span 65), cremophor, PEG-60 hydrogenated castor oil, PEG-40 hydrogenated castor oil, sodium lauryl glutamate or disodium cocoamphodiacetate. However, it is not limited to the examples listed.According to another embodiment of the present invention, there is provided a pharmaceutical composition comprising 10% to 50% by weight of artemisinin or a derivative thereof, 30% to 70% by weight of microcrystalline 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 magnesium stearate.

[0041] In another embodiment of the present invention, pyronaridine or its pharmaceutically acceptable salt and artemisinin or its derivative are prepared together with a pharmaceutically acceptable carrier. In addition to the carrier, the pharmaceutical composition of the present invention may further include a disintegrant for rapid disintegration and dissolution when in contact with an aqueous medium for in vivo administration, a solubilizer or surfactant for increasing dissolution or absorption, and a glidant or lubricant for increasing fluidity or lubricity. The example of a carrier may include lactose, dextrin, starch, pregelatinized starch, microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, polyethylene glycol, silicon dioxide, hydrotalcite, magnesium aluminum silicate, aluminum hydroxide, aluminum silicate, magnesium aluminum metasilicate, bentonite, butylated hydroxytoluene and a mixture thereof. Representative examples of surfactants may include sodium lauryl sulfate and its derivatives, poloxamer and its derivatives, saturated polyglycolized glyceride (also known as gelucire), labrasol, various polysorbates (e.g., polyoxyethylene sorbitan monolaurate (hereinafter referred to as Tween 20), polyoxyethylene sorbitan monopalmitate (hereinafter referred to as Tween 40), polyoxyethylene sorbitan monostearate (hereinafter referred to as Tween 60), polyoxyethylene sorbitan monooleate (hereinafter referred to as Tween 80)), sorbitan esters (e.g., sorbitan monolaurate (hereinafter referred to as Span 20), sorbitan monopalmitate (hereinafter referred to as Span 40), sorbitan monostearate (hereinafter referred to as Span 60), sorbitan monooleate (hereinafter referred to as Span 80), sorbitan trilaurate (hereinafter referred to as Span 80), polyoxyethylene sorbitan monostearate (hereinafter referred to as Span 60), polyoxyethylene sorbitan monooleate (hereinafter referred to as Span 80), polyoxyethylene sorbitan monolaurate (hereinafter referred to as Span 20), polyoxyethylene sorbitan monopalmitate (hereinafter referred to as Span 40), polyoxyethylene sorbitan monostearate (hereinafter referred to as Span 60), polyoxyethylene sorbitan monooleate (hereinafter referred to as Span 80), polyoxyethylene sorbitan monolaurate (hereinafter referred to as Span 8 ...stearate (hereinafter referred to as Span 80), 25), sorbitan trioleate (hereinafter referred to as Span 85), sorbitan tristearate (hereinafter referred to as 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 disintegrants may include cross-linked polyvinylpyrrolidone, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, agar, alginic acid or sodium alginate. Examples of glidants or lubricants may include colloidal silicon dioxide, silicon dioxide, talc, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, stearic acid or silicon dioxide. However, it is not limited to the examples listed.According to another embodiment of the present invention, a pharmaceutical composition is provided, which comprises 15% to 60% by weight of pyronaridine or a pharmaceutically acceptable salt thereof, 5% to 20% by weight of artemisinin or a derivative thereof, 5% to 30% by weight of microcrystalline cellulose, 10% to 40% by weight of cross-linked polyvinylpyrrolidone, 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 butylated hydroxytoluene, 0.1% to 5% by weight of silicon dioxide and 0.5% to 10% by weight of magnesium stearate.

[0042] The pharmaceutical composition of the present invention can be formulated into powders, granules, tablets, capsules, dry syrups, coatings, injections, suppositories, transdermal administration, inhalation administration, and the like.

[0043] In another embodiment of the present invention, the pharmaceutical composition of the present invention can be administered in combination with one or more additional drugs having antiviral efficacy to prevent and treat epidemic RNA virus infection.

[0044] In another embodiment of the present invention, examples of other antiviral agents may include viral replication inhibitors, helicase inhibitors, viral protease inhibitors, and viral cell entry inhibitors, but are not limited thereto. In another embodiment of the present invention, other antiviral agents may be, for example, ribavirin, interferon, niclosamide, or a combination thereof, but are not limited thereto.

[0045] In another embodiment of the present invention, examples of epidemic RNA virus infectious diseases may include, but are not limited to, Zika virus infection, Ebola virus infection, and respiratory diseases caused by novel influenza virus and coronavirus infection. In another embodiment of the present invention, examples of respiratory diseases caused by coronavirus infection may include, but are not limited to, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) or COVID-19. In another embodiment of the present invention, the respiratory disease caused by coronavirus infection may be COVID-19.

[0046] In the present invention, the term "prevention" refers to any action to inhibit or delay the occurrence, spread and recurrence of epidemic RNA virus infection by administering the pharmaceutical composition of the present invention, and the term "treatment" refers to any action to improve or beneficially change the symptoms of the disease by administering the pharmaceutical composition of the present invention.

[0047] In addition, as used herein, the term "therapeutically effective amount" refers to an amount that shows a higher response than a negative control, and preferably refers to an amount sufficient to prevent or treat an epidemic RNA virus infection. The therapeutic dose for patients is generally 50 mg / day to 2,000 mg / day, and more preferably 100 mg / day to 1,000 mg / day, depending on the severity of the condition and whether it is administered alone or in combination, or in combination with other drugs. It can be administered once a day or in divided doses by oral or parenteral routes. However, the therapeutically effective amount can be appropriately changed according to various factors such as the type and severity of the disease, the patient's age, weight, health status and gender, route of administration and treatment cycle.

[0048] Although the present invention describes the prevention and treatment of epidemic RNA virus infections in humans, preferably respiratory diseases caused by coronavirus infection in humans, and more preferably COVID-19 in humans, the present invention can be used to treat infectious RNA viruses in animals and humans, especially viruses in the Coronaviridae family that cause respiratory diseases, and more particularly viruses that cause COVID-19.

[0049] The problems of the present invention are not limited to the above technical problems, and those skilled in the art will clearly understand other unmentioned technical problems through the following description. In addition, the above description does not limit the claimed invention in any way, and in addition, the combination of the discussed features is not absolutely necessary for the solution of the present invention.

[0050] Advantageous Effects of the Invention

[0051] The present invention provides a pharmaceutical composition for preventing or treating epidemic RNA virus infection, comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and / or artemisinin or a derivative thereof as an active ingredient. The pharmaceutical composition according to the present invention can be effectively used for preventing or treating, for example, respiratory diseases caused by coronaviruses such as COVID-19 by effectively inhibiting epidemic RNA virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Concentration-response curves for inhibition and cytotoxicity against SARS-CoV-2 with pretreatment with pyronaridine tetraphosphate, one of the active ingredients of the present invention, measured 24 hours after infection.

[0053] Figure 2 Concentration-response curves for simultaneous treatment with pyronaridine tetraphosphate for inhibition and cytotoxicity against SARS-CoV-2 measured at 24 hours post-infection.

[0054] Figure 3The results of measuring the inhibitory effect of artesunate (one of the active ingredients of the present invention) on SARS-CoV-2 at 24 hours and 48 hours after infection and comparing it with chloroquine as a control are shown.

[0055] Figure 4 Comparative results of the inhibitory effects of a combination of pyronaridine tetraphosphate and artesunate at different ratios on SARS-CoV-2 infected cells are shown, and at the optimal ratio, the inhibitory effects on the virus were compared at 24 and 48 hours after infection.

[0056] Figure 5 Figure 3 Concentration-response curves of inhibitory effects and cytotoxicity against SARS-CoV-2 measured 24 and 48 h after infection of the human lung cell line Calu-3 cells when pyronaridine tetraphosphate or artesunate were co-treated with viral infection, compared with hydroxychloroquine as a control.

[0057] Figure 6 Concentration-response curves for inhibition of SARS-CoV-2 by post-infection treatment of the human lung cell line Calu-3 cells with pyronaridine tetraphosphate or artesunate when measured 48 hours after drug treatment.

[0058] Figure 7 The results show that hamsters were infected with SARS-CoV-2 and orally administered low or high doses of a combination of pyronaridine tetraphosphate and artesunate in a 3:1 ratio once a day for 3 days 1 hour after infection, or a high dose of pyronaridine tetraphosphate alone 25 hours after infection, and then the virus titers in the lungs were analyzed on the 4th day after infection and compared with the untreated virus-inoculated control group. DETAILED DESCRIPTION

[0059] Hereinafter, the constitution and effects of the present invention will be described in more detail by way of examples. However, these examples are merely illustrative, and the scope of the present invention is not limited thereto.

[0060] Example 1: Preparation of a single tablet of pyronaridine tetraphosphate

[0061] Hydroxypropyl cellulose is dissolved in ethanol to prepare a binding solution. After wet granulation of pyronaridine tetraphosphate using the prepared binding solution, the resulting product is dried and granulated. Low-substituted hydroxypropyl cellulose, sodium starch glycolate, microcrystalline cellulose and silicon dioxide are mixed. After lubrication by adding magnesium stearate, tablets are prepared by tableting.

[0062] [Table 1]

[0063]

[0064] Example 2: Preparation of artesunate single tablet

[0065] Silicon dioxide and sodium lauryl sulfate are sieved using a sieve. The sieved silicon dioxide and sodium lauryl sulfate are mixed with artesunate, microcrystalline cellulose, low-substituted hydroxypropyl cellulose and sodium starch glycolate, magnesium stearate is added for lubrication, and then tableting is performed to prepare tablets. The obtained product is coated with a film coating agent.

[0066] [Table 2]

[0067]

[0068] Example 3: Preparation of pyronaridine tetraphosphate / artesunate compound tablets

[0069] Polyethylene glycol as a melt dispersion carrier, butylhydroxytoluene as an active ingredient and artesunate are mixed, heated to melt, then rapidly cooled and finely pulverized. Then, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone and magnesium stearate are mixed to obtain mixture 1. After dissolving hydroxypropyl cellulose in ethanol, pyronaridine tetraphosphate is wet granulated, dried and granulated to obtain mixture 2. Mixture 1, mixture 2, sodium lauryl sulfate, silicon dioxide and cross-linked polyvinylpyrrolidone are mixed, and then magnesium stearate is added thereto to lubricate the mixture, followed by tableting to prepare tablets. The obtained product is coated with a film coating agent.

[0070] [Table 3]

[0071]

[0072] In order to determine whether the pyronaridine or its salt, artemisinin or its derivatives of the present invention have antiviral activity against coronavirus, the agents were treated alone and in combination as in the following experimental examples, and the inhibition rate of viral infection was evaluated.

[0073] Experimental Example 1: Evaluation of the antiviral effect of pyronaridine tetraphosphate (pretreatment)

[0074] In Experimental Example 1, pyronaridine tetraphosphate was pretreated for 1 hour before cells were infected with SARS-CoV-2 (Korean isolate), and the inhibitory effect on viral infection was evaluated.

[0075] 1) Preparation of viruses and host cells

[0076] Vero cells were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO. 2The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC). After virus amplification, the virus titer was determined by plaque assay, which was performed by counting the virus plaques formed in the cells used for virus amplification after virus infection.

[0077] 2) Determine antiviral efficacy using immunofluorescence staining imaging

[0078] Vero cells were plated at 1.2 × 10 4 The cells were seeded at a density of 10 cells in μClear plates and pretreated with a series of 10 drug dilutions in the concentration range of 0.05-50 μM in culture medium for 1 hour 24 hours before the experiment, and then SARS-CoV-2 was inoculated into the cells at a multiplicity of infection (MOI) of 0.0125. 24 hours after infection, the cells were fixed with 4% formaldehyde, and the infected cells were analyzed by immunofluorescence staining using antibodies against the 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 by an imaging analysis program, compared with positive and negative controls. The antiviral effect of the drug is expressed as a concentration-response curve, and the 50% effective concentration (EC 50 , the concentration that inhibits 50% of the cytotoxicity induced by virus infection) and the 50% cytotoxic concentration (CC 50 , the concentration of compound that causes damage in 50% of cells compared to normal cells) was calculated as shown in Equation 1.

[0079] <Equation 1>

[0080] Sigmoidal model, Y = maximum response + (maximum response – minimum response) / (1 + (IC 50 / X) 曲线坡度 )

[0081] The results are as follows Figure 1 As shown, in the case of infection of Vero cells with SARS-CoV-2, a 70% viral inhibition rate was observed at a concentration of 50 μM pyronaridine, but the cytotoxicity was also increased by 17% by drug pretreatment. Vero cells are isolated from the kidney epithelial cells of African green monkeys (Chlorocebus sp.) and are known as type 1 IFN-deficient cells. In previous studies, when measuring the in vitro antiviral efficacy of pyronaridine against Ebola virus in Vero cells, the in vitro antiviral efficacy of pyronaridine against Ebola virus was significantly decreased at a concentration below CC. 50 (CC 50No antiviral activity was observed at a concentration of 1.3 μM, but when inoculated into human Hela cells, CC 50 higher and showed antiviral activity at non-toxic concentrations (EC 50 =0.42-1.12μM, CC 50 =3.1 μM). In addition, pyronaridine significantly inhibited mortality and viral infection rate in a mouse model challenged with Ebola virus (Lane et al., 2015). It is well known that the efficacy of antiviral agents may vary in in vitro or in vivo measurement systems, depending on the differences in the characteristics of the host cells tested and their intracellular immune signaling pathways (Lane et al., 2015), so an assay based on human-derived host cells was additionally established, and the antiviral efficacy of pyronaridine was further confirmed in various cell lines and animal studies.

[0082] Experimental Example 2: Inhibitory Effect of Pyronaridine Tetraphosphate on SARS-CoV-2 Virus (Simultaneous Treatment)

[0083] In Experimental Example 2, when cells were treated simultaneously during infection with SARS-CoV-2 (Korean isolate), the inhibitory effect of pyronaridine on viral infection was evaluated. Chloroquine is known to inhibit the binding of viruses to cells and the glycosylation of SARS-CoV host receptors by increasing the endosomal pH value, thereby showing antiviral efficacy (Vincent et al., 2005). Since pyronaridine with a structure similar to chloroquine is expected to also work through a similar mechanism, its antiviral efficacy was simultaneously treated with pyronaridine during viral infection and measured. By optimizing some experimental conditions used in Experimental Example 1, experiments were performed under relatively low cytotoxic test conditions.

[0084] 1) Preparation of viruses and host cells

[0085] Vero cells were cultured at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC). After viral amplification, the viral titer was determined by measuring the RNA copy number by qRT-PCR.

[0086] 2) Using RNA copy number to measure antiviral efficacy

[0087] After pyronaridine tetraphosphate was dissolved in DMSO, it was diluted to a concentration of 0.033-100 μM using culture medium. SARS-CoV-2 was inoculated with 2×10 4Vero cells were seeded in 96-well plates at a density of 10 cells / well (MOI = 0.01), and culture medium containing each drug dilution was added to each well. 24 hours after infection, cell supernatants were collected, RNA was extracted, and qRT-PCR was performed on the RdRp gene. The antiviral efficacy of the drug was analyzed by comparing the viral RNA copy number of the drug with that of the control. The drug concentration-response curve was drawn using the viral infection inhibition rate (% inhibition) obtained by back-calculating the viral titer from the RNA copy number, and the 50% effective concentration (EC 50 , a concentration that inhibits the virus titer by 50%), as shown in Experimental Example 1.

[0088] 3) Cytotoxicity measurement (% cytotoxicity)

[0089] Cytotoxicity was measured using a tetrazolium salt-based assay (WST-1). WST-1 is converted into a chromogenic substance called formazan by mitochondrial dehydrogenases present only in living cells. After adding 10 μL of WST-1 premix to each well, the cells were incubated for another hour, and the amount of formazan produced was calculated by measuring its absorbance by ELISA. The 50% cytotoxic concentration (CC50, the concentration of the compound that causes damage in 50% of the cells compared to normal cells) was calculated.

[0090] The results are as follows Figure 2 As shown, pyronaridine exhibited antiviral effects in a concentration-dependent manner when treated simultaneously, with cytotoxicity observed at certain high concentrations, but at non-cytotoxic concentrations, the antiviral activity against SARS-CoV-2 virus exceeded 90% inhibition (EC 50 =8.27μM, CC 50 =11.54 μM; selectivity index, SI>1.40).

[0091] Experimental Example 3: Inhibitory effect of artesunate on SARS-CoV-2 (simultaneous treatment)

[0092] In Experimental Example 3, the antiviral efficacy of artesunate was measured under the same experimental conditions as in Experimental Example 2.

[0093] 1) Preparation of viruses and host cells

[0094] The preparation methods of Vero cells and viruses were the same as those in Experimental Example 2.

[0095] 2) Using RNA copy number to measure antiviral efficacy

[0096] Artesunate was dissolved in DMSO and then diluted to concentrations of 3.13 μM, 12.5 μM, and 50 μM using culture medium. SARS-CoV-2 was inoculated at 2×104 The density of cells / well was 0.01 in Vero cells in a 96-well plate, and the medium containing each drug dilution was added to each well. 24 hours and 48 hours after infection, the cell supernatant was collected, and the RdRp gene was subjected to qRT-PCR to calculate the virus titer, and the virus infection inhibition rate (% inhibition rate) was calculated as shown in Experimental Example 2. Chloroquine was used as a control.

[0097] 3) Cytotoxicity measurement (% cytotoxicity)

[0098] The cytotoxicity was measured in the same manner as in Experimental Example 2.

[0099] The results are as follows Figure 3 As shown, artesunate exhibited antiviral activity in a concentration-dependent manner and showed an 83% viral inhibition rate at a concentration of 50 μM. At 12.5 μM, the inhibition rates were 40% and 51% at 24 hours post-infection (24hpi) and 48 hours post-infection (48hpi), respectively. In addition, at 3.13 μM, the inhibition rate was 39% at 48 hours post-infection. There was no significant cytotoxicity under all tested conditions. Compared with pyronaridine or chloroquine used as controls, the EC of artesunate was 2.5477 μM and 2.222 μM, respectively. 50 It is lower and has a slower onset of inhibition of SARS-CoV-2, but has a lasting antiviral effect, showing a pattern in which the viral suppression rate slowly increases over time.

[0100] Experimental Example 4: Inhibitory effect of pyronaridine tetraphosphate / artesunate combination on SARS-CoV-2

[0101] Unlike chloroquine, which did not show antiviral effects in the guinea pig model infected with Ebola virus, pyronaridine significantly improved viral titers and survival rates in the mouse model attacked by Ebola virus. Therefore, it is speculated that chloroquine may also have different mechanisms of action, in which immunomodulatory mechanisms such as type 1 IFN-1 pathways (Lane et al., 2019) are proposed. Artesunate also showed antiviral efficacy against Ebola virus in in vitro tests, but was weaker than pyronaridine (Gignox et al., 2016). Therefore, in Experimental Example 4, the changes in the antiviral efficacy of the two drugs under different combination ratios according to the combination treatment were evaluated.

[0102] 1) Preparation of viruses and host cells

[0103] The preparation methods of Vero cells and viruses were the same as those in Experimental Example 2.

[0104] 2) Using RNA copy number to measure antiviral efficacy

[0105] Pyronaridine tetraphosphate and artesunate were dissolved in DMSO and diluted to various concentrations using culture medium at various ratios of 1:1, 3:1, 10:1, etc. 24 hours before the experiment, SARS-CoV-2 was inoculated with 2×10 4 Vero cells (MOI = 0.01) were inoculated into 96-well plates at a density of cells / well, and the culture medium containing each drug dilution was added to each well. 24 hours after infection, the cell supernatant was collected, qRT-PCR was performed on the RdRp gene, and the viral titer and viral infection inhibition rate (% inhibition rate) were calculated as shown in Experimental Example 2. When 10 μM pyronaridine tetraphosphate was combined with 3.3 μM artesunate, the viral titer was measured at 24 hours and 48 hours after infection, respectively, and compared with chloroquine and lopinavir as controls.

[0106] 3) Cytotoxicity measurement (% cytotoxicity)

[0107] The cytotoxicity was measured in the same manner as in Experimental Example 2.

[0108] The results are as follows Figure 4 As shown, the antiviral effect of the combination of artesunate and pyronaridine was higher than that of artesunate alone, and the antiviral effect increased as the proportion of pyronaridine in the combination increased. Specifically, when 10 μM pyronaridine tetraphosphate and 3.3 μM artesunate were combined (ratio 3:1), the inhibition rate of viral infection reached 90-100%, which was higher than the antiviral effect of chloroquine or lopinavir used as a control. In this case, the inhibition rate of infection can be maintained for up to 48 hours. Figure 4 No significant cytotoxicity was observed in all combination ratios shown, and lower cytotoxicity (49.5% reduction) was observed when 10 μM pyronaridine tetraphosphate and 3.3 μM artesunate were treated in combination compared to 10 μM pyronaridine tetraphosphate alone.

[0109] Experimental Example 5: Inhibitory Effects of Pyronaridine Tetraphosphate or Artesunate on SARS-CoV-2 in Human Lung Cell Line (Simultaneous Treatment)

[0110] It is reported that there may be species differences in the antiviral effects of humans and other animals, such as receptor structure. Therefore, in order to confirm the efficacy in human lung cell lines, in Experimental Example 5, when SARS-CoV-2 (Korean isolate) was inoculated into Calu-3 cells (human lung cell line), it was treated with pyronaridine phosphate or artesunate and the efficacy of inhibiting viral infection was evaluated. When the cells were treated simultaneously during SARS-CoV-2 (Korean isolate) infection, the inhibitory effect of pyronaridine tetraphosphate or artesunate on viral infection was evaluated in Calu-3 cells, a human lung cell line.

[0111] 1) Preparation of viruses and host cells

[0112] Calu-3 cells were cultured at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC).

[0113] 2) Using RNA copy number to measure antiviral efficacy

[0114] After dissolving in DMSO, pyronaridine tetraphosphate was diluted to a concentration of 0.033-100 μM using culture medium. SARS-CoV-2 was inoculated at 2×10 4 Vero cells (MOI = 0.01) were inoculated in 96-well plates at a density of 10 cells / well, and the medium containing each drug dilution was added to each well. At 24 hours and 48 hours after infection, qRT-PCR was performed on the RdRp gene as shown in Experimental Example 2. The virus infection inhibition rate (% inhibition rate) of the virus titer was calculated from the RNA copy number to draw the drug concentration-response curve, and the 50% effective concentration (EC 50 , a concentration that inhibits the virus titer by 50%), as shown in Experimental Example 1.

[0115] 3) Cytotoxicity measurement (% cytotoxicity)

[0116] The cytotoxicity was measured in the same manner as in Experimental Example 2.

[0117] The results are as follows Figure 5 As shown, when treated simultaneously at the time of infection, both pyronaridine and artesunate exhibited antiviral effects in a human lung cell line in a concentration-dependent manner. In addition, they exhibited antiviral activity against SARS-CoV-2 at non-cytotoxic concentrations at 24 hours post-infection (24hpi) and 48 hours post-infection (48hpi), with an inhibition rate of more than 90% (pyronaridine IC 48hpi) 50 =8.58μM, CC 50 >100μM; selectivity index, SI>11.66; IC of artesunate 48 hours after infection 50 =0.45μM, CC 50>100 μM; selectivity index, SI>220.8). Specifically, in the case of artesunate, the effect was significantly increased in the human lung cell line compared to the monkey kidney cell line Vero cells. In contrast, at less than 50 μM, hydroxychloroquine showed no antiviral effect in the human lung cell line, while hydroxychloroquine showed antiviral efficacy in the monkey cell line.

[0118] Experimental Example 6: Inhibitory effect of pyronaridine tetraphosphate or artesunate treatment on SARS-CoV-2 in human lung cell lines after infection

[0119] In Experimental Example 6, Calu-3 cells were treated with pyronaridine tetraphosphate or artesunate at 0, 2, 4, 6, 8, 10, 12, 24, and 36 hours after SARS-CoV-2 (Korean isolate) inoculation, and how long the inhibitory effect of each drug on viral infection lasted was evaluated.

[0120] 1) Preparation of viruses and host cells

[0121] Calu-3 cells and viruses were prepared in the same manner as described in Experimental Example 5.

[0122] 2) Measuring antiviral efficacy using viral plaque assay

[0123] After pyronaridine tetraphosphate and artesunate were dissolved in DMSO, they were diluted to 12.5 μM using culture medium. One hour after inoculation with SARS-CoV-2 (MOI = 0.1), the supernatant was removed, the Calu-3 cells were washed, and then DMEM medium containing 2% bovine serum was added. The medium containing the drug was added at 0, 2, 4, 6, 8, 10, 12, 24 and 36 hours, respectively. 48 hours after each drug treatment, the cell supernatant was collected and a plaque assay was performed, in which plaques produced by infectious virus infection were counted in Vero cells used for virus amplification. A DMEM-F12 culture medium layer containing 2% agarose was spread on the infected Vero cell layer, and after 72 hours of culture, the number of plaques was counted with crystal violet counterstaining. The antiviral efficacy of the drug was analyzed by the viral infection inhibition rate (% inhibition) of the viral titer calculated inversely from the number of plaques formed, and compared with the control.

[0124] The results are as follows Figure 6As shown, the greatest antiviral efficacy was shown when 12.5 μM pyronaridine was treated (>99% inhibition when added up to 6 hours after infection, >94% inhibition when added up to 12 hours after infection, and 90% inhibition when added up to 24 hours after infection). On the other hand, 12.5 μM artesunate treatment showed 92-96% inhibition when added up to 6 hours after infection, >90% inhibition when added up to 12 hours after infection, and 48% inhibition when added up to 24 hours after infection.

[0125] Experimental Example 7: Inhibitory effect of pyronaridine tetraphosphate / artesunate combination on SARS-CoV-2 in COVID-19 animal model

[0126] In Experimental Example 7, pyronaridine tetraphosphate and artesunate (combined in a ratio of 3:1) were orally administered to hamsters infected with SARS-CoV-2 (Korean isolate) to evaluate the antiviral efficacy in animals.

[0127] 1) Preparation of viruses and hamsters for SARS-CoV-2 inoculation

[0128] SARS-CoV-2 virus was provided by the Korea Centers for Disease Control and Prevention (KCDC). As an experimental animal model, Syrian hamsters, which are highly susceptible to SARS-CoV-2 and have less supply restrictions, were used and SARS-CoV-2 (1×10 6 PFU / 100 μL) were inoculated into the two nasal passages of hamsters at a volume of 50 μL.

[0129] 2) Measuring in vivo antiviral efficacy using plaque assay

[0130] One hour after nasal inoculation of SARS-CoV-2, pyronaridine tetraphosphate (180 mg / kg or 360 mg / kg) and artesunate (60 mg / kg or 120 mg / kg) were orally administered once a day at a ratio of 3:1 for 3 consecutive days to evaluate the in vivo antiviral efficacy of the two drug combinations against SARS-CoV-2. As a comparative experimental group, pyronaridine alone was orally administered once at 360 mg / kg 25 hours after infection to evaluate the duration of post-infection efficacy of pyronaridine alone. Both pyronaridine tetraphosphate and artesunate were prepared before use, completely dissolved in 5% sodium bicarbonate, and orally administered. As a control group, a normal control group (Mock) that was not inoculated with the virus and a vehicle control group that was only administered with the solvent at the same time were used. Four days after infection, the left and right lobes of the lungs were removed, the virus was extracted, and the virus titer in the lung tissue was analyzed by plaque assay as described in Experimental Example 6. The lung virus titer quantified by plaque assay was standardized to the total weight (g) of the lung tissue and then converted to log values ​​to calculate the final titer (Log10 Plaque forming unit / g, Log 10 PFU / g).

[0131] The results are as follows Figure 7 As shown, at 4 days after infection, the reduction of infectious virus titer in lung tissue was statistically significant in the 180 / 60 mg / kg and 360 / 120 mg / kg combined administration groups of pyronaridine tetraphosphate (P)-artesunate (A). 10 Median PFU: 8.30 in the virus-challenged vector control group vs 7.22 in the PA180 / 60 mg / kg co-administration group (p<0.001) vs 7.61 in the PA360 / 120 mg / kg co-administration group (p=0.046). On the other hand, when pyronaridine tetraphosphate was orally administered alone, a significant decrease in the infectious virus titer was observed in the lung tissue at a high dose of 360 mg / kg, and a significant inhibitory effect was observed even when administered once 25 hours after infection [median Log of the virus-challenged vector control group vs 7.22 in the PA180 / 60 mg / kg co-administration group (p<0.001) vs 7.61 in the PA360 / 120 mg / kg co-administration group (p=0.046)]. 10 PFU 8.30 vs 7.22 in the single high-dose 25hpi administration group (p<0.001)].

Claims

1. Use of a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating a respiratory disease caused by coronavirus infection, wherein the respiratory disease caused by coronavirus infection is selected from Middle East Respiratory Syndrome (MERS) and COVID-19.

2. Use of a therapeutically effective amount of artemisinin or a derivative thereof in the preparation of a medicament for preventing or treating a respiratory disease caused by coronavirus infection, wherein the respiratory disease caused by coronavirus infection is selected from Middle East Respiratory Syndrome (MERS) and COVID-19, The artemisinin derivative is selected from dihydroartemisinin, artesunate, artemether and arteether.

3. Use of a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof, and artemisinin or a derivative thereof in the preparation of a medicament for preventing or treating a respiratory disease caused by coronavirus infection, wherein the respiratory disease caused by coronavirus infection is selected from Middle East Respiratory Syndrome (MERS) and COVID-19, The artemisinin derivative is selected from dihydroartemisinin, artesunate, artemether and arteether.

4. The use according to claim 1 or 3, wherein the pharmaceutically acceptable salt of pyronaridine is selected from phosphate, sulfate, hydrochloride, acetate, methanesulfonate, benzenesulfonate, toluenesulfonate, maleate and fumarate.

5. The use according to claim 4, wherein the pharmaceutically acceptable salt of pyronaridine is pyronaridine tetraphosphate.

6. The use according to claim 3, wherein The weight ratio of the pyronaridine or a pharmaceutically acceptable salt thereof to the artemisinin or a derivative thereof is 10:1 to 1:

10.

7. The use according to claim 6, wherein The weight ratio of the pyronaridine or a pharmaceutically acceptable salt thereof to the artemisinin or a derivative thereof is 1:1 to 6:

1.

8. The use according to claim 7, wherein The weight ratio of the pyronaridine or a pharmaceutically acceptable salt thereof to the artemisinin or a derivative thereof is 3:

1.

9. The use according to claim 2 or 3, wherein the artemisinin derivative is artesunate.

10. The use according to any one of claims 1 to 3, wherein the medicament further comprises at least one other antiviral agent.

11. The use according to claim 10, wherein the other antiviral agent is selected from the group consisting of a viral replication inhibitor, a helicase inhibitor, a viral protease inhibitor and a viral cell entry inhibitor.

12. The use according to claim 11, wherein the other antiviral agent is selected from ribavirin, interferon, niclosamide and a combination thereof.

13. The method of claim 1 , wherein the respiratory disease caused by coronavirus infection is COVID-19.

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