REDUCTION OF VIRAL INFECTIONS

Abstract

The present invention relates to a buffer composition having a pH of from 6.7 to 7.9 at a temperature of 37° C., for use in the treatment, prophylactic treatment or amelioration of an airborne viral infection, or for use in reducing viral replication in a subject infected with an airborne virus or exposed to an airborne virus capable of causing an airborne viral infection in the subject. The buffer composition may comprise an N- substituted aminosulphonic acid. The present invention also relates to a method of preparing the buffer composition, and to a concentrate of the buffer composition. The buffer composition can be used in a method of treatment, prophylactic treatment or amelioration of an airborne viral infection in a subject, and a method of reducing viral replication in a subject infected with an airborne virus or exposed to an airborne virus capable of causing an airborne viral infection in the subject. Airborne viruses include RNA viruses, such as coronaviruses, such as MERS-CoV, SARS-CoV, and SARS-CoV-2. The buffer composition can be administered by nasal spray, inhaler or nebulizer, or in the form of a cream, gel or emulsion, and the invention therefore also relates to a nasal spray, inhaler, nebulizer, cream, gel or emulsion comprising the buffer composition. The buffer composition can also be applied to a mask or other face covering thereby reducing the risk of viral infection with an airborne virus, and the invention therefore also relates to a spray comprising the buffer composition. The buffer composition can also be used in a receptacle through which an oxygen-containing gas is bubbled prior to inhalation by a subject, and the invention therefore also relates to a receptacle containing the buffer composition.

Claims

1. A buffer composition having a pH of from 6.7 to 7.9 at a temperature of 37° C., for use in the treatment, prophylactic treatment or amelioration of an airborne viral infection, or for use in reducing or preventing viral replication in a subject infected with an airborne virus or exposed to an airborne virus capable of causing an airborne viral infection in the subject.

2. An aqueous composition comprising (i) a Good’s buffer, an aminosulphonic acid, an aminosulphinic acid, a phosphate, a phosphite, a heteroaryl, a phenolic acid, an amino acid, a peptide, a peptide equivalent, a polymeric buffer, an ionic liquid buffer, or a combination thereof; and (ii) hydrogen carbonate ions or an equivalent thereof; wherein the aqueous composition is for use in the treatment, prophylactic treatment or amelioration of an airborne viral infection, or for use in reducing or preventing viral replication in a subject infected with an airborne virus or exposed to an airborne virus capable of causing an airborne viral infection in the subject.

3. The composition of any one of the preceding claims, wherein the composition is an aqueous composition comprising: (i) from 1 to 100 mmoles/L (preferably from 1 to 12 mmoles/L) Good’s buffer, aminosulphonic acid, aminosulphinic acid, phosphate, phosphite, heteroaryl, phenolic acid, amino acid, peptide, peptide equivalent, polymeric buffer, ionic liquid buffer, or a combination thereof; (ii) calcium ions and magnesium ions at a molar concentration ratio of from 5:1 to 1:1, wherein said calcium ions are at a concentration of from 0.1 to 2.5 mmoles/L; TABLE-US-00018 (iii) from 21 to 35 mmoles/L hydrogen carbonate ions or an equivalent thereof; (iv) from 2.5 to 6.2 mmoles/L potassium ions; (v) from 96 to 126 mmoles/L chloride ions; and (vi) from 100 to 150 mmoles/L sodium ions.

4. An aqueous composition comprising (i) a Good’s buffer, an aminosulphonic acid, an aminosulphinic acid, a phosphate, a phosphite, a heteroaryl, a phenolic acid, an amino acid, a peptide, a peptide equivalent, a polymeric buffer, an ionic liquid buffer, or a combination thereof; (ii) hydrogen carbonate ions or an equivalent thereof; and (iii) zinc ions.

5. An aqueous composition comprising (i) a Good’s buffer, an aminosulphonic acid, an aminosulphinic acid, a phosphate, a phosphite, a heteroaryl, a phenolic acid, an amino acid, a peptide, a peptide equivalent, a polymeric buffer, an ionic liquid buffer, or a combination thereof; (ii) hydrogen carbonate ions or an equivalent thereof; and (iii) transferrin and/or iron ions.

6. The composition of any one of the preceding claims, wherein the composition is an aqueous composition comprising: (i) from 1 to 100 mmoles/L (preferably from 1 to 12 mmoles/L) Good’s buffer, aminosulphonic acid, aminosulphinic acid, phosphate, phosphite, heteroaryl, phenolic acid, amino acid, peptide, peptide equivalent, polymeric buffer, ionic liquid buffer, or a combination thereof; (ii) calcium ions and magnesium ions at a molar concentration ratio of from 5:1 to 1:1, wherein said calcium ions are at a concentration of from 0.1 to 2.5 mmoles/L; TABLE-US-00019 (iii) from 21 to 35 mmoles/L hydrogen carbonate ions or an equivalent thereof; (iv) from 2.5 to 6.2 mmoles/L potassium ions; (v) from 96 to 126 mmoles/L chloride ions; (vi) from 100 to 150 mmoles/L sodium ions; and (vii) from 0.1 to 200 .Math.moles/L zinc ions.

7. The composition of any one of the preceding claims, wherein the composition is an aqueous composition comprising: (i) from 1 to 100 mmoles/L (preferably from 1 to 12 mmoles/L) Good’s buffer, aminosulphonic acid, aminosulphinic acid, phosphate, phosphite, heteroaryl, phenolic acid, amino acid, peptide, peptide equivalent, polymeric buffer, ionic liquid buffer, or a combination thereof; (ii) calcium ions and magnesium ions at a molar concentration ratio of from 5:1 to 1:1, wherein said calcium ions are at a concentration of from 0.1 to 2.5 mmoles/L; TABLE-US-00020 (iii) from 21 to 35 mmoles/L hydrogen carbonate ions or an equivalent thereof; (iv) from 2.5 to 6.2 mmoles/L potassium ions; (v) from 96 to 126 mmoles/L chloride ions; (vi) from 100 to 150 mmoles/L sodium ions; (vii) from 1 to 100 .Math.moles/L transferrin; and (viii) from 1 to 100 .Math.moles/L iron ions.

8. The composition of any one of the preceding claims, further comprising one or more of: TABLE-US-00021 (a) from 2 to 11 mmoles/L glucose; (b) from 50 to 150 .Math.moles/L glycerol; (c) from 7 to 15 .Math.moles/L choline ions; (d) from 5 to 400 .Math.moles/L glutamate; (e) from 5 to 200 .Math.moles/L aspartate; (f) from 100 to 2000 .Math.moles/L glutamine; (g) from 20 to 215 .Math.moles/L pyroglutamate; (h) from 20 to 200 .Math.moles/L arginine; (i) from 1 to 250 nmoles/L thiamine pyrophosphate ions; (j) from 40 to 100 .Math.moles/L carnitine; (k) from 5 to 600 mIU/L porcine or human insulin; (l) from 20 to 200 .Math.moles/L hyaluronic acid; (m) from 1 to 100 .Math.moles/L transferrin; (n) from 20 to 250 .Math.moles/L leucine; (o) from 10 to 100 .Math.moles/L linoleic acid; (p) from 200 to 1000 .Math.moles/L cholesterol; (q) from 20 to 500 .Math.moles/L pyridoxal-5-phosphate; or (r) from 10 to 250 .Math.moles/L chitosan.

9. The composition of any one of the preceding claims, further comprising an antibiotic.

10. The composition of any one of claims 2 to 9, having a pH of from 6.7 to 7.9 at a temperature of 37° C.

11. The composition of any one of the preceding claims, wherein the composition is suitable for inhalation into the oral cavity, upper respiratory tract, lower respiratory tract, nasal cavity, pharynx, larynx, trachea, bronchi or lungs.

12. The composition of any one of the preceding claims, wherein the composition is suitable for administration by inhaler, nebulizer, nasal spray, mouth spray, bronchial spray, nasal drops, nasal wash, nasal lavage, nasal packing, mouthwash or gargle, or in the form of a cream, gel, emulsion or ointment.

13. The composition of any one of the preceding claims, wherein the composition is suitable for application to a mask or other face covering, or wherein the composition is suitable for use in a receptacle configured to allow an oxygen-containing gas to be bubbled through the composition prior to inhalation of the gas by a subject, or wherein the composition is suitable for diffusing or spraying into the air prior to inhalation of the air by a subject.

14. The composition of any one of the preceding claims, for use in the treatment, prophylactic treatment or amelioration of an airborne viral infection, or for use in reducing or preventing viral replication in a subject infected with an airborne virus or exposed to an airborne virus capable of causing an airborne viral infection in the subject, wherein the airborne viral infection is caused by (i) an RNA virus, such as a coronavirus (including a coronavirus selected from MERS-CoV, SARS-CoV, and SARS-CoV-2), an influenza virus (including an influenza virus selected from influenza A virus, influenza B virus, influenza C virus, and parainfluenza virus), a rhinovirus, Measles virus, Mumps virus, Rubella virus, or human respiratory syncytial virus, or (ii) a DNA virus, such as Parvovirus B19, an adenovirus, an adeno-associated virus, a herpes virus (including a herpes virus selected from Varicella-Zoster virus (VZV or HHV-3), Epstein-Barr virus (EBV or HHV-4), human herpes virus 6 (HHV-6A and HHV-6B), and human herpes virus 7 (HHV-7)), a polyomavirus (including a polyomavirus selected from BK polyomavirus and WU polyomavirus), or Variola virus.

15. A method of preparing the composition of any one of claims 1 to 14, comprising combining all components with water, optionally making up to the desired volume, optionally filtering and optionally storing in a sealed vessel.

16. A concentrate for the preparation of the composition of any one of claims 1 to 14, comprising water and all components, wherein the concentrate is dilutable with water to form the composition of any one of claims 1 to 14.

17. A concentrate for the preparation of the composition of any one of claims 1 to 14, comprising water and all components except the hydrogen carbonate ions or the equivalent thereof and their countercations, wherein the concentrate is dilutable with water comprising the hydrogen carbonate ions or the equivalent thereof and their countercations to form the composition of any one of claims 1 to 14.

18. A method of treatment, prophylactic treatment or amelioration of an airborne viral infection in a subject, the method comprising administering an effective amount of the composition of any one of claims 1 to 14 to the subject.

19. A method of reducing or preventing viral replication in a subject infected with an airborne virus or exposed to an airborne virus capable of causing an airborne viral infection in the subject, the method comprising administering an effective amount of the composition of any one of claims 1 to 14 to the subject.

20. The method of claim 18 or 19, wherein the airborne viral infection is caused by (i) an RNA virus, such as a coronavirus (including a coronavirus selected from MERS-CoV, SARS-CoV, and SARS-CoV-2), an influenza virus (including an influenza virus selected from influenza A virus, influenza B virus, influenza C virus, and parainfluenza virus), a rhinovirus, Measles virus, Mumps virus, Rubella virus, or human respiratory syncytial virus, or (ii) a DNA virus, such as Parvovirus B19, an adenovirus, an adeno-associated virus, a herpes virus (including a herpes virus selected from Varicella-Zoster virus (VZV or HHV-3), Epstein-Barr virus (EBV or HHV-4), human herpes virus 6 (HHV-6A and HHV-6B), and human herpes virus 7 (HHV-7)), a polyomavirus (including a polyomavirus selected from BK polyomavirus and WU polyomavirus), or Variola virus.

21. A method of reducing the risk of viral infection with an airborne virus in a subject, the method comprising applying the composition of any one of claims 1 to 14 to a mask or other face covering.

22. A method of reducing the risk of viral infection with an airborne virus in a subject, the method comprising bubbling an oxygen-containing gas through a composition of any one of claims 1 to 14 prior to inhalation of the gas by the subject.

23. A method of reducing the risk of viral infection with an airborne virus in a subject, the method comprising diffusing or spraying the composition of any one of claims 1 to 14 into the air prior to inhalation of the air by the subject.

24. A spray, inhaler, nebulizer, nasal spray, mouth spray, bronchial spray, nasal drops, nasal wash, nasal lavage, nasal packing, mouthwash or gargle, receptacle, cream, gel, emulsion or ointment, comprising an aqueous composition comprising (i) a Good’s buffer, an aminosulphonic acid, an aminosulphinic acid, a phosphate, a phosphite, a heteroaryl, a phenolic acid, an amino acid, a peptide, a peptide equivalent, a polymeric buffer, an ionic liquid buffer, or a combination thereof; and (ii) hydrogen carbonate ions or an equivalent thereof.

25. A spray, inhaler, nebulizer, nasal spray, mouth spray, bronchial spray, nasal drops, nasal wash, nasal lavage, nasal packing, mouthwash or gargle, receptacle, cream, gel, emulsion or ointment, comprising the composition of any one of claims 1 to 14.

26. A spray, inhaler, nebulizer, nasal spray, mouth spray, bronchial spray, nasal drops, nasal wash, nasal lavage, nasal packing, mouthwash or gargle, receptacle, cream, gel, emulsion or ointment, comprising (i) the concentrate of claim 16, and (ii) water.

27. A spray, inhaler, nebulizer, nasal spray, mouth spray, bronchial spray, nasal drops, nasal wash, nasal lavage, nasal packing, mouthwash or gargle, receptacle, cream, gel, emulsion or ointment, comprising (i) the concentrate of claim 17, and (ii) water comprising hydrogen carbonate ions or an equivalent thereof and their countercations.

28. A mask or other face covering coated or impregnated with an aqueous composition comprising (i) a Good’s buffer, an aminosulphonic acid, an aminosulphinic acid, a phosphate, a phosphite, a heteroaryl, a phenolic acid, an amino acid, a peptide, a peptide equivalent, a polymeric buffer, an ionic liquid buffer, or a combination thereof; and (ii) hydrogen carbonate ions or an equivalent thereof.

29. A mask or other face covering coated or impregnated with the composition of any one of claims 1 to 14.

Description

FIGURES OF THE INVENTION

[0525] FIG. 1 lists buffers of the invention. All of the buffers listed in FIG. 1 are considered to be Good’s buffers. Good’s buffers may be classified as N-substituted aminosulphonic acid Good’s buffers and non-sulphonic acid Good’s buffers, as indicated in FIG. 1.

[0526] FIG. 2 shows a SARS-CoV-2 growth curve analysis in infected Vero cells treated with test buffer or control medium. 1×10.sup.5 Vero cells per well were pre-treated with test buffer or control medium 1 hour prior to infection with SARS-CoV-2 at moi 0.1. Adsorption was carried out at 4° C. The infected monolayer was then washed twice with cold DMEM + 0.2% BSA, followed by 1-hour incubation at 37° C. either in the presence of test buffer or control medium. After 1 hour, the monolayer was washed again with DMEM-2 (FBS 2%) to remove unbound virus. Cells were then incubated for the indicated times when virus yield was collected and titrated for infectivity. Graph and statistical analysis were performed with Graphpad Prism 6 software. Data was plotted as mean ± SD from 3 replicates; *p < 0.05; ***p < 0.001; ****p < 0.0001.

[0527] FIG. 3 shows the % neutralisation of SARS-CoV-2 spike pseudotype reporter particles by test buffers at various dilutions compared to control. SARS-CoV-2 spike pseudotype reporter particles were incubated with test buffers at various dilutions or control at 37° C. for 1 hour. Then 4 ×10.sup.4 HuH7 cells (human hepatocytes) were added and incubated at 37° C. for 72 hours, after which infection rates were determined by measuring luminescence/luciferase activity.

EXAMPLES

Example 1

[0528] An aqueous buffer solution was prepared by stirring the components set out in Table 1 in 1 L of sterile water.

TABLE-US-00001 Weight Amount Molar Amount Component 1.066 g 5 mmoles N,N-bis(2-hydroxyethyl)-2-aminoethanesulphonic acid (BES) 138.725 mg 1.25 mmoles calcium chloride 42.845 mg 0.45 mmoles magnesium chloride 2.100 g 25 mmoles sodium hydrogen carbonate 372.757 mg 5 mmoles potassium chloride 6.428 g 110 mmoles sodium chloride

Example 2 (Hypothetic)

[0529] An aqueous buffer solution was prepared by stirring the components set out in Table 2 in 1 L of sterile water.

TABLE-US-00002 Weight Amount Molar Amount Component 1.066 g 5 mmoles N,N-bis(2-hydroxyethyl)-2-aminoethanesulphonic acid (BES) 138.725 mg 1.25 mmoles calcium chloride 42.845 mg 0.45 mmoles magnesium chloride 2.100 g 25 mmoles sodium hydrogen carbonate 372.757 mg 5 mmoles potassium chloride 6.428 g 110 mmoles sodium chloride 6.814 mg 50 .Math.moles zinc chloride

Example 3 (Hypothetic)

[0530] The capacity of the subject buffer solutions to prevent or reduce viral infection of human bronchial epithelial cells may be tested using a method such as that described by Harcourt et al (J Vis Exp, 2013, vol 72, e50157). In the disclosed method, a polarized layer of human airway epithelial Calu-3 cells may be prepared in liquid-covered cultures (LCC) in Transwells.

[0531] Polarisation of the LCC may be evaluated by determining the trans-epithelial electrical resistance (TEER) and/or equilibration of sodium fluorescein (both methods as described in Harcourt et al). Viral-induced depolarisation of the cell layer may be evaluated as described in Harcourt et al.

[0532] LCC are washed in serum-free medium, such as serum-free EMEM. The subject buffer solution is added to the basolateral compartments of all test Transwells. For “uninfected” control groups, the subject buffer solution is also added to the apical compartment of the appropriate Transwells. For “mock-infected” control groups, inactivated virus diluted in the subject buffer solution is added to the apical compartment of the appropriate Transwells, and for “virus-infected” test groups, virus diluted in the subject buffer solution is added to the apical compartments of appropriate Transwells.

[0533] The effect of the subject buffer solutions on virus-induced depolarisation of the cell layer may then be determined by measuring the TEER and/or equilibration of sodium fluorescein as described in Harcourt et al.

Example 4

[0534] TABLE-US-00003 Aqueous buffer solutions A, B, C, and D were prepared by stirring the components set out in Table 3 in 1 L of ultrapure water (ATSM Type I, 18.2 MΩ/cm at 25° C.) Component Molecular Buffer A Buffer B Buffer C Buffer D Molar Amount Molar Amount Molar Amount Molar Amount N,N-bis(2-hydoxyethyl)-2-amino-ehtanesulphonic acid (BES) 213.25 5.00 mmoles 5.00 mmoles 5.00 mmoles 5.00 mmoles CaCl.sub.2 .Math. 2 H.sub.2O 147.0146 1.25 mmoles 1.25 mmoles 1.25 mmoles 1.25 mmoles MgCl.sub.2 .Math. 6 H.sub.2O 203.30 0.45 mmoles 0.45 mmoles 0.45 mmoles 0.45 mmoles NaHCO.sub.3 84.01 25.00 mmoles 25.00 mmoles 25.00 mmoles 25.00 mmoles KCl 74.55 5.00 mmoles 5.00 mmoles 5.00 mmoles 5.00 mmoles NaCl 58.50 110.00 mmoles 110.00 mmoles 110.00 mmoles 110.00 mmoles ZnCl.sub.2 136.30 - 0.1 mmoles - 0.1 mmoles D-glucose .Math. H.sub.2O 198.17 10.00 mmoles 10.00 mmoles 10.00 mmoles 10.00 mmoles Glycerol 92.0938 0.11 mmoles 0.11 mmoles 0.11 mmoles 0.11 mmoles L-Glutamic acid 147.1293 0.30 mmoles 0.30 mmoles 0.30 mmoles 0.30 mmoles L-Glutamine 146.146 0.40 mmoles 0.40 mmoles 0.40 mmoles 0.40 mmoles L-Aspartic acid 133.11 0.02 mmoles 0.02 mmoles 0.02 mmoles 0.02 mmoles L-Carnitine ‘inner salt’ 161.20 0.05 mmoles 0.05 mmoles 0.05 mmoles 0.05 mmoles Choline Chloride 139.62 0.01 mmoles 0.01 mmoles 0.01 mmoles 0.01 mmoles Thiamine Pyrophosphate Chloride 460.77 - - 40.00 nmoles 40.00 nmoles Human recombinant insulin - - 28 mIU 28 mIU Measured Osmolarity [mOsmoles/L] 270 272 286 288 Conductivity (mS/cm) 12.33 12.37 12.21 12.25 pH at 37° C. 7.29 7.15 7.30 7.22

Example 5- Antiviral Trial Test With Buffers to Assess Coronavirus Entry Interference

[0535] Host: Vero-CCL81

[0536] Virus: SARS-CoV-2 (stock titer 1.67×10.sup.6 PFU/mL in Vero) (PFU = plaque forming unit)

[0537] I. Objective: Infection of Vero cells with SARS-CoV-2, either in the presence or absence of test buffers designed to prevent (or decrease) endosome acidification and virus entry into cells.

[0538] II. Study Design: [0539] 1. 7e4 cells were seeded into each well of a 24-well plate the day before testing. 24 hours later, right before infection, the number of cells counted per well was 1e5. [0540] 2. Moi (multiplicity of infection) used in the experiment was 0.1 (1 PFU to every 10 cells). [0541] 3. 1 hour prior to infection, cells were pre-treated with test buffer or control medium by replacing overlay media (DMEM-10) with 0.5 mL of test buffer or control medium. [0542] 4. The adsorption was carried out at 4° C., with virus inoculum prepared in test buffer or control medium. Virus inoculum was prepared to have the calculated virus titer in 100 .Math.L/well. [0543] 5. After adsorption, unbound virus was removed by washing the monolayer twice with cold binding buffer (DMEM + 0.2% BSA). [0544] 6. Following adsorption, infected cells were incubated for 1 hour at 37° C. to synchronize infection and to promote entry, either in the presence of test buffer or control medium. [0545] 7. After 1 hour, the monolayer was washed 3 times with DMEM-2 (FBS 2%), an extra step to remove unbound virus. Then the monolayer was covered with 0.5 mL of DMEM-.sub.2 for the times indicated below when virus in the overlay was collected and titrated for infectivity through plaque assay in Vero cells. [0546] 8. Time points: 1 (after 1 hour incubation at 37° C.), 3, 6, 24 and 48 hours post infection (hpi). [0547] 9. Graph and statistical analysis were performed with Graphpad Prism 6 software. Data was plotted as mean ± SD from 3 replicates; *p < 0.05; ***p < 0.001; ****p < 0.0001.

[0548] III. Conditions: [0549] Trial A. medium = serum free low pH = 5.5 adjusted control (negative control) [0550] Forced entry [0551] Trial B. medium = serum free low pH + 100 mM NH.sub.4Cl (positive control) [0552] Blocked entry [0553] Note: Vero cells got rounded and detached from the well after treatment with low pH media. Since the protocol involves many washing steps, too many cells were lost. The remaining cells recovered after replacement of low pH media with DMEM-2, but their total numbers were too different for a statistical comparison with the other groups. The same was not observed with the test buffers, Vero cells showed no cytotoxic signs after treatment with the test buffers. [0554] Trial C. medium = buffer A of example 4 [0555] Trial D. medium = buffer B of example 4 [0556] Trial E. medium = buffer C of example 4 [0557] Trial F. medium = buffer D of example 4 [0558] Trial G. medium = Uninfected control - DMEM-2 [0559] Monolayer of Vero cells was intact and showed no signs of damage or cytotoxicity after treatment with DMEM-2 [0560] Trial H. = Untreated control. Infected moi 0.1 - DMEM-2

TABLE-US-00004 24-well Plate Design (one plate per time point): 1 2 3 4 5 6 A A A A E E E B B B B F F F C C C C G G G D D D D H H H

[0561] IV. Virus: Sars-CoV-2 titrated in Vero cells on the day of experiment. [0562] Plaque counts: 19 PFUs at dilution 10.sup.-4 (10-fold dilution) [0563] Titer on day of infection: 1.9 × 10.sup.6 PFU/ml

V. Results

[0564] The results are represented graphically in FIG. 2 which shows a SARS-CoV-2 growth curve analysis in infected Vero cells treated with test buffer or control medium. As can be seen, all four test buffers of example 4 suppressed viral replication for at least 24 hours to a statistically significant extent compared to untreated control in DMEM-2.

Example 6 - Insight Into Mode of Action

[0565] Host: HuH7 cells (human hepatocytes)

[0566] Virus: SARS-CoV-2 spike pseudotype reporter particle

[0567] I. Objectives: Test for effect of buffers to prevent or decrease viral entry / first cycle of replication. Investigate the effect of reducing ZnCl.sub.2 concentration in buffers (100 .Math.M to 0.39 .Math.M)

II. Study Design

[0568] Entry of coronavirus into host cells is mediated by the spike S protein. SARS-CoV-2 spike pseudotype reporter particles were used as a model that simulates infection by SARS-CoV-2 by replacing the envelope proteins in the vector virus with the spike S protein. The vector virus contains a reporter luminescent gene. By detecting the luminescence in target cells, it is possible to screen the ability of a test buffer to neutralize the virus i.e. the ability of a test buffer to reduce viral infectivity. Therefore this experiment investigates the early stage of infection, i.e. entry and first cycle of replication only.

[0569] Dilution of buffers and incubation with pseudovirus: [0570] 1. In a flat-bottom 96-well plate (ThermoFisher, #136101), dilutions of buffers A and B of example 4 (in duplicate) were prepared to a final dilution of 1/256 of buffer in a total volume of 100 .Math.l per well. [0571] 2. 1×10.sup.5 RLU of SARS-CoV-2 pseudotyped lentiviral particles were added to each well and incubated at 37° C. for 1 hour. [0572] 3. The first column (8 control wells) received SARS-CoV-2 pseudotyped lentiviral particles and cells only (virus control) and the second column received cells only (background control).

[0573] Preparation of HuH7 cells and challenge: [0574] 1. Media was removed from T.sub.75 containing adherent HuH7 cells and the cells were rinsed with PBS. [0575] 2. 2 ml of trypsin was added to the cells and the cells were returned to the incubator for 5 minutes. [0576] 3. It was confirmed using a microscope that cells had become detached and the cells were resuspended in 8 ml of FBS-containing DMEM, pipetting up and down to produce a suspension of single cells. [0577] 4. The concentration of cells was determined using a cell counter. [0578] 5. The cells were diluted in DMEM to a final concentration of 4 ×10.sup.5 cells/ml. [0579] 6. 100 .Math.l of cell suspension containing 4 ×10.sup.4 cells was added to each well of the 96-well plate. [0580] 7. The plate was incubated at 37° C. and 5% CO.sub.2 for 72 hours.

[0581] Assessing neutralisation of infection by buffers: [0582] 1. 150 .Math.l of media/supernatant was removed from each well and 50 .Math.l of Steady-Glo® Luciferase Assay System (Promega) was added. [0583] 2. Luminescence/luciferase activity was measured using CLARIOstar Plate Reader (BMG Labtech).

[0584] Analysis using PRISM 8: [0585] 1. The curves of relative infection rates (in %) versus the buffer dilutions (log10 values) against a negative control of pooled sera collected prior to 2016 (Sigma) and serum of a positive neutraliser were plotted using Prism 8 (GraphPad). [0586] 2. A non-linear regression (curve fit) method was used to determine the dilution fold that neutralised 50%.

III. Results

[0587] The results are represented graphically in FIG. 3 which shows the % neutralisation versus the buffer dilutions (log10 values). As can be seen, buffers A and B of example 4 effectively inhibited the infection of HuH7 cells with SARS-CoV-2 spike pseudotype reporter particles, even at 256x dilution.

Example 7- Synthetic

[0588] Three compositions according to the invention were prepared as follows. Compositions A, B and C vary in the amount of essential oils (ginger oil, eucalyptus oil, basil oil, clove oil) they comprise, namely a total of 2%, 1% and 0.4% respectively.

TABLE-US-00005 components of phosphate buffer Component Quantity KH.sub.2PO.sub.4 0.3532 g Na.sub.2HPO.sub.4 1.4542 g Water for injection Qs Total 100 ml

TABLE-US-00006 Component Quantity Composition A Quantity Composition B Quantity Composition C NaCl 0.6 g 0.6 g 0.6 g KCl 0.0075 g 0.0075 g 0.0075 g MgCl.sub.2 .Math. 6 H.sub.2O 0.009 g 0.009 g 0.009 g NaHCO.sub.3 0.21 g 0.21 g 0.21 g Xylitol 1 g 1 g 1 g EDTA 0.1 g 0.1 g 0.1 g CaCl.sub.2 .Math. 2 H.sub.2O 0.0183 g 0.0183 g 0.0183 g ZnCl.sub.2 0.0001 g 0.0001 g 0.0001 g Glycerol 0.001 g 0.001 g 0.001 g HPMC 0.5 g 0.5 g 0.5 g Ginger oil 0.5 g 0.25 g 0.1 g Eucalyptus oil 0.5 g 0.25 g 0.1 g Basil oil 0.5 g 0.25 g 0.1 g Clove oil 0.5 g 0.25 g 0.1 g Water for injection 40 ml 40 ml 40 ml PEG 400 5 ml 5 ml 5 ml Poloxamer 188 1.2 g 1.2 g 1.2 g Benzalkonium chloride 0.01 g 0.01 g 0.01 g Phosphate Buffer Qs Qs Qs Sodium Hyaluronate 0.2 g 0.2 g 0.2 g Total 100 ml 100 ml 100 ml

[0589] Method of manufacturing: [0590] Phase I: Preparation of phosphate buffer [0591] 1. KH.sub.2PO.sub.4 was dissolved in water for injection (24 ml) to obtain a clear solution. [0592] 2. Na.sub.2HPO.sub.4 was added to the solution and stirred to obtain a clear solution. [0593] 3. The volume was made up to 100 ml with water for injection. [0594] 4. The pH of the buffer was checked and found to be in the range of 7.2 to 7.5. [0595] Phase II: Preparation of aqueous phase [0596] 1. Poloxamer was dissolved in water for injection (10 ml). Then sodium hyaluronate was added and the mixture was allowed to swell to give mixture A. [0597] 2. HPMC was allowed to swell in water for injection (10 ml) to give mixture B. [0598] 3. NaCl, KCl, MgCl.sub.2 .Math. 6 H.sub.2O, NaHCO.sub.3, xylitol, EDTA, CaCl.sub.2 .Math. 2 H.sub.2O, and ZnCl.sub.2, one after the other in this order, were dissolved in water for injection (20 ml) to give mixture C. [0599] Phase III: Preparation of oil phase and mixing [0600] 1. PEG 400 and all essential oils (ginger oil, eucalyptus oil, basil oil, clove oil) were added together to give mixture D. [0601] 2. Mixtures A and B were mixed together, and then mixture C was added to form a blend. [0602] 3. Mixture D was added to this blend, and then glycerol and benzalkonium chloride were added. [0603] 4. The volume was made up to 100 ml with the previously prepared phosphate buffer. [0604] 5. This mixture was homogenized at 8000-9000 rpm for 15-20 minutes. [0605] 6. The homogenized composition was filtered through a Whatman filter (paper of size 11 .Math.m). [0606] 7. The pH of the composition was checked and found to be in the range of 7.2 to 7.7. The buffer capacity with respect to 1M HCl was found to be 0.02. [0607] 8. The composition was dispensed into spray bottles.

Example 8 - Antimicrobial / Germ Kill Assay

[0608] This study was conducted to evaluate the antimicrobial activity of the compositions of examples 7B and 7C. The assay measured the changes in a population of aerobic microorganisms within a specified sampling time (30 or 60 seconds) when Test Item (compositions of examples 7B and 7C) was present.

[0609] Test Item (composition of example 7B or 7C) was brought into contact with a known population of microorganisms for a specified period of time (30 or 60 seconds) at room temperature. Then the sample was neutralised to quench the antimicrobial activity of the Test Item, and the surviving microorganisms were enumerated. The percent reduction was calculated by comparison with the microbial population before treatment.

TABLE-US-00007 composition of example 7B Microorganisms % reduction after 30 sec % reduction after 60 sec Salmonella abony 99.998 99.999 Staphylococcus aureus 99.874 99.895 Escherichia coli 99.912 99.942 Aspergillus brasiliensis 99.954 99.980 Candida albicans 99.979 99.984 Listeria monocytogenes 99.852 99.891 Staphylococcus epidermidis 99.953 99.967

TABLE-US-00008 composition of example 7C Microorganisms % reduction after 30 sec % reduction after 60 sec Salmonella abony 99.992 99.999 Staphylococcus aureus 99.998 99.998 Escherichia coli 99.998 99.999 Aspergillus brasiliensis 93.239 94.376 Candida albicans 99.999 99.999 Listeria monocytogenes 99.999 99.999 Staphylococcus epidermidis 99.998 99.999

[0610] The study results are summarised in Tables 6 and 7. Compositions of examples 7B and 7C both showed substantial antimicrobial activity against bacteria (such as Salmonella abony, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Staphylococcus epidermidis) as well as and fungi (such as Aspergillus brasiliensis and Candida albicans).

Example 9 - Virucidal Effect Against SARS-CoV-2 and Influenza A (H1N1)

[0611] This study was conducted to evaluate the virucidal activity of the composition of example 7A against SARS-COV-2 and Influenza A (H1N1) virus in vitro. Test Item (composition of example 7A) was tested for virucidal activity by liquid-liquid contact of virus solution with Test Item. Test Item at two test concentrations was incubated with the causative agent of novel coronavirus (SARS-CoV-2 strain USA-WA1/2020) and influenza A (H1N1 pdmo9) for 5 minutes. Subsequently, virus incubated with Test Item was neutralized and added to a confluent layer of host cells. Surviving virus was quantified by standard end-point dilution assay. The Reed-Muench method was used to determine end-point titers (50% cell culture infectious dose, CCID50) of the samples, and the log reduction value (LRV) of the Test Item compared to the negative (water) control was calculated (LRV<1 indicates no virucidal activity, LRV>1 indicates virucidal activity).

Study Design

[0612] Host cells - SARS-COV-2: VeroE6, Influenza: MDCK

[0613] Pre-Incubation of Test Item with virus - 5 minutes

[0614] Incubation of cells post infection with virus - 5 days [0615] SARS-CoV-2 virus stock was prepared by growing virus in VeroE6 cells. Influenza A (H1N1) virus stock was prepared by growing virus in MDCK cells. Test medium used was MEM supplemented with 10 U/mL trypsin, 1 .Math.g/mL EDTA, and 50 .Math.g/mL gentamicin. [0616] Test Item was mixed with virus solution at two concentrations (called 90% and 50%) and incubated together at room temperature for 5 minutes. 90% - Test Item was mixed with virus solution, so that there was 90% composition of example 7A and 10% virus by volume. 50% - Test Item was mixed with virus solution, so that there was 50% composition of example 7A and 50% virus by volume. [0617] Following the contact period, solutions were neutralized by ⅒ dilution in test medium. [0618] Surviving virus was quantified by standard end-point dilution assay. [0619] Samples were serially diluted using eight 10-fold dilutions in test medium and added to host cells. [0620] Plates were incubated at 37° C. with 5% CO.sub.2. [0621] On day 5 post-infection, plates were scored for the presence or absence of viral cytopathic effect (CPE). The Reed-Muench method was used to determine end-point titers (50% cell culture infectious dose, CCID50) of the samples, and the log reduction value (LRV) of the Test Item.

Results

[0622] The study results are summarised in Tables 8 and 9.

TABLE-US-00009 Concentration (% v/v) of Test Item incubated with SARS-CoV-2 virus Final essential oil concentration LRV % Virucidal effect 90% 1.8% >1.8 log 90% 50% 1% >1.8 log 90%

TABLE-US-00010 Concentration (% v/v) of Test Item incubated with influenza A virus Final essential oil concentration LRV % Virucidal effect 90% 1.8% >3.8 log 99.9% 50% 1% 1.8 log 90%

[0623] The composition of example 7A demonstrated virucidal activity against SARS-CoV-2 and influenza A (H1N) when tested at two different concentrations for 5 minutes.

Example 10 - Protease Inhibition In Vitro

[0624] In this study, the effect of the composition of example 7B was studied on inhibition of proteases such as cathepsin L,3CL, furin and DPP.sub.4, which are relevant in COVID-19, using cell-free assays. Cathepsin L mediates the cleavage of the S1 subunit of the coronavirus surface spike glycoprotein and thus facilitates coronavirus entry into human host cells, virus and host cell endosome membrane fusion, and viral RNA release for next round of replication. 3C-like protease (3CLpro) is essential for SARS-CoV replication. DPP.sub.4 interacts with spike glycoprotein S1b domain to promote virus entry. Furin plays a role in the cleavage of SARS-CoV-2 and its entry into the host cell. Purified proteases were incubated with Test Item (composition of example 7B) / Positive Control and corresponding fluorogenic substrates were used to evaluate the inhibitory effect of Test Item / Positive Control.

Study Design

Procedure

[0625] Protease inhibition assays were performed using cell-free biochemical kits from BPS Bioscience, US, as per manufacturer’s protocol. [0626] Positive controls (PC) were provided in the kits. [0627] The composition of example 7B was diluted with assay buffer from the kit to obtain the desired final % concentrations of essential oils as indicated in tables 10-13. [0628] Percentage inhibition in fluorescence values was determined in comparison with enzyme control (i.e. without Test Item / Positive Control).

TABLE-US-00011 3CL protease Sample Concentration Percentage inhibition GC376 (.Math.M) (PC) 0.1 24.2 ** 1 65.4 ** 10 82.4 ** 100 79.1 ** Composition of example 7B (essential oil %) 0.001 -16.6 0.005 -1.9 0.01 0.0 0.1 -1.9 0.2 16.0 ** (** represent significant values, where p<0.001, as compared to control)

TABLE-US-00012 DPP.sub.4 protease Sample Concentration Percentage inhibition Sitagliptin (.Math.M) (PC) 0.001 32.4 ** 0.01 76.2 ** 0.1 87.1 ** 1 99.5 ** Composition of example 7B (essential oil %) 0.001 18.4 * 0.005 16.1 * 0.01 15.4 * 0.025 14.4 * 0.05 9.4 (* and ** represent significant values, where p<0.01 and P<0.001 respectively, as compared to control)

TABLE-US-00013 cathepsin L protease Sample Concentration Percentage inhibition E-64 (.Math.M) (PC) 0.0001 -15.5 0.001 -1.1 0.01 17.2 ** 0.1 56.3 ** Composition of example 7B (essential oil %) 0.001 -93.1 0.01 43.7 ** 0.025 66.7 ** 0.05 48.9 ** 0.1 31.0 ** (** represent significant values, where p<0.001, as compared to control)

TABLE-US-00014 furin protease Sample Concentration Percentage inhibition Chloromethylketone (.Math.M) (PC) 0.001 45.6 ** 0.01 95.3 ** 0.05 100.6 ** Composition of example 7B (essential oil %) 0.001 -13.4 0.01 -3.2 0.025 10.3 ** 0.05 26.2 ** 0.1 45.7 ** (** represent significant values, where p<0.001, as 3compared to control)

Results

[0629] The study results are summarised in Tables 10-13. The composition of example 7B resulted in 16%, 18.4%, 66.7% and 45.7% inhibition of 3CL, DPP.sub.4, cathepsin L and furin protease respectively. The results demonstrated that the composition of example 7B led to significant inhibition (p<0.01 or p<0.001) of 3CL, DPP.sub.4, cathepsin L and furin protease as compared to enzyme control.

Example 11 - Inhibition of Spike S1-ACE2 Interaction

[0630] SARS-CoV2 enters the human body via SpikeS1 protein that binds to ACE2 receptors present on cells of the nasal mucosa and lungs. Inhibition of binding of SpikeS1 protein and ACE2 receptors has been widely considered as a preventive strategy for COVID-19. In this study, the effect of the composition of example 7B was studied on binding inhibition of SpikeS1 protein with ACE2 receptors, using an ELISA-like colorimetric kit in a cell-free assay. Different concentrations of Test Item (composition of example 7B) and Positive Control along with ACE2 Inhibitor Screening reagent were added in a 96-well plate (pre-coated with Rabbit Fc-tagged SARS-Cov-2 Spike S1 RBD) and incubated. Further, Spike Inhibitor Screening reagent was added to the plate and incubated. Finally, the plate was treated with Anti-His-HRP Conjugate, followed by addition of TMB Substrate to produce absorbance, which was then measured using a spectrophotometer at 450 nm wavelength.

Study Design

Procedure

[0631] The inhibition of SpikeS1-ACE2 interaction was studied using a cell-free assay kit, SARS-CoV-2 Spike-ACE2 Interaction Inhibitor Screening Assay kit from Cayman Chemical Company, US, as per manufacturer’s protocol. [0632] Emodin was used as Positive Control (PC). [0633] The composition of example 7B was diluted with serum free medium to obtain the desired final % concentrations of essential oils as indicated in table 14. [0634] SARS-CoV2 Inhibitor Control was used as internal kit control (provided in the kit). [0635] Percentage inhibition in SpikeS1-ACE2 interaction was determined as compared to 100% initial activity.

TABLE-US-00015 Sample Concentration Percentage inhibition (wrt 100% initial activity) 100% Initial Activity 0.0 SARS-CoV2 Inhibitor 81.9 ** Emodin (PC) (.Math.g/ml) 0.01 35.5 ** 0.1 38.9 ** 1 44.3 ** 10 48.9 ** 50 62.8 ** Composition of example 7B (essential oil %) 0.001 48.5 ** 0.005 52.1 ** 0.01 42.8 ** 0.05 47.5 ** 0.1 55.2 ** 0.33 63.9 ** ** represent significant values, where p<0.001, as compared to control

Results

[0636] The composition of example 7B significantly (p<0.001) inhibited SpikeS1-ACE2 binding by 63.9% as compared to control. Based on these results, it can be concluded that the composition of example 7B inhibits binding of SpikeS.sub.1 protein and ACE2 receptors.

Example 12

[0637] This example was conducted to show that BES (as used in example 4) can be replaced by taurine in the buffer compositions of the present invention.

[0638] Aqueous buffer solutions A to F were prepared by stirring the components set out in Table 15 in 1L of ultrapure water (ATSM Type I, 18.2 MΩ/cm at 25° C.).

TABLE-US-00016 Component Molecular weight Buffer A Buffer B Buffer C Buffer D Buffer E Buffer F Molar Amount Molar Amount Molar Amount Molar Amount Molar Amount Molar Amount BES 213.25 5 mmoles 10 mmoles 20 mmoles - - - Taurine 125.14 - - - 5 mmoles 10 mmoles 20 mmoles NaHCO.sub.3 84.01 25 mmoles 25 mmoles 25 mmoles 25 mmoles 25 mmoles 25 mmoles pH at 37° C. 7.18 6.99 6.81 7.70 7.57 7.43

[0639] It was found that all six buffer solutions A to F are buffer compositions having a suitable pH for use in the treatments of the present invention.

Example 13 - Manufacturing Example

[0640] Thiamine pyrophosphate chloride was prepared as a 0.4 mg/mL stock solution in MilliQ endotoxin-free purified water and stored frozen in dark glass vials. Choline chloride was prepared as a 17.45 mg/mL stock solution in MilliQ endotoxin-free purified water and stored frozen in glass vials. Human recombinant insulin was prepared as a 0.5 mIU/mL stock solution in MilliQ endotoxin-free purified water acidified to pH 2.4 with 0.1N hydrochloric acid and stored frozen in glass vials.

[0641] In the following preparations, MilliQ endotoxin-free purified water was used throughout, both in the initial stirring, and in the final dilution.

[0642] For the preparation, a stainless steel container was filled with 8 litres of MilliQ endotoxin-free purified water and the following components were added while constantly stirring, in the following order: 642.96 grams of sodium chloride, 37.28 grams of potassium chloride, 18.38 grams of calcium chloride dihydrate, 9.14 grams of magnesium chloride hexahydrate, 1.363 grams zinc chloride, 106.61 grams of N,N-bis(2-hydroxyethyl)-2-aminoethanesulphonic acid (BES), optionally 1.84 milligrams of thiamine pyrophosphate chloride (using 4.6 mL of the stock solution), 0.9899 grams of L-carnitine, 0.1396 grams of choline chloride (using 8 mL of the stock solution), 1.013 grams of glycerol, optionally 2.8 mIU of human recombinant insulin (using 5.6 mL of the stock solution), 0.310 grams of L-aspartate sodium salt, 180.2 grams of anhydrous D-glucose, 5.07 grams of L-glutamate sodium salt and 5.84 grams of L-glutamine. The mixture was stirred until completely dissolved and then the final volume of 10 litres was produced by adding further MilliQ endotoxin-free purified water. The solution was filtered through a sterile filter (0.2 .Math.m Sartobran PH) into 100 mL sterile sealed glass bottles. This solution is a 10x concentrate of the solution intended for use. The concentrate can be stored under dark conditions at 3-8° C. for up to five years.

[0643] For use, 100 mL of the concentrate was diluted with 900 mL of double deionised or MilliQ endotoxin-free purified water to 1 litre with the addition of 2.1 grams of endotoxin-free sodium bicarbonate and stored at 8-10° C. prior to use. For storage stability, it is preferable not to add sodium bicarbonate to the concentrate before it is stored.

[0644] It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention, which is defined by the following claims only.

[0645] SEQ ID NO: 1 is the following sequence, wherein Xaa can be any naturally occurring amino acid:

TABLE-US-00017 Ser Leu Thr His Arg Lys Phe Gly Gly Ser Gly Gly Ser Pro Phe 1               5                   10                  15 Ser Gly Leu Ser Ser Ile Ala Val Arg Ser Gly Ser Tyr Leu Asp                 20                  25                  30 Xaa Ile Ile Ile Asp Gly Val His His Gly Gly Ser Gly Gly Asn                 35                  40                  45 Leu Ser Pro Thr Phe Thr Phe Gly Ser Gly Glu Tyr Ile Ser Asn                 50                  55                  60 Met Thr Ile Arg Ser Gly Asp Tyr Ile Asp Asn Ile Ser Phe Glu                 65                  70                  75 Thr Asn Met Gly Arg Arg Phe Gly Pro Tyr Gly Gly Ser Gly Gly                 80                  85                  90 Ser Ala Asn Thr Leu Ser Asn Val Lys Val Ile Gln Ile Asn Gly                 95                  100                 105 Ser Ala Gly Asp Tyr Leu Asp Ser Leu Asp Ile Tyr Tyr Glu Gln                 110                 115                 120 Tyr