PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING COVID-19 COMPRISING NANO-SIZED GRAPHENE OXIDE COMPOSITE AND METHOD USING SAME

Abstract

Provided are a pharmaceutical composition for preventing or treating a coronavirus infection or infectious disease, a composition for disinfection, and a health functional food or feed composition, all including nano-sized graphene oxide, and a method of using the same. Since the nano-sized graphene oxide or a complex thereof has anti-coronavirus activity, the graphene oxide may be used for prevention or treatment of a coronavirus infection, or disinfection of coronaviruses.

Claims

1-12. (canceled)

13. A method for preventing or treating a coronavirus infection or infectious disease, comprising administering a pharmaceutical composition comprising nano-sized graphene oxide or a complex thereof to a subject.

14. The method for preventing or treating a coronavirus infection or infectious disease of claim 13, wherein the graphene oxide has an average size of about 1 nm to about 50 nm, and a thickness of about 0.3 nm to about 10 nm.

15. The method for preventing or treating a coronavirus infection or infectious disease of claim 13, wherein the graphene oxide is present as a homogenized dispersion.

16. The method for preventing or treating a coronavirus infection or infectious disease of claim 13, wherein the coronavirus is selected from the group consisting of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses.

17. The method for preventing or treating a coronavirus infection or infectious disease of claim 13, wherein the coronavirus is porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCoV or BCV), avian infectious bronchitis virus (IBV), transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), or a combination thereof.

18. The method for preventing or treating a coronavirus infection or infectious disease of claim 13, wherein the coronavirus infectious disease is coronavirus cold, coronavirus enteritis, coronavirus diarrhea, coronavirus pneumonia, severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), acute respiratory distress syndrome (ARDS), avian infectious bronchitis, or a combination thereof.

19. A method for disinfecting coronavirus, comprising spraying, applying, scattering, sprinkling, washing, or a combination thereof with a composition for anti-coronavirus disinfection comprising nano-sized graphene oxide or a complex thereof in the air, on a surface of an object, or on a mammal.

20. The method for disinfecting coronavirus of claim 19, wherein the graphene oxide has an average size of about 1 nm to about 50 nm, and a thickness of about 0.3 nm to about 10 nm.

21. The method for disinfecting coronavirus of claim 19, wherein the graphene oxide is present as a homogenized dispersion.

22. The method for disinfecting coronavirus of claim 19, wherein the coronavirus is selected from the group consisting of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses.

23. The method for disinfecting coronavirus of claim 19, wherein the coronavirus is porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCoV or BCV), avian infectious bronchitis virus (IBV), transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), or a combination thereof.

24. The method for disinfecting coronavirus of claim 19, wherein the coronavirus infectious disease is coronavirus cold, coronavirus enteritis, coronavirus diarrhea, coronavirus pneumonia, severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), acute respiratory distress syndrome (ARDS), avian infectious bronchitis, or a combination thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0077] FIG. 1 is a diagram schematically showing a method of synthesizing nanoGO according to the present disclosure.

[0078] FIG. 2 is a diagram showing particle size distribution calculated from an SEM image of nanoGO.

[0079] FIG. 3 is a diagram showing an SEM image of nanoGO.

[0080] FIG. 4 is a diagram showing a TEM image of nanoGO.

[0081] FIG. 5 is a diagram showing antiviral activity against PEDV and BCoV of nanoGO prepared by using various dilution factors. Marked letters indicate statistically significant differences between different dilution factors within each group (p<0.05), and asterisks indicate significant differences between the groups (*: p<0.05; **: p<0.01).

[0082] FIG. 6 is a diagram showing replication efficiency of PEDV and BCoV after nanoGO incubation through immunofluorescence assay (IFA). Cells with a fluorescent signal (marked with arrows) indicate that the cells are infected with a virus, and the greater the number of fluorescent cells, the greater the amount of viral replication.

[0083] FIG. 7 is a diagram showing antiviral activity of nanoGO at various concentrations against SARS-CoV-2, based on test results of cytopathic effects (CPE).

MODE OF DISCLOSURE

[0084] Hereinafter, the disclosure will be described in more detail through embodiments. However, these embodiments are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these embodiments.

EXAMPLE 1. PREPARATION OF NANO GRAPHENE OXIDE (NANOGO)

[0085] An uncontaminated graphene oxide (GO) was synthesized by using the modified Taylor's method (FIG. 1).

[0086] Specifically, high-purity graphite, sodium nitrate (NaNO.sub.3) and sulfuric acid (H.sub.2SO.sub.4) were mixed, and potassium permanganate (KMnO.sub.4) was mixed with the mixture on an ice water bath, and the mixture was rotated at a constant rotational speed (swelling effect of graphite occurs due to the rotation) for a certain period of time. After that, in order to apply a shearing force to the expanded graphite, a constant rotational force was applied for a certain time to facilitate separation between the layers. Then, hydrogen peroxide (H.sub.2O.sub.2) was added to the mixture and centrifuged to produce a graphite oxide. Distilled water solution (3 mg/ml) was added to the graphite oxide, and interlayer separation was performed by tip-sonicating for at least 1 hour to prepare a graphene oxide. The prepared graphene oxide was freeze-dried to prepare a dried graphene oxide.

[0087] The prepared graphene oxide was vacuum-filtered with a cellulose nitrate membrane filter (0.45 μm, GE Healthcare) to prepare a nano graphene oxide (nanoGO). In this example, a nanoGO solution (3 mg/mL) at a concentration of 1% was prepared as a standard, and a concentration (%) thereof was adjusted as necessary.

EXAMPLE 2. CHARACTERIZATION OF NANO GRAPHENE OXIDE (NANOGO)

[0088] In order to identify a size and shape of the nanoGO prepared in Example 1, scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging analyses were performed.

[0089] As a result of the analyses, it was confirmed that the size of the nanoGO prepared in Example 1 exhibited a distribution of about 15 nm to about 45 nm (FIG. 2), and the shape was analyzed through the SEM imaging and TEM imaging (FIGS. 3 and 4).

EXAMPLE 3. IDENTIFICATION OF CYTOTOXICITY OF NANO GRAPHENE OXIDE (NANOGO)

[0090] In order to identify cytotoxicity of the nanoGO prepared in Example 1, the following experiment was performed.

[0091] Specifically, nanoGO solutions prepared by using various dilution factors were added to Vero E6 cells (ATCC-1586), and cytotoxicity was measured by a CCK8 assay.

[0092] As a result, as shown in Table 1 below, it was confirmed that cytotoxicity did not appear at 1% nanoGO solution and all dilution factors thereof.

TABLE-US-00001 TABLE 1 Dilution factor 1x 10x 20x 40x 80x 160x 320x 640x Nano- Δ Δ Δ — — — — — oxide graphene Δ Δ Δ — — — — — (Δ: Slightly cytotoxic. The experiments were conducted in duplicate)

EXAMPLE 4. CONFIRMATION OF ANTIVIRAL EFFECT OF NANO GRAPHENE OXIDE (NANOGO) AGAINST PEDV AND BCOV

[0093] In order to confirm antiviral activity of the nano graphene oxide prepared in Example 1 against coronavirus, experiments were conducted on porcine epidemic diarrhea virus (PEDV) and bovine coronavirus (BCoV), which are well-known coronaviruses that cause a disease in animals.

[0094] First, the 1% nanoGO solution prepared in Example 1 was diluted by 50 to 800 times in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% FBS. Each dilution was mixed with an equal volume of PEDV (DR13 strain) or BCoV (BC94 strain) having a titer of 10.sup.7 TCID.sub.50/ML and incubated at room temperature for 60 minutes. PEDV and BCoV treated with nanoGO were titrated by using Vero cells. The maximum dilution factor at which the virus titer in the nanoGO-treated group decreased to 4.0 log 10 TCID.sub.50/ml or more (death and inactivation) compared to virus infectivity in the control group was determined as an effective dilution factor (according to Korean Animal and Plant Quarantine Agency Notice No. 2018-16 (2018. 5.31) Efficacy Test Guidelines). In addition, the antiviral effect of nanoGO was expressed as an inhibition rate (%) and was calculated as follows: [log 10 (TCID.sub.50/ml of virus)−log 10 (TCID.sub.50/ml of nanoGO-treated group)]/(log 10 (TCID.sub.50/ml of virus)×100%.

[0095] As a result, as the dilution factor of nanoGO increased (1/50 to 1/800), a titer of PEDV/BCoV was observed to be increased from 0.0 to 6.3/6.4 log 10 TCID.sub.50, and to gradually approach the titer of the control group (mock) not treated with nanoGO (6.6 log 10 TCID.sub.50), and it was confirmed that 100-fold diluted nanoGO blocked virus replication more efficiently. Specifically, the virus titer of the nanoGO-treated groups (1.8 for PEDV, 2.5 for BCoV) was reduced by at least 4 log 10 compared to the control group (Table 2). Therefore, it may be seen that nanoGO exhibits antiviral activity against PEDV/BCoV in a dose-dependent manner.

TABLE-US-00002 TABLE 2 NanoGo PEDV BCoV Condition of dilution Average titer Average titer treatment factor (log10 TCID.sub.50) (log10 TCID.sub.50) Virus + 1/50  0.0 0.0 nanoGO 1/100 1.8 2.5 1/150 2.8 3.9 1/200 3.9 4.8 1/250 4.4 5.2 1/300 5.1 5.5 1/400 5.6 5.8 1/500 6.0 6.3 1/600 6.2 6.3 1/800 6.3 6.4 Virus only NA* 6.6 6.6 [*NA: Not applicable: the underlined indicate the maximum dilution factor]

[0096] Next, as a result of calculating inhibition rates based on the results of the antiviral inhibitory effect, it was confirmed that the highest antiviral activity of nanoGO against PEDV and BCoV was 72.1% and 61.9%, respectively, when diluted at a rate of 1/100-fold, and when the concentration of the nanoGO solution was diluted to 1/800, antiviral activity against PEDV and BCoV was hardly observed (p>0.05). In addition, nanoGO showed a more effective antiviral activity against PEDV than BCoV up to 1/300-fold dilution (p<0.01) (FIG. 5).

[0097] Next, to analyze/detect replication of live viruses of PEDV and BCoV after a nanoGO treatment, immunofluorescence assays (IFA) were performed. Specifically, nanoGO diluted by a specific factor (1/50, 1/100, and 1/800) was treated to PEDV or BCoV, and the viruses were inoculated into Vero cells, respectively. An untreated control group without PEDV or BCoV was used as a negative control (mock), and a group inoculated with only PEDV or BCoV without nanoGO treatment was used as a positive control (virus). 24 hours after the virus inoculation, an immunofluorescence analysis was performed by using a PEDV IFA kit (Median Diagnostics) and BCoV primary antibodies (Median Diagnostics), and a statistical analysis was performed by using GraphPad Prism version 8.0.2.

[0098] As a result, PEDV and BCoV-infected cells (green fluorescence) were not observed at low dilutions (1/50) of nanoGO (A and F of FIG. 6), and viruses could not be completely inactivated from dilution factors of 100 or more, however, it was confirmed that the viruses were reduced compared to the positive control (B to C of FIG. 6, and G to H of FIG. 6).

EXAMPLE 5. CONFIRMATION OF ANTIVIRAL EFFECT OF NANO GRAPHENE OXIDE (NANOGO) AGAINST SARS-COV-2

[0099] In order to confirm an antiviral activity of the nano graphene oxide prepared in Example 1 against SARS-CoV-2, the corona virus that causes COVID-19, the following experiment was performed.

[0100] Specifically, a neutralization test was performed to evaluate the antiviral activity of nanoGO against SARS-CoV-2. First, the 1% nanoGO solution prepared in Example 1 was serially two-fold diluted with DMEM supplemented with 5% FBS (2-fold to 4096-fold dilution). Then, SARS-CoV-2 (BetaCoV/Korea/KCDC03/2020) of 25 TCID.sub.50/ml was mixed with the diluted nanoGO in the same volume, and the mixture was incubated at 37° C. for 60 minutes. After that, Vero E6 cell monolayers were infected with 0.1 ml of a nanoGO mixture, respectively, and the presence or absence of cytopathic effect (CPE) was monitored daily for 5 days. A neutralizing titer was expressed as a reciprocal of the highest dilution factor that induces inhibition of CPE. All experiments related to SARS-CoV-2 were performed at the BL3 facility located at Chonbuk National University's Research Center for Infectious Diseases Common for Humans and Animals.

[0101] As a result, it was confirmed that nanoGO completely inhibited SARS-CoV-2 replication at a dilution rate of 1/2 to 1/8 (CPE was not found) (FIG. 7). Therefore, it may be seen that a nano graphene oxide has an anti-COVID-19 coronavirus effect.

[0102] The above description of the present disclosure is for illustrative purposes, and those skilled in the art to which the present disclosure belongs will be able to understand that the examples and embodiments can be easily modified without changing the technical idea or essential features of the disclosure. Therefore, it should be understood that the above examples are not limitative, but illustrative in all aspects.