RU2843665C1 - Pharmaceutical composition for preventing or treating an epidemic rna-viral infectious disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the use of a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing or treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19), wherein the pharmaceutically acceptable pyronaridine salt is selected from the group consisting of phosphate, sulphate, hydrochloride, acetate, methanesulphonate, benzenesulphonate, toluenesulphonate, maleate and fumarate, also relates to the use of a therapeutically effective amount of artemisinin or a derivative thereof in the manufacture of a medicament for preventing or the treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19), where the artemisinin derivative is selected from the group consisting of dihydroartemisinin, artesunate, artemether and athether, also relates to the use of a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and artemisinin or a derivative thereof in the manufacture of a medicament for the prevention or treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19), wherein the pharmaceutically acceptable pyronaridine salt is selected from the group consisting of phosphate, sulphate, hydrochloride, acetate, methanesulphonate, benzene sulphonate, toluene sulphonate, maleate and fumarate; and wherein the artemisinin derivative is selected from the group consisting of dihydroartemisinin, artesunate, artemether and arthiester.

EFFECT: group of inventions provides the use of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof for the prevention or treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19).

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Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to the use of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof for the prevention or treatment of epidemic RNA viral infections. More specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of epidemic RNA viral infections, in particular coronavirus disease 2019 (COVID-19), comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof in combination with a pharmaceutically acceptable carrier.

PREREQUISITES FOR THE CREATION OF THE INVENTION

RNA viruses, which have RNA genomes, have a higher mutation rate than DNA viruses and readily generate mutants adapted to changes in the host and environment. Because of this ability, RNA viruses are difficult to control with antiviral agents or prophylactic vaccines. In addition, RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that synthesizes RNA using the RNA of the viral genome as a template, and host cell RNA polymerases that synthesize RNA but use DNA as a template are unable to affect the replication of RNA viruses (RNA viruses). RNA viruses are classified into single-stranded positive-sense (+) strand, single-stranded negative-sense (-) strand, and double-stranded dsRNA viruses (dsRNA) depending on the polarity of the genome and whether the genomic RNA has the same polarity as the mRNA.

Acute viral infections that have recently spread worldwide and caused a global public health crisis spread rapidly through transport and trade from the country of origin of the virus to other countries, creating a significant global demand for the development of therapeutic agents. In particular, the 2009 H1N1 influenza, 2014 Ebola epidemics in West Africa and the Democratic Republic of Congo in 2019, and Zika in 2016 are infections caused by RNA viruses.

Coronaviruses are viruses that belong to the family of positive-strand RNA viruses, such as the Zika virus, have a positive-sense, single-stranded RNA genome of 25-32 kbp and are zoonotic viruses that can infect both human and animal cells, such as birds and mammals. Coronaviruses have a structure in which their outer shell contains protruding, characteristic club-shaped spike proteins. Coronaviruses 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), which emerged in 2003, Middle East respiratory syndrome (MERS), which most recently emerged in Saudi Arabia in 2012, or coronavirus disease 2019 (COVID-19, 2019-nCoV infection), recently declared a public health emergency of international concern (PHEIC) by the World Health Organization (WHO).

SARS-CoV causes severe acute respiratory syndrome, which initially emerged in China in 2002 and has spread worldwide with a reported mortality rate of about 10% in 8,096 patients. The syndrome typically presents with high fever and myalgia, followed by a dry cough without sputum after 2-7 days, causing respiratory failure in 10-20% of patients. Since appropriate treatment has not yet been developed, antibacterial drugs in combination with antiviral drugs such as oseltamivir or ribavirin or steroids can be used to treat atypical pneumonia.

MERS-CoV is thought to be a virus transmitted from animal hosts such as camels to humans, causing severe acute respiratory syndrome and renal failure, the virus causes about 2000 infections in 26 countries, including the Middle East, with a mortality rate of 35.6% (WHO, 2016). The incubation period is about 5 days, with fever, cough, shortness of breath, pneumonia. The virus is predominantly spread by limited transmission among family members or members of health care settings, and usually progresses to severe disease in people with underlying medical conditions such as diabetes.

SARS-CoV-2 (2019-nCoV) is the virus that causes COVID-19, with the first case reported in Wuhan, China, in 2019. After an incubation period of 1 to 14 days, mild to severe respiratory symptoms occur, such as cough, fever, malaise, shortness of breath, pneumonia or acute respiratory distress syndrome, and rarely sputum, sore throat, and diarrhea. Since there is no selective antiviral agent for this virus, symptomatic treatment alone or in combination with antiviral agents previously indicated for other viral diseases is used.

In particular, COVID-19, as shown by the most recent viral outbreak, spreads very rapidly as there is no treatment or vaccine currently available (Li et al., 2020), and a suitable cell or animal assay system for this disease has not yet been established. Currently, the drugs that have been reported as being used in clinical settings or suggested in expert recommendations in China and Korea include chloroquine, remdesivir (Wang et al., 2020), lopinavir, favipiravir, ribavirin, interferon, etc., and more than 80 clinical trials are currently underway (Maxmen et al., 2020). In particular, since SARS-CoV-2 belongs to the coronavirus family, reagents, including those such as niclosamide (Xu et al., 2020), which were previously known to have antiviral activity against MERS-CoV or SARS-CoV, which share approximately 79.5% nucleotide sequence homology with SARS-CoV-2, have been in the focus of attention (Zhou et al., 2020).

It is noted that in cases of SARS or COVID-19, in close contact with a patient, the risk of infection is very high: the viruses are quite transmissible and are transmitted to a significant number of people in a densely populated environment by airborne droplets. In addition, acute viral infections caused by these coronaviruses spread quickly through transport and trade from the country of origin of the virus to other countries, thereby causing a global public health crisis.

However, the development of appropriate treatment regimens to effectively inhibit, treat or prevent such epidemic-causing respiratory viral pathogens is currently insufficient. Thus, there is an urgent need to develop a drug to counteract such diseases for the health and well-being of people worldwide.

In addition, although the clinical symptoms of respiratory infections of these viruses are somewhat similar and the viruses belong to the same RNA virus family, there are still differences between the viruses at the genetic and structural levels, and these differences are reported to affect the sensitivity and efficacy of antiviral drugs. In addition, these molecular genetic viral differences result in differences in the transmission routes, host receptors for virus binding, transmission rate, incubation period, and/or modes of infection, resulting in different clinical symptoms and therapeutic efficacy, and thus necessitate the development and use of appropriate reagents against the target virus.

Furthermore, given the rapid transmission, high mortality and global health and economic risks caused by respiratory infectious diseases, in addition to the development of new drugs and vaccines that takes from one to several years, drug repositioning approaches based on drugs whose safety is assured by previous clinical trials and practical experience of such developments may prove to be a very effective and cost-effective strategy for exploring the possibility of preventing, improving or treating RNA viral infections, in particular coronavirus respiratory diseases.

DISCLOSURE OF INVENTION

TECHNICAL PROBLEM

The aim of the present invention is to provide the use of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof for the prevention or treatment of epidemic RNA viral infections.

Another aim of the present invention is to create a pharmaceutical composition for the prevention or treatment of epidemic RNA viral infections, containing a therapeutically effective amount of pyronaridine or its pharmaceutically acceptable salt and/or artemisinin or its derivative in combination with a pharmaceutically acceptable carrier.

Another aim of the present invention is to provide a method for the prevention or treatment of epidemic infections caused by an RNA virus using pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof.

SOLVING THE PROBLEM

To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of epidemic RNA viral infections, containing a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof in combination with a pharmaceutically acceptable carrier.

Furthermore, the present invention relates to the use of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof for the prevention or treatment of epidemic RNA viral infections.

Furthermore, the present invention relates to a method for the prevention or treatment of epidemic RNA viral infections, which comprises administering to a subject in need thereof a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof.

The present invention is described in detail below.

According to one aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of epidemic RNA viral infections, comprising a therapeutically effective amount of pyronaridine having Formula 1 (below) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier:

[Formula 1]

According to another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of epidemic infections caused by an RNA virus, comprising a therapeutically effective amount of artemisinin having Formula 2 (below) or a derivative thereof in combination with a pharmaceutically acceptable carrier:

[Formula 2]

According to another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of epidemic RNA viral infections, comprising a therapeutically effective amount of pyronaridine of the above Formula 1 or a pharmaceutically acceptable salt thereof and artemisinin of the above Formula 2 or a derivative thereof in combination with a pharmaceutically acceptable carrier.

In one embodiment of the present invention, examples of the pharmaceutically acceptable salt of pyronaridine may include, but are not limited to, an acid addition salt formed from phosphoric acid, sulfuric acid, hydrochloric acid, acetic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, maleic acid, or fumaric acid. In another embodiment of the present invention, the pharmaceutically acceptable salt of pyronaridine may be pyronaridine tetraphosphate.

In another embodiment of the present invention, examples of the artemisinin derivative may include, but are not limited to, dihydroartemisinin, artesunate, artemether, and artiefether. In another embodiment of the present invention, the artemisinin derivative may be artesunate.

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 and artemisinin or a derivative thereof in combination with a pharmaceutically acceptable carrier, a suitable weight ratio of pyronaridine or a pharmaceutically acceptable salt thereof to artemisinin or a derivative thereof may be from 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 from 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 from 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.

The term "pharmaceutically acceptable" as used herein refers to a degree of toxicity that is physiologically acceptable, does not inhibit the action of the active ingredient when administered to humans, and generally does not cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions. The pharmaceutical composition with a pharmaceutically acceptable carrier according to the present invention can be formulated in various ways depending on the route of administration known in the art. The route of administration can be oral or parenteral, without being limited to these. Parenteral routes of administration include various routes such as transdermal, nasal, intraperitoneal, intramuscular, subcutaneous, intravenous, etc.

When the pharmaceutical composition of the present invention is administered orally, the pharmaceutical composition together with a suitable carrier for oral administration of the present invention can be prepared into a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer, injection, suppository and the like according to a method known in the art. Examples of suitable carriers 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 carboxymethylcellulose, hydroxypropyl methylcellulose and low-substituted hydroxymethylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, if necessary, crospovidone, sodium starch glycolate, sodium croscarmellose, sodium carboxymethylcellulose, agar, alginic acid or sodium alginate may be added as a disintegrant. In addition, the pharmaceutical composition may additionally include a glidant, an antiplatelet agent, a plasticizer, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, etc.

In addition, for parenteral administration, the pharmaceutical composition of the present invention can be prepared in the form of an injection, a suppository, a preparation for transdermal administration and a nasal inhalation according to methods known in the art, in combination with a suitable carrier for parenteral administration. In the case of injection, the composition must be sterilized and protected from contamination by 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, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof, and/or vegetable oil. More preferably, suitable carriers include Hank's solution, Ringer's solution, phosphate buffered saline (PBS) or sterile water for injection containing triethanolamine, 10% ethanol, 40% propylene glycol and isotonic solutions such as 5% glucose. To protect the injection from microbial contamination, it may further contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In addition, in most cases, the injection may further contain an isotonic agent such as sugar or sodium chloride.

These compounds are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, a widely read reference book of pharmaceutical chemistry.

In the case of inhalation administration, the compound of the present invention can be conveniently delivered by means of an aerosol spray from a pressurized container or a nebulizer using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of using an aerosol spray from a pressurized container, the unit dosage can be determined by providing a valve for delivering a metered amount. For example, capsules and cartridges for use in inhalers or insufflators can contain a powder mixture on a suitable powder base.

Other pharmaceutically acceptable carriers include those described in the following document (Remington's Pharmaceutical Sciences, 19th Edition, 1995 Mack Publishing Company, Easton, Pennsylvania).

In another embodiment of the present invention, pyronaridine or a pharmaceutically acceptable salt thereof is prepared in combination with pharmaceutically acceptable carriers.

As described above, the "pharmaceutically acceptable carrier" that can be used in the present invention may be any commonly used in the pharmaceutical field. Representative examples 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, aluminosilicate, magnesium aluminum metasilicate, bentonite and a mixture thereof. In addition to the carrier, the pharmaceutical composition of the present invention may further comprise a disintegrant for rapid disintegration and dissolution upon contact with an aqueous medium when administered in vivo ; a solubilizer or surfactant for enhancing dissolution or absorption; and a glidant or lubricant to improve slip or increase flowability. Examples of the disintegrant may include crospovidone, sodium starch glycolate, sodium croscarmellose, sodium carboxymethylcellulose, agar, alginic acid or sodium alginate. Examples of the glidant or lubricant may include, but are not limited to, colloidal silicon dioxide, silicon dioxide, talc, magnesium stearate, calcium stearate, zinc stearate, sodium stearate fumarate, stearic acid or silicon dioxide. According to another embodiment of the present invention, there is provided a pharmaceutical composition comprising from 40 to 80% by weight of pyronaridine or a pharmaceutically acceptable salt thereof, from 1 to 30% by weight of microcrystalline cellulose, from 0.1 to 5% by weight of silicon dioxide. , from 1 to 10% by weight of hydroxypropyl cellulose, from 1 to 10% by weight of low-substituted hydroxypropyl cellulose, from 2 to 20% by weight of sodium starch glycolate and from 1 to 10% by weight of magnesium stearate.

In another embodiment of the present invention, artemisinin or a derivative thereof is formulated in combination with pharmaceutically acceptable carriers. In addition to the carrier, the pharmaceutical composition of the present invention may further comprise a disintegrant for rapid disintegration and dissolution upon contact with an aqueous medium when administered in vivo , a solubilizer or surfactant for enhancing dissolution or absorption, and a glidant or lubricant for improving slip or increasing fluidity. Examples of the carrier 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 the disintegrant may include crospovidone, sodium starch glycolate, sodium croscarmellose, sodium carboxymethylcellulose, agar, alginic acid, or sodium alginate. Examples of a glidant or lubricant 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 the surfactant may include sodium lauryl sulfate and its derivative, poloxamer and its derivative, saturated polyglycolized glyceride (also known as gelucir), 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 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 not limited to such. According to another embodiment of the present invention, a pharmaceutical composition is provided, comprising from 10 to 50% by weight of artemisinin or a derivative thereof, from 30 to 70% by weight of microcrystalline cellulose, from 2 to 20% by weight of low-substituted hydroxypropyl cellulose, from 2 to 20% by weight of sodium starch glycolate, from 0.1 to 5% by weight of silicon dioxide, from 0.5 to 15% by weight of sodium lauryl sulfate and from 0.1 to 5% by weight of magnesium stearate.

In another embodiment of the present invention, pyronaridine or a pharmaceutically acceptable salt thereof and artemisinin or a derivative thereof were prepared in combination with pharmaceutically acceptable carriers. In addition to the carrier, the pharmaceutical composition of the present invention may further comprise a disintegrant for rapid disintegration and dissolution upon contact with an aqueous medium upon in vivo administration, a solubilizer or surfactant for increasing dissolution or absorption, and a glidant or lubricant for improving slip or increasing fluidity. Examples of the 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, butylhydroxytoluene, and a mixture thereof. Representative examples of the surfactant may include sodium lauryl sulfate and its derivative, poloxamer and its derivative, saturated polyglycolized glyceride (also known as gelucir), 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 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 not limited to such. Examples of a disintegrant may include crospovidone, sodium starch glycolate, sodium croscarmellose, sodium carboxymethylcellulose, agar, alginic acid or sodium alginate. Examples of a glidant or lubricant may include colloidal silicon dioxide, silicon dioxide, talc, magnesium stearate, calcium stearate, zinc stearate, sodium stearate fumarate, stearic acid or silicon dioxide, but not limited to such. According to another embodiment of the present invention, there is provided a pharmaceutical composition comprising from 15 to 60% by weight of pyronaridine or a pharmaceutically acceptable salt thereof, from 5 to 20% by weight of artemisinin or a derivative thereof, from 5 to 30% by weight of microcrystalline cellulose, from 10 to 40% by weight of crospovidone, from 2 to 15% by weight of low-substituted hydroxypropyl cellulose, from 1 to 10% by weight of sodium lauryl sulfate, from 5 to 30% by weight of polyethylene glycol, from 0.1 to 5% by weight of hydroxypropyl cellulose, from 0.001 to 1% by weight of butylhydroxytoluene, from 0.1 to 5% by weight of silicon dioxide and from 0.5 to 10% by weight of magnesium stearate.

The pharmaceutical composition of the present invention can be prepared in the form of powder, granules, tablets, capsules, dry syrup, preparation for external administration, injections, suppositories, preparation for transdermal administration, inhalation administration, etc.

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 for the prevention and treatment of epidemic infections caused by an RNA virus.

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

In another embodiment of the present invention, examples of an epidemic infectious disease caused by an RNA virus may include, but are not limited to, Zika virus infection, Ebola virus infection, and respiratory diseases caused by a novel influenza virus and coronavirus infection. In another embodiment of the present invention, examples of respiratory diseases caused by 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 present invention, a respiratory disease caused by a coronavirus infection may be coronavirus disease 2019 (COVID-19).

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread and recurrence of epidemic RNA viral infections by administering the pharmaceutical composition of the present invention, and the term "treatment" refers to any action in which the symptoms of the disease are improved or beneficially modified by administering the pharmaceutical composition of the present invention.

In addition, the term "therapeutically effective amount" as used herein refers to an amount that shows a significant response compared to a negative control, and preferably refers to an amount sufficient to prevent or treat epidemic RNA virus infections. The therapeutic dose for a patient is usually 50 to 2000 mg/day, and more preferably 100 to 1000 mg/day, depending on the severity of the condition and whether the compound is administered alone or in combination, or in combination with other drugs. This amount can be administered once a day or in divided doses by oral or parenteral routes. However, the therapeutically effective amount may be appropriately varied depending on various factors such as the type and severity of the disease, the age, body weight, health condition and sex of the patient, the route of administration and the period of treatment.

Although the present invention describes the prevention and treatment of epidemic RNA viral infections in humans, preferably human respiratory diseases caused by coronavirus infections, and more preferably human COVID-19, the present invention may be useful for the treatment of infections caused by RNA viruses, in particular Coronaviridae viruses causing respiratory diseases, and more particularly the virus causing COVID-19, in animals and humans.

The objectives of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned above will be clear to a person skilled in the art from the following description. In addition, the above description does not limit the claimed invention in any way, moreover, a specific combination of the discussed features is not absolutely necessary for the proposed invention.

ADVANTAGES OF THE INVENTION

The present invention provides a pharmaceutical composition for the prevention or treatment of epidemic RNA viral infections, comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and/or artemisinin or a derivative thereof as an active ingredient(s). The pharmaceutical composition of the present invention can be effectively used for the prevention or treatment by effectively inhibiting epidemic RNA viral infections, such as respiratory diseases such as COVID-19 caused by coronaviruses.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a concentration-response curve of the inhibitory effect against SARS-CoV-2 and cytotoxicity upon pretreatment with pyronaridine tetraphosphate, which is one of the active ingredients of the present invention, as measured 24 hours after challenge/infection.

Fig. 2 is a concentration-response curve of the inhibitory effect against SARS-CoV-2 and cytotoxicity of co-treatment with pyronaridine tetraphosphate measured 24 hours after virus challenge.

Fig. 3 shows the results of measuring the inhibitory effect against SARS-CoV-2 by co-treatment with artesunate, one of the active ingredients of the present invention, at 24 and 48 hours after infection, compared with the effects of chloroquine used as a control.

Fig. 4 shows the results of comparison of the inhibitory effect of combinations with different ratios of pyronaridine tetraphosphate and artesunate in cells infected with SARS-CoV-2, with the optimal ratio comparing the inhibitory effects against viruses 24 hours and 48 hours after infection.

Fig. 5 shows the concentration-response curves of the inhibitory effect against SARS-CoV-2 and cytotoxicity measured 24 hours and 48 hours after infection of human lung Calu-3 cells with co-administration of pyronaridine tetraphosphate or artesunate at the time of viral infection, and a comparison of these with the data using hydroxychloroquine as a control.

Fig. 6 is a curve showing the inhibitory effect against SARS-CoV-2 when co-administered post-infection (post-infection treatment) with pyronaridine tetraphosphate or artesunate in human lung cell line Calu-3 cells, measured 48 hours after drug treatment.

Figure 7 shows the results of experiments in which hamsters were infected with SARS-CoV-2 and then treated orally with low or high doses of pyronaridine tetraphosphate and artesunate in a 3:1 ratio at 1 hour post-infection once daily for 3 days, or with high doses of pyronaridine tetraphosphate alone at 25 hours post-infection, and shows the viral titers in the lungs at day 4 post-infection compared to a control group inoculated with the virus but not treated with the drug.

EXAMPLES OF INVENTION

The composition and effects of the present invention will now be described in more detail by way of examples. It should be understood that these examples are merely illustrative and do not limit the scope of the present invention.

Example 1: Preparation of a monotablet of pyronaridine tetraphosphate

Hydroxypropyl cellulose was dissolved in ethanol to prepare a binder solution. After wet granulation of pyronaridine tetraphosphate using the prepared binder solution, the resulting product was dried and granulated. Low-substituted hydroxypropyl cellulose, sodium starch glycolate, microcrystalline cellulose and silicon dioxide were mixed. After adding magnesium stearate lubricant, tablets were prepared by tabletting.

[Table 1]

Ingredient Content (mg/product) Pyronaridine tetraphosphate
Microcrystalline cellulose
Silicon dioxide
Hydroxypropyl cellulose
Low substituted hydroxypropyl cellulose
Sodium starch glycolate
Magnesium stearate 360
48
6
12
18
24
12

Example 2: Preparation of a monotablet of artesunate

Silicon dioxide and sodium lauryl sulfate were sieved through a sieve. The sieved silicon dioxide and sodium lauryl sulfate were mixed with artesunate, microcrystalline cellulose, low-substituted hydroxypropyl cellulose and sodium starch glycolate, lubricated with magnesium stearate and then tabletted to prepare tablets. The resulting product was coated with a film-forming agent.

[Table 2]

Ingredient Content (mg/product) Artesunate
Microcrystalline cellulose
Low substituted hydroxypropyl cellulose
Sodium starch glycolate
Silicon dioxide
Sodium Lauryl Sulfate
Magnesium stearate
Film coating agent (Opadry) 100
228
30
20
5
12
5
12

Example 3: Preparation of a pyronaridine tetraphosphate/artesunate combination tablet

Polyethylene glycol as a melting dispersing carrier, butylhydroxytoluene and artesunate as an active ingredient were mixed, melted by heating, and then rapidly cooled and finely ground. Then, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crospovidone and magnesium stearate were mixed thereto to obtain Mixture 1. After dissolving hydroxypropyl cellulose in ethanol, 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 magnesium stearate was added to the mixture to lubricate the mixture, followed by tabletting to obtain tablets. The resulting product was coated with a film-forming agent.

[Table 3]

Ingredient Content (mg/product) Artesunate
Pyronaridine tetraphosphate
Microcrystalline cellulose
Crospovidone
Low substituted hydroxypropyl cellulose
Sodium Lauryl Sulfate
Polyethylene glycol
Hydroxypropyl cellulose
Butylated hydroxytoluene
Silicon dioxide
Magnesium stearate
Film coating agent (Opadry) 60
180
93
120
38
23
90
6
0.12
4.5
16.5
20

In order to determine whether pyronaridine or a salt thereof and artemisinin or a derivative thereof according to the present invention have antiviral activity against coronavirus, the reagents were administered individually and in combination as shown in the following experimental examples, and the degree of inhibition of viral infection was evaluated.

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

In Experimental Example 1, before infecting cells with SARS-CoV-2 (Korean isolate), the cells were pretreated with pyronaridine tetraphosphate for 1 hour and then its inhibitory efficacy against viral infection was evaluated.

1) Preparation of viruses and host cells

Vero cells were purchased from American Type Culture Collection (ATCC) and incubated at 37°C with 5% _CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC). After virus amplification, viral titers were determined by plaque assay by counting viral plaques formed in the cells used for virus amplification upon virus infection.

2) Determination of antiviral efficacy using immunofluorescent staining

Vero cells were seeded at 1.2 x 10 ⁴ cells/well in µClear plates and 24 h before the experiment, the cells were pretreated for 1 h with a serial 10-fold dilution of the drug in culture media in the range of 0.05-50 μM, and then the cells were inoculated with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.0125. At 24 h post-infection, the cells were fixed with 4% formaldehyde and infected cells were analyzed by immunofluorescence staining using antibodies against the SARS-CoV-2 N protein. The infection rate was calculated as the ratio of the number of infected cells to the total number of cells compared to positive and negative controls using image analysis software. The antiviral effect of the drug is presented as a concentration-response curve, and using the Graph Prism analysis program (Ver. 8), the 50% effective concentration (EC _{50 -} the concentration that inhibits the cytotoxicity caused by viral infection by 50%) and 50% cytotoxic concentration (CC _{50 -} the concentration of the compound that causes damage to 50% of the cells compared to normal cells) were calculated as shown in Equation 1.

<Equation 1>

Sigmoid curve model,

Y= Lower value + (Upper - Lower)/(1 + (IC ₅₀ /X) ^{slope of the curve} )

The result is shown in Fig. 1: In the case of Vero cells infected with SARS-CoV-2, 70% virus inhibition was observed at 50 μM pyronaridine, but the cytotoxicity was also increased by 17% when pre-treated with the drug. Vero cells were isolated from African green macaque (Chlorocebus sp.) kidney epithelial cells and are known to be IFN-1 deficient cells. In a previous study, when measuring the in vitro antiviral efficacy of pyronaridine against Ebola virus in Vero cells, no antiviral activity was observed at a concentration below CC ₅₀ (CC ₅₀ = 1.3 μM), but when inoculated into human Hela cells, the CC ₅₀ was found to be higher and the antiviral activity was exhibited at a non-toxic concentration (EC ₅₀ = 0.42-1.12 μM, CC ₅₀ = 3.1 μM). In addition, pyronaridine significantly reduced mortality and viral infection rates in Ebola virus-infected mouse models (Lane et al., 2015). It is well known that the efficacy of antiviral agents may vary in in vitro or in vivo assay systems depending on the differences in the properties of the host cells tested and their intracellular immune signaling pathways (Lane et al., 2015), and therefore, assays in human-derived host cells were further performed and the antiviral efficacy of pyronaridine was further confirmed in various cell lines and animal studies.

Experimental Example 2: Inhibitory effect of pyronaridine tetraphosphate on the SARS-CoV-2 virus (administration at the time of infection)

In Experimental Example 2, the inhibitory effect of pyronaridine on viral infection was evaluated by co-treating the cells at the time of SARS-CoV-2 (Korean isolate) infection. It is known that chloroquine exhibits antiviral efficacy by increasing endosomal pH, which leads to inhibition of virus binding to cells and glycosylation of host cell receptors to SARS-CoV (Vincent et al., 2005). Since pyronaridine, which has a similar structure to chloroquine, was expected to act by a similar mechanism, pyronaridine was administered simultaneously (with the virus) at the time of viral infection and its antiviral efficacy was measured. By optimizing some of the experimental conditions used in Experimental Example 1, the experiment was conducted under test conditions with relatively low cytotoxicity.

1) Preparation of viruses and host cells

Vero cells were incubated at 37°C with 5% _CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC). After virus amplification, viral titers were determined by qRT-PCR measuring the number of RNA copies.

2) Measurement of antiviral efficacy by RNA copy number analysis

After dissolving pyronaridine tetraphosphate in DMSO, it was diluted to a concentration of 0.033-100 μM in the culture medium. 24 hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2 × 10 ⁴ cells / well (MOI = 0.01), and the culture medium containing different dilutions of the drug was added to each well. 24 hours after infection, the cell supernatant was collected, RNA was isolated and qRT-PCR was performed against the RdRp gene. The antiviral efficacy of the drug was analyzed by comparing the number of viral RNA copies in the drug with that in the control. A concentration-response curve was constructed showing the dependence of the degree of inhibition of viral infection (% inhibition) on the viral titer calculated from the number of RNA copies, and the 50% effective concentration (EC ₅₀ , the concentration that inhibits the viral titer by 50%) was calculated using the Graph Prism analysis program (Ver. 8), as in Experimental Example 1.

3) Cytotoxicity measurement (% cytotoxicity)

Cytotoxicity was measured using a tetrazolium salt assay (WST-1). WST-1 is converted to 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 an additional 1 hour and the amount of formazan formed was calculated by measuring the optical density using ELISA. The 50% cytotoxic concentration (CC ₅₀ , the concentration of the compound that causes damage to 50% of the cells compared to normal cells) was calculated.

As a result, as shown in Fig. 2, pyronaridine exhibited an antiviral effect in a concentration-dependent manner when treated at the time of virus inoculation, and cytotoxicity was observed at some high concentrations, but the antiviral activity against SARS-CoV-2 virus resulting in more than 90% inhibition was observed at non-cytotoxic concentrations (EC ₅₀ = 8.27 μM, CC ₅₀ = 11.54 μM; selectivity index, SI> 1.40).

Experimental Example 3: Inhibitory effect of artesunate on SARS-CoV-2 (co-administration)

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

1) Preparation of the 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 by RNA copy number analysis

Artesunate was dissolved in DMSO and then diluted to concentrations of 3.13, 12.5, and 50 μM in the culture medium. 24 hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2 × 10 ⁴ cells/well (MOI = 0.01), and the culture medium containing different dilutions of the drug was added to each well. At 24 hours and 48 hours after infection, the cell supernatant was collected and qRT-PCR against the RdRp gene was performed to calculate the virus titer and the inhibition rate of virus infection (% inhibition), as shown in Experimental Example 2, chloroquine was used as a control.

3) Cytotoxicity measurement (% cytotoxicity)

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

As a result, as shown in Fig. 3, artesunate exhibited antiviral activity in a concentration-dependent manner and showed a virus inhibition rate of 83% at a concentration of 50 μM. At 12.5 μM, the inhibition rate was 40% and 51% at 24 hpi (24 hours post-infection) and 48 hpi (48 hours post-infection), respectively. In addition, at 3.13 μM, the inhibition rate was 39% at 48 hpi. No significant cytotoxicity was observed under all the conditions tested. Artesunate had a lower EC ₅₀ and a slower onset of SARS-CoV-2 inhibition compared with pyronaridine or chloroquine used as controls, but had a longer lasting antiviral effect, showing a pattern in which the degree of viral inhibition slowly increased over time.

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

In contrast to chloroquine, which showed no antiviral effect in Ebola virus-infected guinea pig models, pyronaridine significantly improved viral titer and survival in Ebola virus-infected mouse models. Thus, it was suggested that chloroquine may have a mechanism of action different from those for which immunomodulatory mechanisms have been proposed, such as the type 1 IFN-1 pathway (Lane et al., 2019). Artesunate also demonstrated antiviral efficacy against Ebola virus in in vitro assays, but weaker than pyronaridine (Gignox et al., 2016). Therefore, Experimental Example 4 assessed the changes in antiviral efficacy depending on the combined administration of the two drugs at different ratios.

1) Preparation 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 by RNA copy number analysis

Pyronaridine tetraphosphate and artesunate were dissolved in DMSO, diluted to different concentrations in the culture medium in different ratios, creating combinations such as 1:1, 3:1, 10:1, etc. 24 hours before the start of the experiment, Vero cells seeded in a 96-well plate at a density of 2×10 ⁴ cells/well (MOI=0.01) were inoculated with SARS-CoV-2, and then culture medium containing different dilutions of the drug was added to each well. At 24 hours post-infection, the cell supernatant was collected and qRT-PCR was performed against the RdRp gene to calculate the virus titer and the inhibition rate of virus infection (% inhibition), as shown in Experimental Example 2. When a combination containing 10 μM pyronaridine tetraphosphate and 3.3 μM artesunate was administered, the virus titers were measured at 24 and 48 hours post-infection, and compared with chloroquine and lopinavir used as controls.

3) Cytotoxicity measurement (% cytotoxicity)

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

As a result, as shown in Fig. 4, the antiviral effect of artesunate administered in combination with pyronaridine was higher than that of artesunate administered alone, and the antiviral effect increased as the ratio of pyronaridine in the combination increased. Specifically, when 10 μM pyronaridine tetraphosphate and 3.3 μM artesunate were used together (ratio of 3:1), an inhibition rate of viral infection of 90-100% was achieved, which was higher in antiviral effect than chloroquine or lopinavir used as a control. In this case, the inhibition rate of infection was maintained for up to 48 hours. No significant cytotoxicity was observed at all the ratios in the combinations shown in Fig. 4, and when 10 μM pyronaridine tetraphosphate and 3.3 μM artesunate were administered in combination, lower cytotoxicity was observed compared to treatment with 10 μM pyronaridine tetraphosphate alone (49.5% reduction).

Experimental Example 5: Inhibitory effect of pyronaridine tetraphosphate or artesunate on SARS-CoV-2 in human lung cell lines (co-administration)

It has been reported that there may be species differences in the antiviral response between humans and other mammals, such as receptor structure. Therefore, in order to confirm the efficacy in human lung cell lines, in Experimental Example 5, SARS-CoV-2 (Korean isolate) was inoculated into Calu-3 cells (human lung cell line) and treated with pyronaridine phosphate or artesunate, and the efficacy in inhibiting viral infection was evaluated. The inhibitory effect of pyronaridine tetraphosphate or artesunate on viral infection was evaluated in human lung cell lines such as Calu-3 when co-administered to the cells at the time of SARS-CoV-2 (Korean isolate) virus infection.

1) Preparation 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 fetal bovine serum (FBS) and antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC).

2) Measuring antiviral efficacy using RNA copy number

After dissolution in DMSO, pyronaridine tetraphosphate was diluted to a concentration of 0.033-100 μM in culture medium. 24 hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2×10 ⁴ cells/well (MOI=0.01), and culture medium containing different dilutions of the drug was added to each well. At 24 hours and 48 hours post-infection, qRT-PCR against the RdRp gene was performed as shown in Experimental Example 2. A concentration-response curve was plotted showing the degree of inhibition of viral infection (% inhibition) from the viral titer calculated from the RNA copy number, and the 50% effective concentration (EC ₅₀ , the concentration that inhibits the viral titer by 50%) was calculated using Graph Prism analysis program (Ver. 8), as shown in Experimental Example 1.

3) Cytotoxicity measurement (% cytotoxicity)

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

As a result, as shown in Fig. 5, both pyronaridine and artesunate exhibited antiviral effects in human lung cell lines in a concentration-dependent manner when co-administered at the time of infection. Moreover, at both 24 h post infection (24 hpi) and 48 h post infection (48 hpi), they exhibited antiviral activity against SARS-CoV-2, showing more than 90% inhibition at non-cytotoxic concentrations (for pyronaridine 48 h post infection IC ₅₀ = 8.58 μM, CC ₅₀ > 100 μM, selectivity index, SI > 11.66, for artesunate 48 h post infection IC ₅₀ = 0.45 μM, CC ₅₀ > 100 μM, selectivity index, SI > 220.8). In particular, in the case of artesunate, the effect was significantly enhanced in human lung cell lines compared to that in macaque kidney cell lines (Vero cells). In contrast, hydroxychloroquine showed no antiviral activity at concentrations below 50 μM in human lung cell lines, but hydroxychloroquine showed antiviral activity in 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 were administered separately into Calu-3 cells at 0, 2, 4, 6, 8, 10, 12, 24, and 36 hours after inoculation with SARS-CoV-2 virus (Korean isolate), and the inhibitory effect of each drug on viral infections was evaluated.

1) Preparation of viruses and host cells

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

2) Determination of antiviral efficacy by viral plaque assay

After dissolving pyronaridine tetraphosphate and artesunate in DMSO each separately, they were diluted to 12.5 μM with culture medium. At 1 hour after inoculation of SARS-CoV-2 (MOI=0.1) into the cells, the supernatant was removed and the Calu-3 cells were washed, followed by the addition of DMEM culture medium containing 2% bovine serum. The drug-containing culture media were added at 0, 2, 4, 6, 8, 10, 12, 24, and 36 hours each separately. At 48 hours after treatment with each drug, the cell supernatants were collected and plaque assay was performed, in which plaques formed by infectious virus infection were counted in Vero cells used for virus amplification. A layer of DMEM-F12 medium containing 2% agarose was applied to a layer of infected Vero cells and after incubation for 72 hours, the number of plaques was counted using counterstaining with crystal violet. The antiviral efficacy of the drug was analyzed by analyzing the degree of inhibition of viral infection (% inhibition) depending on the virus titer calculated from the number of plaques formed and compared with the control.

As a result, as shown in Fig. 6, the maximum antiviral efficacy was shown by treatment with 12.5 μM pyronaridine (>99% inhibition when added at 6 hours post infection, >94% inhibition when added at 12 hours post infection, >90% inhibition when added at 24 hours post infection). On the other hand, treatment with 12.5 μM artesunate showed 92-96% inhibition when added at 6 hours post infection, >90% inhibition when added at 12 hours post infection, and 48% inhibition when added at 24 hours post infection.

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

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

1) Preparing viruses and hamsters for SARS-CoV-2 infection

The SARS-CoV-2 virus was provided by the Korea Centers for Disease Control and Prevention (KCDC). Syrian hamsters, which have shown high susceptibility to SARS-CoV-2 and are widely available from suppliers, were used as experimental animal models. SARS-CoV-2 (1×10 ⁶ PFU/100 μL) was inoculated into each of the hamster's nasal cavities in an amount of 50 μL.

2) Measurement of antiviral efficacy in vivo by plaque assay

At 1 hour after nasal inoculation with SARS-CoV-2, pyronaridine tetraphosphate (180 mg/kg or 360 mg/kg) and artesunate (60 mg/kg or 120 mg/kg) were orally administered in combination at a ratio of 3:1 once a day for 3 days, and the antiviral efficacy of the combination of the two drugs against SARS-CoV-2 in vivo was evaluated. For the comparative experimental group, pyronaridine at a dose of 360 mg/kg was orally administered once 25 hours after inoculation to evaluate the duration of post-infection efficacy of pyronaridine alone. Both pyronaridine tetraphosphate and artesunate were prepared immediately before use, completely dissolved in 5% sodium bicarbonate, and administered orally. A normal control group (Mock), which was not inoculated with the virus, and a control group that was simultaneously treated with only the vehicle were used as control groups. On day 4 post-infection, both left and right lung lobes were excised, virus was isolated, and virus titers in lung tissues were analyzed by plaque assay as described in Experimental Example 6. The virus titer in lungs determined by plaque assay was normalized to the total weight (g) of lung tissues and then log-transformed to calculate the endpoint titer (Log ₁₀ plaque-forming units/g, Log ₁₀ PFU/g, PFU-plaque forming unit).

As a result, as shown in Fig. 7, on the 4th day after inoculation, the reduction in infectious virus titer in lung tissues was statistically significant: for both pyronaridine tetraphosphate (P)-artesunate (A) 180/60 mg/kg and 360/120 mg/kg co-administration groups [median Log10PFU: 8.30 for virus-infected 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 was administered orally alone, a significant reduction in infectious virus titer in lung tissue was observed at a high dose of 360 mg/kg, and a significant inhibitory effect was observed even at a single administration at 25 hpi [median Log10PFU 8.30 for virus-infected control group treated with vehicle alone vs. 7.22 for the group receiving a single high dose at 25 hpi, (p<0.001)].

Claims (10)

1. Use of a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19), wherein the pharmaceutically acceptable salt of pyronaridine is selected from the group consisting of phosphate, sulfate, hydrochloride, acetate, methanesulfonate, benzenesulfonate, toluenesulfonate, maleate and fumarate. 2. Use of a therapeutically effective amount of artemisinin or a derivative thereof in the manufacture of a medicament for the prevention or treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19), wherein the artemisinin derivative is selected from the group consisting of dihydroartemisinin, artesunate, artemether and artieether. 3. Use of a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof and artemisinin or a derivative thereof in the manufacture of a medicinal product for the prevention or treatment of Middle East respiratory syndrome (MERS) or coronavirus disease 2019 (COVID-19), wherein the pharmaceutically acceptable salt of pyronaridine is selected from the group consisting of phosphate, sulfate, hydrochloride, acetate, methanesulfonate, benzenesulfonate, toluenesulfonate, maleate and fumarate; and wherein the artemisinin derivative is selected from the group consisting of dihydroartemisinin, artesunate, artemether and artieether. 4. The use according to claim 1 or 3, wherein the pharmaceutically acceptable salt of pyronaridine is pyronaridine tetraphosphate. 5. Use according to claim 3, wherein the weight ratio of pyronaridine or its pharmaceutically acceptable salt to artemisinin or its derivative is from 10:1 to 1:10. 6. Use according to claim 5, wherein the weight ratio of pyronaridine or its pharmaceutically acceptable salt to artemisinin or its derivative is from 1:1 to 6:1. 7. The use according to claim 6, wherein the weight ratio of pyronaridine or its pharmaceutically acceptable salt to artemisinin or its derivative is 3:1. 8. The use according to claim 2 or 3, wherein the artemisinin derivative is artesunate.