PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING EPIDEMIC RNA VIRAL INFECTIOUS DISEASE

Abstract

The present invention relates to a use of pyronaridine or a pharmaceutically acceptable salt thereof, and/or artemisinin or a derivative thereof for preventing or treating an epidemic RNA viral infectious disease, and more specifically, to a pharmaceutical composition for preventing or treating an epidemic RNA viral infectious disease, in particular, Coronavirus Disease 2019 (COVID-19), the composition comprising a therapeutically effective amount of pyronaridine or a pharmaceutically acceptable salt thereof, and/or artemisinin or a derivative thereof, together with a pharmaceutically acceptable carrier.

Claims

1.-32. (canceled)

33. A method for treating an epidemic RNA virus infection comprising: administering to a subject in need thereof a therapeutically effective amount of (i) pyronaridine or a pharmaceutically acceptable salt thereof, or (ii) artemisinin or a pharmaceutically acceptable salt thereof.

34. The method of claim 33, comprising administering to a subject in need thereof both (i) pyronaridine or a pharmaceutically acceptable salt thereof, and (ii) artemisinin or a pharmaceutically acceptable salt thereof.

35. The method of claim 33, wherein the pharmaceutically acceptable salt of pyronaridine is selected from the group consisting of phosphate, sulfate, hydrochloride, acetate, methanesulfonate, benzenesulfonate, toluenesulfonate, maleate and fumarate.

36. The method of claim 35, wherein the pharmaceutically acceptable salt of pyronaridine is pyronaridine tetraphosphate.

36. The method of claim 34, wherein a weight ratio of the pyronaridine or a pharmaceutically acceptable salt thereof to the artemisinin or a derivative thereof is 10:1 to 1:10.

37. The method of claim 36, 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.

38. The method of claim 37, wherein the weight ratio of the pyronaridine or a pharmaceutically acceptable salt thereof to the artemisinin or a derivative thereof is 3:1.

39. The method of claim 33, wherein the artemisinin derivative is selected from the group consisting of dihydroartemisinin, artesunate, artemether and arteether.

40. The method of claim 39, wherein the artemisinin derivative is artesunate.

41. The method of claim 33, which at least one other antiviral agent is further administered.

42. The method of claim 34, which at least one other antiviral agent is further administered.

43. The method of claim 41, 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.

44. The method of claim 43, wherein the other antiviral agent is selected from the group consisting of ribavirin, interferon, niclosamide and a combination thereof.

45. The method of claim 34, wherein the epidemic RNA virus infection is selected from the group consisting of Zika virus infection, Ebola virus infection, respiratory diseases caused by novel influenza virus or coronavirus infections.

46. The method of claim 45, wherein the respiratory disease caused by coronavirus infection is selected from the group consisting of Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Coronavirus Disease 2019 (COVID-19).

47. The method of claim 46, wherein the respiratory disease caused by coronavirus infection is Coronavirus Disease 2019 (COVID-19).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0045] FIG. 1 is a concentration-response curve regarding the inhibitory effects against SARS-CoV-2 and cytotoxicity by the pretreatment with pyronaridine tetraphosphate—which is one of the active ingredients of the present invention—when measured at 24 hours post-infection.

[0046] FIG. 2 is a concentration-response curve regarding the inhibitory effects against SARS-CoV-2 and cytotoxicity by the co-treatment with pyronaridine tetraphosphate when measured at 24 hours post-infection.

[0047] FIG. 3 shows the results in which the inhibitory effects against SARS-CoV-2 by co-treatment of artesunate—one of the active ingredients of the present invention—were measured at 24 hours and 48 hours post-infection and compared with those by chloroquine as a control.

[0048] FIG. 4 shows the results comparing the inhibitory effects by the combination in various ratios of pyronaridine tetraphosphate and artesunate in SARS-CoV-2-infected cells by the combinations, and at the optimal ratio, the inhibitory effects against viruses at 24 hours and 48 hours post-infection were compared.

[0049] FIG. 5 is concentration-response curves regarding the inhibitory effects against SARS-CoV-2 and cytotoxicity measured 24 hours and 48 hours post-infection in the human lung cell line, Calu-3 cells, in comparison with those by hydroxychloroquine as a control, when pyronaridine tetraphosphate or artesunate was treated simultaneously with virus infection.

[0050] FIG. 6 is a concentration-response curve regarding the inhibitory effects against SARS-CoV-2 by post-infection treatment with pyronaridine tetraphosphate or artesunate in the human lung cell line, Calu-3 cells, when measured 48 hours after drug treatment.

[0051] FIG. 7 shows the results in which hamsters were infected with SARS-CoV-2 and orally administered with low or high doses of pyronaridine tetraphosphate and artesunate in combination at 3:1 ratio at 1 hour post-infection once a day for 3 days, or with high dose of pyronaridine tetraphosphate alone at 25 hours post-infection, and then the viral titers in the lungs were analyzed at day 4 post-infection and compared the virus-inoculated control group in which the drugs were not administered.

MODE FOR THE INVENTION

[0052] Hereinafter, the constitutions and effects of the present invention will be described in more detail through examples. However, these examples are only illustrative, and the scope of the present invention is not limited thereto.

Example 1: Preparation of Pyronaridine Tetraphosphate Monotablet

[0053] Hydroxypropyl cellulose was dissolved in ethanol to prepare a binding solution. After wet granulation of pyronaridine tetraphosphate using the prepared binding solution, the obtained product was dried and granulated. Low-substituted hydroxypropyl cellulose, sodium starch glycolate, microcrystalline cellulose and silicon dioxide were mixed. After lubrication by adding magnesium stearate, tablets were prepared by tableting.

TABLE-US-00001 TABLE 1 Ingredient content (mg/preparation) Pyronaridine tetraphosphate 360 Microcrystalline cellulose 48 Silicon dioxide 6 Hydroxypropyl cellulose 12 Low-substituted hydroxypropyl cellulose 18 Sodium starch glycolate 24 Magnesium stearate 12

Example 2: Preparation of Artesunate Monotablet

[0054] Silicon dioxide and sodium lauryl sulfate were sieved using 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 by adding magnesium stearate, and then tableted to prepare tablets. The obtained product was coated with a film-coating agent.

TABLE-US-00002 TABLE 2 Ingredient content (mg/preparation) Artesunate 100 Microcrystalline cellulose 228 Low-substituted hydroxypropyl cellulose 30 Sodium starch glycolate 20 Silicon dioxide 5 Sodium lauryl sulfate 12 Magnesium stearate 5 Film-coating agent (Opadry) 12

Example 3: Preparation of Pyronaridine Tetraphosphate/Artesunate Combination Tablet

[0055] 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 pulverized. 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 thereto to lubricate the mixture, followed by tableting to prepare tablets. The obtained product was coated with a film-coating agent.

TABLE-US-00003 TABLE 3 Ingredient content (mg/preparation) Artesunate 60 Pyronaridine tetraphosphate 180 Microcrystalline cellulose 93 Crospovidone 120 Low-substituted hydroxypropyl cellulose 38 Sodium lauryl sulfate 23 Polyethylene glycol 90 Hydroxypropyl cellulose 6 Butylhydroxytoluene 0.12 Silicon dioxide 4.5 Magnesium stearate 16.5 Film-coating agent (Opadry) 20

[0056] In order to determine whether pyronaridine or a salt thereof, and artemisinin or a derivative thereof of the present invention have antiviral activity against coronavirus, the reagents were treated alone and in combination as in the following Experimental Examples, and the inhibitory rates against viral infection were evaluated.

Experimental Example 1: Evaluation of Antiviral Effects of Pyronaridine Tetraphosphate (Pretreatment)

[0057] In Experimental Example 1, before infecting cells with SARS-CoV-2 (a Korean isolate), pyronaridine tetraphosphate was pretreated for 1 hour, and the inhibitory efficacy against virus infection was evaluated.

[0058] 1) Preparation of Viruses and Host Cells

[0059] Vero cells were purchased from the American Type Culture Collection (ATCC) and incubated at 37° C. with 5% CO.sub.2 in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and an antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC). After virus amplification, the viral titers were determined by a plaque assay by counting viral plaques formed in the cells used for virus amplification upon infection with the virus.

[0060] 2) Determination of Antiviral Efficacy Using Immunofluorescence Staining Imagings

[0061] Vero cells were seeded at 1.2×10.sup.4 cells per well in μClear plates, and 24 hours prior to the experiment cells were pre-treated for 1 hour with a series of 10 dilutions of drugs in culture media in the range of 0.05-50 μM, and then SARS-CoV-2 was inoculated to the cells at a multiplicity of infection (MOI) of 0.0125. Twenty-four hours after infection, the cells were fixed with 4% formaldehyde, and the infected cells were analyzed by immunofluorescence staining using an antibody against N protein of SARS-CoV-2. The infection rate was calculated as the ratio of the number of infected cells to the total number of cells compared to the positive and negative controls through the imaging analysis program. The antiviral effect of the drug is represented as a concentration-response curve, and using the Graph Prism (Ver. 8) analysis program, 50% effective concentration (EC.sub.50, concentration that inhibits virus infection-induced cytotoxicity by 50%) and 50% cytotoxic concentration (CC.sub.50, the concentration of the compound that causes damage in 50% of cells in comparison with normal cells) was calculated as shown in Equation 1.

Sigmoidal model,Y=Bottom+(Top−Bottom)/(1+(IC.sub.50/X).sup.Hillslope)  <Equation 1>

[0062] As a result, as shown in FIG. 1, in the case of Vero cells infected with SARS-CoV-2, 70% virus inhibition rate was observed at a concentration of 50 μM of pyronaridine, but cytotoxicity was also increased by 17% by drug pretreatment. The Vero cells were isolated from the kidney epithelial cells of African green monkeys (Chlorocebus sp.) and have been known as type-1 IFN-deficient cells. In the previous study, when measuring the in vitro antiviral efficacy of pyronaridine against Ebola virus in Vero cells, no antiviral activity was observed at a concentration below CC.sub.50 (CC.sub.50=1.3 μM), but when inoculated into human-derived Hela cells, it was reported that the CC.sub.50 is higher and the antiviral activity showed at a non-toxic concentration (EC.sub.50=0.42-1.12 μM, CC.sub.50=3.1 μM). In addition, pyronaridine significantly inhibited mortality and viral infection rate in mouse models challenged with Ebola virus (Lane et al., 2015). It is well known that the efficacy of antiviral agents may differ in in vitro or in vivo assay systems depending on the differences in characteristics of the host cells tested and their intracellular immune signaling pathways (Lane et al., 2015), and thus human-derived host cell-based assays were additionally established, and the antiviral efficacy of pyronaridine was further confirmed in various cell lines and animal studies.

Experimental Example 2: Inhibitory Effects of Pyronaridine Tetraphosphate Against SARS-CoV-2 Virus (Co-Treatment)

[0063] In Experimental Example 2, the inhibitory effects of pyronaridine against viral infection were evaluated when co-treated cells at the time of SARS-CoV-2 (a Korean isolate) infection. Chloroquine is known to exhibit antiviral efficacy by increasing endosomal pH leading to inhibition of viral binding to the cells and glycosylation of host receptors to SARS-CoV (Vincent et al., 2005). Since pyronaridine—which has a structure similar to chloroquine—was also expected to act via a similar mechanism, pyronaridine was simultaneously treated at the time of viral infection and its antiviral efficacy was measured. By optimizing some experimental conditions used in Experimental Example 1, the experiment was carried out under test conditions with relatively low cytotoxicity.

[0064] 1) Preparation of Viruses and Host Cells

[0065] Vero cells were incubated at 37° C. with 5% CO.sub.2 in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and an antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC). After viral amplification, the viral titers were determined through qRT-PCR measuring RNA copy numbers.

[0066] 2) Measurement of Antiviral Efficacy Using RNA Copy Numbers

[0067] After dissolving pyronaridine tetraphosphate in DMSO, it was diluted to a concentration of 0.033-100 μM using culture media. Twenty-four hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2×10.sup.4 cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. Twenty-four hours after infection, the cell supernatant was collected, RNA was extracted, and qRT-PCR was performed against the RdRp gene. The antiviral efficacy of the drug was analyzed by comparing the copy number of viral RNA with the drug with that with the control. A drug concentration-response curve was drawn with the viral infection inhibition rate (% inhibition) from the virus titer inversely calculated from the RNA copy number, and 50% effective concentration (EC.sub.50, the concentration that inhibits virus titer by 50%) was calculated using the Graph Prism (Ver. 8) analysis program, as in Experimental Example 1.

[0068] 3) Measurement of Cytotoxicity (% Cytotoxicity)

[0069] Cytotoxicity was measured using a tetrazolium salts-based assay (WST-1). WST-1 is converted into a chromogenic substance called formazan by mitochondrial dehydrogenases, which are present only in living cells. After adding 10 μL of WST-1 premix to each well, cells were incubated for 1 additional hour, and the amount of formazan produced was calculated with its absorbance measured by ELISA. The 50% cytotoxic concentration (CC.sub.50, the concentration of compound that causes damage in 50% of cells compared with normal cells) was calculated.

[0070] As a result, as shown in FIG. 2, pyronaridine exhibited the antiviral effect in a concentration-dependent manner when co-treated, and cytotoxicity was observed at some high concentrations, but antiviral activity against SARS-CoV-2 virus, more than 90% inhibition, at non-cytotoxic concentrations (EC.sub.50=8.27 μM, CC.sub.50=11.54 μM; selectivity index, SI>1.40).

Experimental Example 3: Inhibitory Effects of Artesunate Against SARS-CoV-2 (Co-Treatment)

[0071] In Experimental Example 3, the antiviral efficacy of artesunate was measured under the same experimental conditions as in Experimental Example 2.

[0072] 1) Preparation of Virus and Host Cell

[0073] Vero cells and viruses were prepared in the same manner as shown in Experimental Example 2.

[0074] 2) Measurement of Antiviral Efficacy Using RNA Copy Numbers

[0075] Artesunate was dissolved in DMSO and then diluted to concentrations of 3.13, 12.5, and 50 μM using media. Twenty-four hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2×10.sup.4 cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. At 24 hours and 48 hours after infection, the cell supernatant was collected, and qRT-PCR was performed against the RdRp gene to calculate the virus titer and to calculate the virus infection inhibition rate (% inhibition) as shown in Experimental Example 2. As the control, chloroquine was used.

[0076] 3) Measurement of Cytotoxicity (% Cytotoxicity)

[0077] Cytotoxicity was measured in the same manner as shown in Experimental Example 2.

[0078] As a result, as shown in FIG. 3, artesunate exhibited the antiviral activity in a concentration-dependent manner and showed 83% inhibition rate against virus at a concentration of 50 μM. At 12.5 μM, the inhibition rates were 40% and 51% at 24 hours post-infection (24 hpi) and 48 hours post-infection (48 hpi), respectively. In addition, at 3.13 μM, the inhibition rate was 39% at 48 hours post-infection. There was no significant cytotoxicity in all conditions tested. Artesunate had a lower EC.sub.50 and slower onset time for SARS-CoV-2 inhibition compared to pyronaridine or chloroquine used as the control, but had a long-lasting antiviral effect, showing a pattern in which the virus inhibition rate increased slowly over time.

Experimental Example 4: Inhibitory Effects of Pyronaridine Tetraphosphate/Artesunate Combination Against SARS-CoV-2

[0079] Unlike chloroquine, which did not show antiviral effect in the guinea pig models infected with Ebola virus, pyronaridine significantly improved the virus titer and survival rate in Ebola virus-challenged mouse models. As such, it was assumed that chloroquine may have different mechanisms of action in addition, among which immunomodulatory mechanisms such as type 1 IFN-1 pathway were suggested (Lane et al., 2019). Artesunate also showed an antiviral efficacy against Ebola virus in in vitro assays but weaker than pyronaridine (Gignox et al., 2016). Therefore, in Experimental Example 4, the changes in antiviral efficacy according to treatment in combination at different combination ratio of the two drugs were evaluated.

[0080] 1) Preparation of Virus and Host Cells

[0081] Vero cells and virus were prepared in the same manner as shown in Experimental Example 2.

[0082] 2) Measurement of Antiviral Efficacy Using RNA Copy Numbers

[0083] Pyronaridine tetraphosphate and artesunate were dissolved in DMSO, diluted to various concentrations using media at various combination ratios such as 1:1, 3:1, 10:1, etc. Twenty-four hours before the experiments, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2×10.sup.4 cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. At 24 hours after infection, the cell supernatant was collected, and qRT-PCR was performed against the RdRp gene to calculate the virus titer and the virus infection inhibition rate (% inhibition) as shown in Experimental Example 2. When 10 μM of pyronaridine tetraphosphate and 3.3 μM of artesunate were treated in combination, virus titers were measured at 24 and 48 hours after infection each, and compared with chloroquine and lopinavir used as the controls.

[0084] 3) Measurement of Cytotoxicity (% Cytotoxicity)

[0085] Cytotoxicity was measured in the same manner as shown in Experimental Example 2.

[0086] As a result, as shown in FIG. 4, the antiviral effect of artesunate treated in combination with pyronaridine was higher than that of artesunate treated alone, and the antiviral effect increased as the ratio of pyronaridine in the combination was higher. Specifically, when 10 μM of pyronaridine tetraphosphate and 3.3 μM of artesunate were combined (ratio 3:1), it exhibited the inhibition rate of 90-100% against virus infection, which is a higher antiviral effect than that of chloroquine or lopinavir used as the controls. In this case, the inhibition rate against infection was maintained up to 48 hours. No significant cytotoxicity was observed in all combination ratios shown in FIG. 4, and when 10 μM of pyronaridine tetraphosphate and 3.3 μM of artesunate were treated in combination, lower cytotoxicity was observed in comparison to the treatment with 10 μM of pyronaridine tetraphosphate alone (49.5% decrease).

Experimental Example 5: Inhibitory Effects of Pyronaridine Tetraphosphate or Artesunate Against SARS-CoV-2 in Human Lung Cell Lines (Co-Treatment)

[0087] It has been reported that there may be species difference among the antiviral actions in humans and other animals, such as receptor structures. Therefore, in order to confirm the efficacy in human lung cell lines, in Experimental Example 5, when SARS-CoV-2 (a Korean isolate) was inoculated into Calu-3 cells (human lung cell line), pyronaridine phosphate or artesunate was treated and evaluated for efficacy in inhibiting viral infection. the inhibitory effects of pyronaridine tetraphosphate or artesunate against viral infection were evaluated in human lung cell lines, Calu-3 cells, when co-treated cells at the time of SARS-CoV-2 (a Korean isolate) infection.

[0088] 1) Preparation of Viruses and Host Cells

[0089] Calu-3 cells were incubated at 37° C. with 5% CO.sub.2 in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and an antibiotic. SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC).

[0090] 2) Measurement of Antiviral Efficacy Using RNA Copy Numbers

[0091] After dissolving in DMSO, pyronaridine tetraphosphate was diluted to a concentration of 0.033-100 μM using media. Twenty-four hours before the experiment, SARS-CoV-2 was inoculated into Vero cells seeded in a 96-well plate at a density of 2×10.sup.4 cells/well (MOI=0.01), and the culture media containing various dilutions of drug were added to each well. At 24 hours and 48 hours after infection, qRT-PCR was performed against the RdRp gene as shown in Experimental Example 2. A drug concentration-response curve was drawn with the viral infection inhibition rate (% inhibition) from the virus titer inversely calculated from the RNA copy number, and 50% effective concentration (EC.sub.50, the concentration that inhibits virus titer by 50%) was calculated using the Graph Prism (Ver. 8) analysis program, as shown in Experimental Example 1.

[0092] 3) Measurement of Cytotoxicity (% Cytotoxicity)

[0093] Cytotoxicity was measured in the same manner as in Experimental Example 2.

[0094] 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-treated at the time of infections. In addition, at both 24 hours post-infection (24 hpi) and 48 hours post-infection (48 hpi), they exhibited antiviral activities against SARS-CoV-2, more than 90% inhibition, at non-cytotoxic concentrations (pyronaridine 48 hours post-infection IC.sub.50=8.58 μM, CC.sub.50>100 μM; selectivity index, SI>11.66; artesunate 48 hours post-infection IC.sub.50=0.45 μM, CC.sub.50>100 μM; selectivity index, SI>220.8). Specifically, in the case of artesunate, the effect was significantly increased in the human lung cell lines compared to that in the monkey kidney cell lines, Vero cells. Contrary to those, hydroxychloroquine showed no antiviral effect at less than 50 μM in the human lung cell lines, whereas hydroxychloroquine showed antiviral efficacy in the monkey cell lines.

Experimental Example 6: Inhibitory Effects of Post-Infection Treatment with Pyronaridine Tetraphosphate or Artesunate Against SARS-CoV-2 in Human Lung Cell Lines

[0095] In Experimental Example 6, pyronaridine tetraphosphate or artesunate was treated each in Calu-3 cells at 0, 2, 4, 6, 8, 10, 12, 24 and 36 hours after SARS-CoV-2 (a Korean isolate) inoculation, and evaluated how long hours the inhibitory effect of each drug were retained against virus infections.

[0096] 1) Preparation of Viruses and Host Cells

[0097] Calu-3 cells and viruses were prepared in the same manner as shown in Experimental Example 5.

[0098] 2) Determination of Antiviral Efficacy Using Viral Plaque Assay

[0099] After dissolving pyronaridine tetraphosphate and artesunate in DMSO each, it was diluted to 12.5 μM using media. At 1 hour after the inoculation with SARS-CoV-2 (MOI=0.1), the supernatant was removed, and Calu-3 cells were washed, followed by the addition of DMEM culture media containing 2% bovine serum. The culture media containing drugs were added at 0, 2, 4, 6, 8, 10, 12, 24 and 36 hours each. At 48 hours after each drug treatment, cell supernatants were harvested and a plaque assay—in which the plaques generated by infectious virus infection were counted in Vero cells, cells used for virus amplification—was performed. The DMEM-F12 medium layer containing 2% agarose was laid on the layer of infected Vero cells, and the number of plaques was counted by using counter-staining with crystal violet, after incubation for 72 hours. The antiviral efficacy of the drug was analyzed with the viral infection inhibition rate (% inhibition) from the virus titer inversely calculated from the number of plaques formed and compared with the control.

[0100] As a result, as seen in FIG. 6, the maximum antiviral efficacy was shown when 12.5 μM pyronaridine was treated (>99% inhibition when added at up to 6 hours post-infection, >94% inhibition when added at up to 12 hours post-infection, and 90% inhibition when added at up to 24 hours post-infection). On the other hand, 12.5 μM artesunate treatment showed 92-96% inhibition when added at up to 6 hours post-infection, >90% inhibition when added at up to 12 hours post-infection, and 48% inhibition when added at up to 24 hours post-infection.

Experimental Example 7: Inhibitory Effects of Pyronaridine Tetraphosphate/Artesunate Combination Against SARS-CoV-2 in COVID-19 Animal Models

[0101] In Experimental Example 7, pyronaridine tetraphosphate and artesunate (combination in a 3:1 ratio) were orally administered to hamsters infected with SARS-CoV-2 (a Korean isolate) to evaluate in vivo antiviral efficacy in animals.

[0102] 1) Preparation of Viruses and Hamsters for SARS-CoV-2 Inoculation

[0103] SARS-CoV-2 virus was provided by the Korea Centers for Disease Control and Prevention (KCDC). As experimental animal models, Syrian hamsters—which showed high susceptibility to SARS-CoV-2 and had low restriction in supply—were used, and SARS-CoV-2 (1×10.sup.6 PFU/100 μL) was inoculated to each of both nasal passages of the hamster with an amount of 50 μL.

[0104] 2) In Vivo Antiviral Efficacy Measurement Using Plaque Assay

[0105] 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 as the combination of 3:1 ratio once a day for 3 days, and in vivo antiviral efficacy of the combination of two drugs against SARS-CoV-2 was evaluated. As the comparative experimental group, pyronaridine 360 mg/kg alone was orally administered once at 25 hours after infection to evaluate the duration of post-infection efficacy of pyronaridine alone. Both pyronaridine tetraphosphate and artesunate were prepared just before use, completely dissolved in 5% sodium bicarbonate, and orally administered. As the control groups, a normal control group (Mock) in which virus was not inoculated and a vehicle control group in which only a solvent was administered at the same time were used. At 4-day post-infection, both the left and right lobes of the lungs were excised, the virus was extracted, and the virus titers in the lung tissues were analyzed by a plaque assay as described in Experimental Example 6. The viral titer in the lungs quantified by a plaque assay was normalized to the total weight (g) of the lung tissues, and then converted to log value to calculate the final titer (Log.sub.10 plaque forming unit/g, Log.sub.10 PFU/g).

[0106] As a result, as shown in FIG. 7, at 4-day post-infection, the reduction in the titer of the infectious virus in the lung tissues was statistically significant in both the pyronaridine tetraphosphate (P)-artesunate (A) 180/60 mg/kg and 360/120 mg/kg co-administration groups [median Log.sub.10 PFU: 8.30 for virus-challenged vehicle control group vs. 7.22 for PA 180/60 mg/kg Co-administration group (p<0.001); vs. 7.61 for PA 360/120 mg/kg co-administration group (p=0.046)]. On the other hand, when pyronaridine tetraphosphate alone was orally administered, a significant decrease in the infectious virus titer was observed in the lung tissue when high dose of 360 mg/kg was administered, and a significant inhibitory effect was observed even if it was administered once at 25 hours post-infection [median Log.sub.10 PFU 8.30 for virus-challenged vehicle control group vs. single high-dose 25 hpi administration group 7.22 (p<0.001)].