COMPOSITIONS HAVING SYNERGISTIC ANTI-VIRAL ACTION AND METHODS FOR TREATING CORONAVIRUS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Ser. No. 63/296,750, entitled “COMPOSITIONS HAVING SYNERGISTIC ANTI-VIRAL ACTION”, filed January 5, 2022 and U.S. Provisional Application Ser. No. 63/296,761 entitled “METHODS FOR TREATING CORONAVIRUS”, filed January 5, 2022, the above-noted applications are incorporated herein in their entireties by references thereto.

FIELD OF THE DISCLOSED SUBJECT MATTER

[0002] The present invention relates to Luteolin compositions having synergistic antiviral activity and methods of their use in treating or ameliorating viral infections caused by coronaviruses such as SARS-CoV-2. More particularly, the present invention relates to the synergistic interaction of Luteolin and magnesium, manganese or zinc salts exhibiting enhanced antiviral action useful for treating or ameliorating “Middle East respiratory syndrome-related coronavirus” (such as MERS-CoV) and “Severe respiratory syndrome-related coronaviruses” (such as SARS-CoV-1 and SARS-CoV-2).

BACKGROUND OF THE DISCLOSED SUBJECT MATTER

[0003] Respiratory syndrome-related coronaviruses are a group of related betacoronaviruses, a genus in the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The name coronavirus is derived from the Latin corona, meaning "crown" or "halo", which refers to the characteristic appearance reminiscent of a solar corona around the virions (virus particles) when viewed under two-dimensional transmission electron microscopy, due to the surface being covered in club- shaped protein spikes.

[0004] The overall structure of betacoronavirus genome is similar to that of other Coronaviruses, with an ORFlab replicase polyprotein (rep, pplab) preceding other elements. This polyprotein is cleaved into many nonstructural proteins (including a SC- like protease - 3CLpro). [0005] SARS-CoV-1 was identified in 2002 in Hong Kong resulting in several thousand deaths, but did not result in pandemic. But in December 2019, a highly aggressive and lethal form of infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, the capital of China's Hubei province, which has successfully spread globally, resulting in the coronavirus pandemic ongoing since 2019. This pandemic is characterized by the emergence of multiple infectious variants (Alpha, Beta, Gamma, Delta and Omicron), faster spread, and greater infectious potential than its previous counterpart SARS-CoV-1 that emerged in 2003. The disease has been given the name Coronavirus disease 2019 or COVID-19. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include muscle pain, sputum production, diarrhea, sore throat, loss of smell, and abdominal pain. While the majority of cases result in mild symptoms, some progress to bilateral pneumonia and multi-organ failure. As of December 13, 2021, COVID-19 has resulted in 270,430,963 infections, 5,322,092 deaths, and 243,147,220 recoveries globally.

[0006] SARS-CoV-2 is a positive-sense single-stranded RNA virus with approximately 29,900 nucleotides. Similar to the genome of other betacoronaviruses, SARS-CoV-2’ s genome contains two flanking untranslated regions (UTRs) and a single long open reading frame encoding a polyprotein. The 5'-terminal two- thirds of the genome encodes a polyprotein, pplab, which is further cleaved into 16 nonstructural proteins that are involved in genome transcription and replication. The 3' terminus encodes structural proteins, including envelope glycoproteins spike (S), envelope (E), membrane (M) and nucleocapsid (N).

[0007] After SARS-CoV-2 infection, the open reading frame, ORFlab, of the viral genome is translated into a polyprotein (the orflab polyprotein having 7096 amino acid residues), from which sixteen non-structural proteins (Nspl-Nspl6), including the RNA-dependent RNA polymerase (RdRp, Nspl2), helicase (Nspl3), papain-like protease (Nsp3) and SC-like proteinase (3CL/?ro, main protease or M/?ro, Nsp5), among other Nsps involved in the transcription and replication of the virus, are released by extensive proteolytic processing. This is primarily achieved by the 306- amino acid 3C-like protease (3CLpro orMpro), a Cys protease, which is the main protease of the virus.

[0008] SARS-CoV-2 is the official name of the virus responsible for the COVID-19 pandemic, which has resulted in thousands of deaths worldwide and unprecedented levels of economic and social disruption. In the United States alone, well over 800,000 deaths have been attributed to COVID-19, with 50 million total infections since 2019, 40 million recoveries and 9 million active cases currently, as of December 14, 2021 (Worldometer Coronavirus, 2021). The virus was previously referred to as the “Wuhan virus”, “Wuhan coronavirus”, “China coronavirus”, “novel coronavirus” and subsequently “2019-nCoV”. The “SARS” part of the name refers to the new coronavirus’ genetic link to the virus that caused the 2003 SARS outbreak. The virus is highly contagious and has a relatively high mortality rate in comparison to its genetically-related counterpart SARS-CoV-1 that emerged in 2003. The pandemic has resulted in severe social distancing, stay at home orders and other measures to mitigate spread of the virus, which have resulted in significant disruptions of social and economic activity worldwide. Several variants (Alpha, Beta, Gamma, Delta and Omicron) have been identified since the pandemic began, with some having high transmissibility, high infectious potential and disease altering ability.

[0009] People may be infected with the virus for 1 to 14 days before developing symptoms. The most common symptoms of coronavirus disease (COVID-19) are fever, tiredness, and dry cough. Most people (about 80% or more) recover from the disease without needing special treatment. However, the disease can be serious and even fatal. Older people, and people with other medical conditions or comorbidities (such as asthma, diabetes, or heart disease), may be more vulnerable to becoming severely ill. Respiratory dysfunction, also termed as Acute Respiratory Distress Syndrome (ARDS), is caused by the virus and can result in extended treatment with ventilators, and in most cases prove fatal. The long-term effects of coronavirus disease are still under investigation.

[0010] Efforts to develop treatments for SARS-CoV-2 (and other coronaviruses) has focused on vaccination, which now requires third or subsequent boosters, including bivalent boosters to better protect against later-developed variants. Many oral antivirals have not proven effective in the current pandemic and have given variable results. There is an unmet current need for orally administered compositions with demonstrated potent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as other coronaviruses which may emerge. The present invention may provide important treatment options for COVID-19 patients, including those exposed to the virus by household contacts, those with diagnosed infections treated in the outpatient setting, and those hospitalized with moderate to severe infection. SUMMARY OF THE DISCLOSED SUBJECT MATTER

[0011] The present invention is based on the recognition of the surprising synergism of Luteolin when combined with one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts (or a combination thereof), in particular in cooperating to enhance the antiviral activity of Luteolin against coronaviruses such as SARS-CoV-2.

[0012] The compositions disclosed herein have been found to demonstrate enhanced antiviral activity against coronavirus, such as but not limited to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this regard, the compositions have been found to exhibit suppression of at least one of Nsp5 (Mpro) of SARS-CoV-2 and PL/?ro of SARS-CoV-2. In this regard, another aspect of the present invention provides a method for inhibiting replication of a virus, for example, in an infected individual. The method comprises the step of suppressing a first viral protease, a second viral protease or both the first viral protease and the second viral protease. The first and second viral proteases may be Nsp5 (M/?ro) of SARS-CoV-2 and PL/?ro of SARS- CoV-2 suppressed by administration of the compositions comprising Luteolin or a pharmaceutically acceptable salt thereof; and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts, and combinations thereof.

[0013] In one aspect, the present invention provides a composition comprising: Luteolin or a pharmaceutically acceptable salt thereof; and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts, and hydrates and combinations thereof, wherein the one or more salts enhances the antiviral activity of Luteolin against coronavirus.

[0014] Embodiments include the following, alone or in any combination.

[0015] The composition may comprise Luteolin or a pharmaceutically acceptable salt thereof; and one or more magnesium salts.

[0016] The composition may comprise Luteolin or a pharmaceutically acceptable salt thereof; and one or more zinc salts.

[0017] The composition may comprise Luteolin or a pharmaceutically acceptable salt thereof; and one or more manganese salts.

[0018] The composition may comprise Luteolin or a pharmaceutically acceptable salt thereof, one or more magnesium salts, and one or more zinc salts.

[0019] The composition may comprise Luteolin or a pharmaceutically acceptable salt thereof, one or more manganese salts, and one or more zinc salts. [0020] The one or more magnesium salts, zinc salts, and manganese salts may have anions selected from the group consisting of acetate, bromide, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycinate, glycolate, glycollylarsanilate, hexanoate, hydrabamine, hydroxynaphthoate, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl sulfate, mucate, napsylate, nitrate, octanoate, oleate, oxide, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, sulfate, tartrate, teoclate, isethionate, tosylate, and triethiiodide anions.

[0021] The one or more manganese salts may be selected from the group consisting of manganese chloride, manganese sulfate, and manganese sulfate monohydrate.

[0022] The one or more zinc salts may be selected from the group consisting of zinc chloride, zinc sulfate, and zinc oxide.

[0023] The composition may further comprise vitamin C, vitamin D, vitamin B12 or combinations thereof.

[0024] The composition may further comprise one or more additional components selected from the group consisting of a carrier, an excipient, a binder, a colorant, a flavoring agent, a preservative, a buffer, a diluent, and any combination thereof.

[0025] The composition may be in a dosage form selected from the group consisting of a capsule, a cachet, a pill, a tablet, a powder, a granule, a pellet, a bead, a particle, a troche, a lozenge, and a gel.

[0026] The composition may be a therapeutic supplement.

[0027] The composition may be a pharmaceutical composition.

[0028] The composition is useful for treating an individual infected by coronavirus; such as SARS-CoV-2 or variant thereof; or such as Severe Acute Respiratory Syndrome-Corona Virus 1 (SARS-CoV-1), Middle East Respiratory Syndrome virus (CoV-MERS), human HCoV-229E, HCoV-OC43, HCoV-NL63 or HCoV-HKUl.

[0029] Another aspect provides a composition comprising Luteolin or a pharmaceutically acceptable salt thereof; one or more magnesium salts or hydrates thereof; and one or more manganese salts or hydrates thereof; wherein the one or more magnesium salts and the one or more manganese salts enhance the antiviral activity of Luteolin.

[0030] Embodiments of the composition include the following, alone or in any combination. [0031] The one or more magnesium salts may be selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium glycinate and magnesium citrate.

[0032] The one or more manganese salts may be selected from the group consisting of manganese chloride, manganese sulfate, and manganese sulfate monohydrate.

[0033] The composition exhibits suppression of a first viral protease and suppression of a second viral protease. The first viral protease may be Nsp5 (M/?ro) of SARS-CoV-2. The second viral protease may be PL/?ro of SARS-CoV-2.

[0034] The composition may further comprise vitamin C, vitamin D, vitamin B12 or combinations thereof.

[0035] The composition may further comprise one or more additional components selected from the group consisting of a carrier, an excipient, a binder, a colorant, a flavoring agent, a preservative, a buffer, a diluent, and any combination thereof.

[0036] The composition may be in a dosage form selected from the group consisting of a capsule, a cachet, a pill, a tablet, a powder, a granule, a pellet, a bead, a particle, a troche, a lozenge, and a gel.

[0037] The composition may be a therapeutic supplement.

[0038] The composition may be a pharmaceutical composition.

[0039] The composition is useful for treating an individual infected by coronavirus; such as SARS-CoV-2 or variant thereof; or such as Severe Acute Respiratory Syndrome-Corona Virus 1 (SARS-CoV-1), Middle East Respiratory Syndrome virus (CoV-MERS), human HCoV-229E, HCoV-OC43, HCoV-NL63 or HCoV-HKUl.

[0040] The composition of any of claims 30-32, wherein the composition inhibits replication of the coronavirus in the individual.

[0041] In another aspect, a method is disclosed for increasing the antiviral potency of Luteolin. The method includes preparing a composition comprising Luteolin (or a pharmaceutically acceptable salt thereof) and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts (or a combination thereof) as described above, administering a therapeutically effective amount to a human or animal of a composition comprising Luteolin and at least one of magnesium salts, manganese salts, zinc salts (or a combination thereof). The term “therapeutically effective amount” as used herein means a nontoxic but sufficient amount of the corresponding compound to provide the desired effect. [0042] In yet another aspect, the present invention provides a method of treating or ameliorating coronavirus. The method comprises administering to an individual in need thereof a composition containing a therapeutically effective amount of Luteolin or a pharmaceutically acceptable salt thereof and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts, and a combination thereof, as described above.

[0043] The method comprises administering a therapeutically effective amount of a composition comprising Luteolin or a pharmaceutically acceptable salt thereof; and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts, and hydrates and combinations thereof, as described above.

[0044] Embodiments of the method included the following, alone or in any combination.

[0045] The method is useful for treating an individual infected by coronavirus; such as SARS-CoV-2 or variant thereof; or such as Severe Acute Respiratory Syndrome-Corona Virus 1 (SARS-CoV-1), Middle East Respiratory Syndrome virus (CoV-MERS), human HCoV-229E, HCoV-OC43, HCoV-NL63 or HCoV-HKUl.

[0046] Administration of the composition exhibits suppression of a first viral protease and suppression of a second viral protease. The first viral protease may be Nsp5 (Mpro) of SARS-CoV-2. the second viral protease may be PL/?ro of SARS-CoV-2.

[0047] The composition inhibits replication of the coronavirus in the individual.

[0048] The one or more magnesium salts, zinc salts, and manganese salts may have anions selected from the group consisting of acetate, bromide, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycinate, glycolate, glycollylarsanilate, hexanoate, hydrabamine, hydroxynaphthoate, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl sulfate, mucate, napsylate, nitrate, octanoate, oleate, oxide, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, sulfate, tartrate, teoclate, isethionate, tosylate, and triethiiodide anions.

[0049] The composition may comprise one or more magnesium salts or hydrates thereof; and one or more manganese salts or hydrates thereof.

[0050] The composition may include one or more magnesium salts selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium glycinate and magnesium citrate. [0051] The composition may include one or more manganese salts are selected from the group consisting of manganese chloride, manganese sulfate, and manganese sulfate monohydrate.

[0052] The composition may include one or more zinc salts selected from the group consisting of zinc chloride, zinc sulfate, and zinc oxide.

[0053] The composition may further comprise vitamin C, vitamin D, vitamin B12 or combinations thereof.

[0054] The composition may comprise a unit dose comprising 75-100 mg of Luteolin. The composition may further comprise 200 to 600 mg of magnesium chloride; 2 to 10 mg of manganese sulfate monohydrate and 5 to 10 mg of zinc oxide. The composition may further comprise 1000- 2000 mg of vitamin C. The composition may further comprise 1000 International Units (IU) of vitamin D3.

[0055] The composition may be in a dosage form selected from the group consisting of a capsule, a cachet, a pill, a tablet, a powder, a granule, a pellet, a bead, a particle, a troche, a lozenge, and a gel.

[0056] The composition may further comprise one or more additional components selected from the group consisting of a carrier, an excipient, a binder, a colorant, a flavoring agent, a preservative, a buffer, a diluent, and any combination thereof.

[0057] The composition may be an over-the-counter therapeutic supplement.

[0058] The composition may be a pharmaceutical composition.

[0059] The composition may be administered in the form of a tablet, the tablet comprising 5-10% of the composition; 80% of fillers, disintegrants, lubricants, glidants, and/or binders; and 10% of additives capable of providing easy disintegration, disaggregation, and dissolution of the tablet in the stomach or the intestine of the individual.

[0060] The one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts and a combination thereof, is present in an amount effective for cooperating to enhance the antiviral activity of Luteolin against coronavirus. In one example, the coronavirus is SARS-CoV-2 or variant thereof. In other embodiments, the coronavirus is Severe Acute Respiratory Syndrome-Corona Virus 1 (SARS-CoV-1), Middle East Respiratory Syndrome virus (CoV-MERS), human HCoV-229E, HCoV-OC43, HCoV-NL63 or HCoV-HKUl. In another embodiment, the individual has been diagnosed with COVID-19. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The disclosed aspects will hereinafter be described in conjunction with the appended drawings, provided to illustrate and not to limit the disclosed aspects.

[0062] Figure 1A shows chemical structures of compounds tested as possible inhibitors of the SARS-CoV-2 protease enzyme 3CL/?ro The reactions were done at 30°C in 50 mM Hepes, 150 mM NaCl, 1 mM TCEP, 1 mM EDTA in 20% DMSO. Figure IB shows a bar plot of the inhibition of 3CL/?ro or M/VY> by the compounds shown in Figure 1A.

[0063] Figures 2A-2D show aspects of the characterization of the inhibition pattern of Luteolin against 3CL/?ro or Mpro enzyme of the SARS-CoV-2. Figure 2A shows the ICso curve activities of luteolin toward 3CL/?ro SARS-CoV-2. Figure 2B shows double-reciprocal plots exhibiting competitive inhibition by luteolin was obtained upon varying the Luteolin concentration 40 pM, 95 pM, 170 pM, 225 pM, 300 pM, and 400 pM at several fixed concentrations of the peptide substrate 50 pM, 80 pM, 120 pM, 300 pM, and 400 pM . The proteolytic rates were measured in 50 mM Hepes, 150 mM NaCl, 1 mM TCEP, 1 mM EDTA in the presence of 20% DMSO at 30 °C. The points are experimental as the mean of n=3 independent experiments, while the lines are theoretical based on a fit to eq. 1. Figure 2C shows ICso calculated from antiviral cell-based assay. Figure 2D shows the titration of Luteolin at concentrations of 125uM, 250uM, 500uM and lOOuM against the whole virus in a cellular assay. These are bright field images that show that Luteolin was well tolerated by the Hek293T cells which were used for infecting with SARS-CoV-2 in a cell-culture-based antiviral assay. A high content fluorescence microscopy approach was used to assess the ability of Luteolin to inhibit SARS-CoV-2 infection in permissive cells. Hek293T cells stably expressing human ACE2 and TMPRSS2 (HEKAT24). Bright field images showing the effect of Luteolin on viral cytopathicity at 125, 250, 500 and 1000 pM concentrations, with parallel images without the virus SARS-CoV-2 and the effect of 20% (v/v) DMSO in which Luteolin was re-suspended.

[0064] Figures 3A-3I show aspects of the determination of the ICso of Luteolin in combination with various metal ions. Figures 3A-3C shows titration of chloride salts of zinc, manganese, and magnesium to determine the ICso values of the different metal ions against Mpro from SARS-CoV-2. Figure 3D-3F shows the ICso of zinc, manganese, and magnesium was determined in the presence of luteolin that was kept fixed at 90 pM. The ICso of manganese, and magnesium decreased in the presence of luteolin, but it did not change with zinc. Figure 3G-3I shows the ICso of luteolin was determined in the presence of different metal ions to further test their synergetic effect. The presence of 2.3 mM of manganese did not affect the ICso of luteolin. However, the addition of 1.3 mM zinc and 24 mM magnesium, decrease the ICso of luteolin by three- and fiftyfold, respectively. In all enzyme assays conducted here, the Mpro and peptide substrate concentrations were fixed at 3 pM and 60 pM, respectively. The enzyme rate was measured in a buffer contains 20 mM Hepes pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO at 30°C. Data are mean ± SD, n= 3. Figures 3J and 3K show bar plots summarizing these results.

[0065] Figures 4A-4I show aspects of the determination of the ICso of Luteolin in combination with various metal ions. Figures 4A-4C show titration of sulfate salts of zinc, manganese, and magnesium to determine the ICso values of the different metal ions against Mpro from SARS-CoV-2. Figures 4D-4F show the ICso of zinc, manganese, and magnesium was determined in the presence of luteolin that was kept fixed at 90 pM. The ICso of manganese, and zinc decreased in the presence of luteolin, but increased with magnesium. Figures 4G-4I show the ICso of luteolin determined in the presence of different metal ions to further test their synergetic effect. The addition of decrease the ICso of luteolin from 78 pM in the absence of metal ions to 23 pM, 49 pM, and 33 pM in the presence of 0.3 mM zinc, 1.85 mM manganese, and 3.5 mM magnesium sulfate salts with a decrease in the ICso of luteolin between two- and three-fold, respectively. In all enzyme assays conducted here, the Mpro and peptide substrate concentrations were fixed at 3 pM and 60 pM, respectively. The enzyme rate was measured in a buffer contains 20 mM Hepes pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO at 30°C. Data are mean ± SD, n= 3.

[0066] Figures 5A-5C show aspects of the thermodynamic stability of Mpro. Figure 5A shows DSC thermal scans of Mpro in the absence of luteolin (black line and presence of 25 pM (red line), 50 pM (blue light), 100 pM (green) and 250 pM (dark blue) luteolin acquired at pH 7.0 and 20% (v/v) DMSO. The sample was heated at a rate of 1 ,0°C/min. Figure 5B shows a bar plot of AH cal calculated from the area under the thermographic peak in absence and presence of luteolin. Figure 5C shows a bar plot of Tm calculated at the apex of melting peak in absence and presence of luteolin. Data presented in B and C are shown as the mean and SD, n=3.

[0067] Figures 6A and 6B show aspects of the docking of Mpro with Luteolin in the catalytic active site. Figure 6A shows the interaction between the luteolin and the amino acids from catalytic site. Figure 6B shows the competitive interaction between the substrate and luteolin by the catalytic pocket.

[0068] Figures 7A and 7B show aspects of molecular docking results for 3CL/?ro with the peptide substrate, in absence and presence of Luteolin. In particular, Figures 7A and 7B show aspects of docking representation of the 3CL/?ro substrate (blue) interacting with the 3CL/?ro dyad amino acids (red sticks) and other active site amino acids (red surface).

[0069] Figures 8A and 8B show aspects of dynamic simulation for 3CL/?ro with Luteolin in the catalytic active site. Figure 8A shows RMSD variations for the 50 ns molecular dynamics simulation for the single complex between luteolin and 3CL/?ro, as well as with luteolin and 3CLpro structure complexed with substrate. Figure 8B shows 3CL/?ro active site amino acids are indicated with black arrows, * indicates catalytic dyad amino acid.

[0070] Figure 9 shows aspects of protein cluster highlighting interactions between Nsp5 (3CL/?ro) with core proteins, their first-degree interactions, as well as the first-degree interactions for Luteolin. The largest circles correspond to central proteins (Hub -bottleneck- black hand pointing, bottleneck- red arrow), these interactions were also highlighted with the wide and gray connectors. Smaller circles correspond to second degree interactions (2 ^nd Nsp5 neighborhood) with Nsp5. Proteins with first-degree interaction with Luteolin are represented with an *. The solid red connectors highlight pathways that can connect Luteolin to Nsp5, white the red dotted connector represents the inhibitory potential of Nsp5 by Luteolin.

DETAILED DESCRIPTION OF THE DISCLOSED SUBJECT MATTER

[0071] Aspects of the present specification disclose, in part, a composition having synergistic antiviral activity against coronaviruses, such as such as SARS-CoV-2 or variant thereof, SARS-CoV-1, CoV-MERS, human HCoV-229E, HCoV-OC43, HCoV-NL63 or HCoV-HKUl. In one aspect, the composition comprises Luteolin and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts and a combination thereof. The inventors have surprisingly found that combinations of Luteolin with these metal salts have enhanced effectiveness at inhibiting proteases that cause replication of coronavirus, such as but not limited to the SARS-CoV-2 virus. Thus, the present invention provides enhanced antiviral activity compared to Luteolin alone. In particular, the disclosure demonstrates that combinations of Luteolin and magnesium salts, zinc salts, manganese salts and any combination of thereof were surprisingly effective at inhibiting 3CL/?ro or Mpro and PL/?ro proteases of SARS-CoV-2.

[0072] In one embodiment, the one or more magnesium salts, zinc salts, manganese salts may be selected from acetate, bromide, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycinate, glycolate, glycollylarsanilate, hexanoate, hydrabamine, hydroxynaphthoate, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl sulfate, mucate, napsylate, nitrate, octanoate, oleate, oxide, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, sulfate, tartrate, teoclate, isethionate, tosylate, triethiiodide, or other anions. Hydrates of such salts are also contemplated. In a preferred embodiment, magnesium salts are magnesium chloride, magnesium sulfate, magnesium glycinate or magnesium citrate. In a preferred embodiment, the manganese salts are manganese chloride, manganese sulfate, or manganese sulfate monohydrate. In a preferred embodiment, the preferred zinc salts are zinc chloride, zinc sulfate, Zinc sulfate heptahydrate or zinc oxide.

[0073] Referring to Figure 1A, chemical structures of potential inhibitors of the 3CL/?ro or Mpro protease enzymes of the SARS-CoV-2 are shown.

[0074] Figure IB shows a bar plot of the inhibition of 3CL/?ro (Mpro) by the compounds shown in Figure 1A, compared to a control that was not treated with a test compound (100%). The plot summarizes the inhibition exhibited by 25 pM of the different inhibitors against Mpro- SARS-CoV-2, and shows that Luteolin, Pl l in Figure 1A, was the most effective of the compounds tested at inhibiting 3CL/?ro (Mpro) (solid bar). The reactions were done at 30°C in 50 mM Hepes, 150 mM NaCl, 1 mM TCEP and 1 mM EDTA in 20% DMSO.

[0075] Figures 2A-2D show aspects of the characterization of the inhibition pattern of Luteolin against 3CL/?ro SARS-CoV-2. Figure 2A shows ICso curve activities of Luteolin toward 3CL/?ro SARS-CoV-2. Figure 2B shows double-reciprocal plots exhibiting competitive inhibition by Luteolin was obtained upon varying the Luteolin concentration 40 pM, 95 pM, 170 pM, 225 pM, 300 pM, and 400 pM at several fixed concentrations of the peptide substrate 50 pM, 80 pM, 120 pM, 300 pM, and 400 pM. The proteolytic rates were measured in 50 mM Hepes, 150 mM NaCl, 1 mM TCEP, and 1 mM EDTA in the presence of 20% DMSO at 30 °C. The points are experimental as the mean of n=3 independent experiments, while the lines are theoretical based on a fit to equation 1.

[0076] Figure 2C shows the IC50 calculated from the antiviral assay, indicating that Luteolin provides an IC50 of 90 + 11 pM. Luteolin was titrated into Hek293T cells previously infected with SARS-CoV-2. Figure 2D shows bright field images of the assays.

[0077] Figures 3A-3H show aspects of the determination of the IC50 of Luteolin in combination with various metal ions. Figures 3A-3C are plots showing titration of chloride salts of zinc, manganese, and magnesium to determine the IC50 values of the different metal ions against Mpro from SARS-CoV-2. Similar to panels in Figures 3A-3C, Figures 3D-3F show the IC50 of zinc chloride, manganese chloride, and magnesium chloride determined in the presence of Luteolin that was kept fixed at 90 pM. The IC50 of manganese chloride, and magnesium chloride decreased in the presence of Luteolin, but it did not change with zinc chloride.

[0078] Figures 3G-3I illustrate the determination of the IC50 of Luteolin in the presence of different metal ions to further test their synergetic effect. The presence of 2.3 mM of manganese did not affect the IC50 of Luteolin. However, the addition of 1.3 mM zinc and 24 mM magnesium, decrease the IC50 of Luteolin by 4-fold and 50-fold, respectively, with IC50 values of Luteolin decreased from 78.0 ± 3.0 pM to 21.2 ± 0.4 pM and 1.5 ± 0.1 pM in the presence of zinc and manganese, respectively. In all enzyme assays conducted here, the Mpro enzyme and peptide substrate concentrations were fixed at 3 pM and 60 pM, respectively. The enzyme rate was measured in a buffer contains 20 mM Hepes pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO at 30°C. Data are mean ± SD, n= 3. Figures 3J and 3K show bar plots summarizing these results.

[0079] Figures 4A-4I show aspects of the determination of the IC50 of Luteolin in combination with various metal ions. Figures 4A-4C show titration of sulfate salts of zinc, manganese, and magnesium to determine the IC50 values of the different metal ions against Mpro from SARS-CoV-2. Similar to panels in Figures 4A-4C, the IC50 of zinc, manganese, and magnesium was determined in the presence of Luteolin that was kept fixed at 90 pM (Figures 4D-4F). The IC50 of manganese, and zinc decreased in the presence of Luteolin, but increased with magnesium. Figures 4G-4I show the determination of the IC50 of Luteolin in the presence of different metal ions to further test their synergetic effect. In all enzyme assays conducted here, the Mpro and peptide substrate concentrations were fixed at 3 pM and 60 pM, respectively. The enzyme rate was measured in a buffer contains 20 mM Hepes pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO at 30°C. Data are mean ± SD, n= 3.

[0080] Figures 5A-5C show aspects of the thermodynamic stability of Mpro. Figure 5A shows differential scanning calorimetry (DSC) thermal scans of Mpro in the absence of Luteolin (black line) and presence of 25 pM (red line), 50 pM (blue line), 100 pM (green line) and 250 pM (dark blue line) Luteolin acquired at pH 7.0 and 20% (v/v) DMSO. The sample was heated at a rate of 1.0°C/min. Figure 5B shows a bar plot of AHcai calculated from the area under the thermographic peak in absence and presence of Luteolin. Figure 5C shows a bar plot of Tm calculated at the apex of the melting peak in absence and presence of Luteolin. Data presented in B and C are shown as the mean and SD, n=3.

[0081] Figures 6A and 6B show aspects of the docking of Mpro with Luteolin in the catalytic site. Figure 6A shows the interaction between the Luteolin and the amino acids from the catalytic site. Figure 6B shows the competitive interaction between the substrate and Luteolin by the catalytic pocket.

[0082] Figures 7A and 7B show aspects of molecular docking results for 3CL/?ro with the peptide substrate, in absence and presence of Luteolin. Figure 7A is a docking representation of the 3CL/?ro substrate (blue) interacting with the 3CL/?ro dyad amino acids (red sticks) and other active site amino acids (red surface). Figure 7A is a docking representation of 3CL/?ro substrate (blue) with Luteolin (green), interacting with the 3CL/?ro dyad amino acids (red sticks) and other active side amino acids (red surface).

[0083] Figures 8A and 8B show aspects of dynamic simulation for 3CL/?ro with Luteolin in the catalytic site. Figure 8A shows RMSD variations for the 50 ns molecular dynamics simulation for the single complexes between Luteolin and 3CL/?ro, as well as with Luteolin and 3CL/?ro structure complexed with substrate. In Figure 8B, 3CL/?ro active site amino acids are indicated with black arrows, catalytic dyad amino acids are indicated with an asterisk (*).

[0084] Figure 9 shows aspects of protein clustering between Nsp5 (3CLpro) with core proteins, highlighting interactions between Nsp5 (3CL/?ro) with core proteins, their first-degree interactions, as well as the first-degree interactions for Luteolin. The largest circles correspond to central proteins (Hub-bottleneck- black hand pointing, bottleneck- red arrow). These interactions were also highlighted with the wide and gray connectors. Smaller circles correspond to second degree interactions (second Nsp5 neighborhood) with Nsp5. Proteins with first-degree interaction with Luteolin are represented with an *. The solid red connectors highlight pathways that can connect Luteolin to Nsp5, while the red dotted connector represents the inhibitory potential of Nsp5 by Luteolin.

[0085] Optionally, the compositions or combinations may further comprise vitamin C, vitamin D and/or vitamin B12. Vitamin C is beneficial for the health of leukocytes, a type of white blood cells that help fight infections. Supplement of 1000 mg twice every day is recommended normally. Vitamin D may play a considerable role in reducing inflammation and enhancing immunity to coronaviruses such as SARS-CoV-2. Vitamin B12 (cobalamin/cyanocobalamin) may regulate chemokine/cytokine formation and mediate the communication among immune cells in pathophysiological pathways and thus protect against various bacterial and viral infections, including CO VID-19. Combination of Luteolin with Vitamin D, Vitamin C and vitamin B12 may provide better immune support, than Luteolin alone, needed during viral infections.

Exemplary Embodiments

[0086] The various embodiments described by way of example below do not limit the scope of the invention. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art.

[0087] Embodiment 1. A composition or combination comprising Luteolin or a pharmaceutically acceptable salt thereof; and one or more salts selected from the group consisting of magnesium salts, manganese salts, zinc salts, and combinations thereof.

[0088] Embodiment 2. The composition or combination of Embodiment 1 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[0089] Embodiment s. The composition or combination of Embodiment 1 comprising Luteolin and a magnesium salt.

[0090] Embodiment 4. The composition or combination of Embodiment 3 wherein the magnesium salt comprises magnesium chloride, magnesium glycinate or magnesium citrate.

[0091] Embodiment 5. The composition or combination of Embodiment 1 comprising Luteolin and magnesium chloride. [0092] Embodiment 6. The composition or combination of Embodiment 1 comprising

Luteolin and magnesium glycinate.

[0093] Embodiment 7. The composition or combination of Embodiment 1 comprising

Luteolin and magnesium citrate.

[0094] Embodiment 8. The composition or combination of Embodiment 3 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[0095] Embodiment 9. The composition or combination of Embodiment 8 comprising

Luteolin, a magnesium salt and vitamin C.

[0096] Embodiment 10. The composition or combination of Embodiment 8 comprising

Luteolin, a magnesium salt, and vitamin D.

[0097] Embodiment 11. The composition or combination of Embodiment 8 comprising

Luteolin, a magnesium salt and vitamin B12.

[0098] Embodiment 12. The composition or combination of Embodiment 8 comprising

Luteolin, a magnesium salt, vitamin C and vitamin D.

[0099] Embodiment 13. The composition or combination of Embodiment 8 comprising

Luteolin, a magnesium salt, vitamin C, vitamin D and vitamin B12.

[00100] Embodiment 14. The composition or combination of Embodiment 1 comprising

Luteolin and a zinc salt.

[00101] Embodiment 15. The composition or combination of Embodiment 1 comprising

Luteolin and zinc chloride or zinc oxide.

[00102] Embodiment 16. The composition or combination of Embodiment 1 comprising

Luteolin and zinc chloride.

[00103] Embodiment 17. The composition or combination of Embodiment 1 comprising

Luteolin and zinc oxide.

[00104] Embodiment 18. The composition or combination of Embodiment 10 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[00105] Embodiment 19. The composition or combination of Embodiment 18 comprising

Luteolin, zinc chloride or zinc oxide, and vitamin C.

[00106] Embodiment 20. The composition or combination of Embodiment 18 comprising

Luteolin, zinc chloride or zinc oxide, and vitamin D. [00107] Embodiment 21. The composition or combination of Embodiment 18 comprising Luteolin, zinc chloride or zinc oxide, and vitamin B12.

[00108] Embodiment 22. The composition or combination of Embodiment 18 comprising Luteolin, zinc chloride or zinc oxide, and vitamin C and vitamin D.

[00109] Embodiment 23. The composition or combination of Embodiment 18 comprising Luteolin, zinc chloride or zinc oxide, and vitamin C, vitamin D and vitamin B12.

[00110] Embodiment 24. The composition or combination of Embodiment 1 comprising a manganese salt.

[00111] Embodiment 25. The composition or combination of Embodiment 24 wherein the manganese salt comprises manganese sulfate.

[00112] Embodiment 26. The composition or combination of Embodiment 24 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[00113] Embodiment 27. The composition or combination of Embodiment 26 comprising

Luteolin, manganese sulfate, and vitamin C.

[00114] Embodiment 28. The composition or combination of Embodiment 26 comprising

Luteolin, manganese sulfate, and vitamin D.

[00115] Embodiment 29. The composition or combination of Embodiment 26 comprising

Luteolin, manganese sulfate, and vitamin B12.

[00116] Embodiment 30. The composition or combination of Embodiment 26 comprising

Luteolin, manganese sulfate, vitamin C and vitamin D.

[00117] Embodiment 31. The composition or combination of Embodiment 26 comprising

Luteolin, manganese sulfate, vitamin C, vitamin D and vitamin B12.

[00118] Embodiment 32. The composition or combination of Embodiment 1 comprising a magnesium salt and a manganese salt.

[00119] Embodiment 33. The composition or combination of Embodiment 32 wherein the magnesium salt comprises magnesium chloride, magnesium glycinate or magnesium citrate.

[00120] Embodiment 34. The composition or combination of Embodiment 32 wherein the magnesium salt comprises magnesium chloride.

[00121] Embodiment 35. The composition or combination of Embodiment 32 wherein the magnesium salt comprises magnesium glycinate. [00122] Embodiment 36. The composition or combination of Embodiment 32 wherein the magnesium salt comprises magnesium citrate.

[00123] Embodiment 37. The composition or combination of Embodiment 32 wherein the manganese salt comprises manganese sulfate.

[00124] Embodiment 38. The composition or combination of Embodiment 3 comprising Luteolin, magnesium chloride and manganese sulfate.

[00125] Embodiment 39. The composition or combination of Embodiment 3 comprising Luteolin, magnesium glycinate and manganese sulfate.

[00126] Embodiment 40. The composition or combination of Embodiment 3 comprising

Luteolin, magnesium citrate and manganese sulfate.

[00127] Embodiment 41. The composition or combination of Embodiment 32 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[00128] Embodiment 42. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, and vitamin C.

[00129] Embodiment 43. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, and vitamin D.

[00130] Embodiment 44. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, and vitamin B12.

[00131] Embodiment 45. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, vitamin C and vitamin D.

[00132] Embodiment 46. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, vitamin C, vitamin D and vitamin B12.

[00133] Embodiment 47. The composition or combination of Embodiment 1 comprising a magnesium salt, a manganese salt and a zinc salt.

[00134] Embodiment 48. The composition or combination of Embodiment 47 wherein the magnesium salt comprises magnesium chloride, magnesium glycinate or magnesium citrate.

[00135] Embodiment 49. The composition or combination of Embodiment 47 wherein the magnesium salt comprises magnesium chloride.

[00136] Embodiment 50. The composition or combination of Embodiment 47 wherein the magnesium salt comprises magnesium glycinate. [00137] Embodiment 51. The composition or combination of Embodiment 47 wherein the magnesium salt comprises magnesium citrate.

[00138] Embodiment 52. The composition or combination of Embodiment 47 wherein the manganese salt comprises manganese sulfate.

[00139] Embodiment 53. The composition or combination of Embodiment 47 wherein the zinc salt comprises zinc chloride.

[00140] Embodiment 54. The composition or combination of Embodiment 47 wherein the zinc salt comprises zinc oxide.

[00141] Embodiment 55. The composition or combination of Embodiment 47 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[00142] Embodiment 56. The composition or combination of Embodiment 55 comprising Luteolin, a magnesium salt, a manganese salt, a zinc salt and vitamin C.

[00143] Embodiment 57. The composition or combination of Embodiment 41 comprising Luteolin, a magnesium salt, a manganese salt, a zinc salt and vitamin D.

[00144] Embodiment 58. The composition or combination of Embodiment 41 comprising Luteolin, a magnesium salt, a manganese salt, a zinc salt and vitamin B12.

[00145] Embodiment 59. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, a zinc salt vitamin C and vitamin D.

[00146] Embodiment 60. The composition or combination of Embodiment 41 comprising

Luteolin, a magnesium salt, a manganese salt, a zinc salt vitamin C, vitamin D and vitamin B12. [00147] Embodiment 61. The composition or combination of Embodiment 1 comprising Luteolin, magnesium chloride, manganese sulfate, and zinc oxide.

[00148] Embodiment 62. The composition or combination of Embodiment 61 further comprising vitamin C, vitamin D, vitamin B12 or combinations thereof.

[00149] Embodiment 63. The composition or combination of Embodiment 1 that is a food supplement.

[00150] Embodiment 64. The composition or combination of Embodiment 1 that is a pharmaceutical composition.

[00151] Embodiment 65. The composition or combination of Embodiment 64 further comprising a pharmaceutically acceptable additive. [00152] Embodiment 66. The composition or combination of Embodiment 64 further comprising a pharmaceutically acceptable carrier.

[00153] The embodiments described in this disclosure may be formulated with one or more pharmaceutically acceptable additives. The additive may be a carrier, an excipient, a binder, a colorant, a flavoring agent, a preservative, a buffer, a diluent or dilutant, and/or combinations thereof. Other pharmaceutically acceptable additives include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, or similar oils, water or saline aqueous solutions, aqueous dextrose and glycerol solutions. The pharmaceutically acceptable additive useful for formulating a dosage form comprising the composition of this disclosure will depend, among other factors, on the elected administration route.

[00154] The combination may be administered in a dosage form selected from the group consisting of a capsule, a cachet, a pill, a tablet, a powder, a granule, a pellet, a bead, a particle, a troche, a lozenge, and a gel.

[00155] The composition may be in solid or liquid form for oral administration. Solid forms include tablets, caplets, pill, capsules, gelcaps, powders, granules, gummies and the like. A tablet or pill is usually a compressed preparation that may contain, for example but not limitation, 5-10% of the Luteolin and metal salts; 80% of fillers, disintegrants, lubricants, glidants, and/or binders; and 10% of compounds that provide easy disintegration, disaggregation, and dissolution of the tablet in the stomach or the intestine. The dissolution time can be modified for a rapid effect or for sustained release. Special coatings can make the tablet resistant to the stomach acids such that it only disintegrates in the duodenum, jejunum and/or colon as a result of enzymatic action or alkaline pH. Pills can be coated with sugar, varnish, shellac, or wax to disguise the taste.

[00156] A caplet is a compressed mixture of ingredients, similar to a tablet, which is formed into a capsule shape. It often has a film or gelatin coating to mask the taste and make it easier to swallow. Capsules generally comprise hard shells that can contain powders or granules, or liquids in some embodiments. A soft gel capsule or gelcap comprises a thicker, softer shell that may be easier to swallow. Gelcaps often comprise liquid or paste compositions inside the gelatin shell.

[00157] Gummies are chewable formulations comprising the active ingredients in a chewable gelatin base. They may come in a variety of flavors, colors, and shapes and may be desirable for a subject that may not like swallowing pills or capsules. [00158] In some embodiments, the composition may be in the form of a biodegradable capsule containing the composition. In one embodiment, the composition is in the form of a supplement. In another embodiment, it is in the form of a pharmaceutical composition. In another embodiment, the compositions may be formulated with liposomes, polymeric micelles, microspheres or nanoparticles

[00159] In some embodiments, such as for a nutritional supplement, the composition can be formulated as a powder that can be added to a liquid, for example a beverage, to prepare a drinkable solution or suspension of the combination. Liquid formulations may be formulated for oral, topical or percutaneous applications.

[00160] Any of these forms may be formulated to provide a unit dose of the combination. A unit dose is the amount of a medication administered to an individual in a single dose. A unit dose for example may comprise 75-100 mg of Luteolin; 200 to 600 mg, such as 500 mg, of magnesium chloride; 2 to 10 mg, such as 3.5 mg, of manganese, as manganese sulfate monohydrate; and 5 to 10 mg, such as 7.5 mg, of zinc, as zinc oxide. Optionally, the unit dose may further comprise vitamin C, vitamin D and/or vitamin B12. The unit dose may comprise 1000-2000 mg of vitamin C. The unit dose may comprise 1000 International Units (IU) of vitamin D3.

[00161] The formulated compositions may be manufactured according to conventional methods known by the skilled person in the art. For example, the compositions may be prepared using standard methods such as those described or referred to in the US Pharmacopoeias and similar reference texts.

[00162] In particular, the compositions of the present invention can be administered as a pharmaceutical composition or an over-the-counter therapeutic supplement, either of which comprises the composition described herein and one or more pharmaceutically acceptable additives.

[00163] The compounds for use according to the invention may be administered as the sole active ingredient or in combination with other active ingredients. In a particular embodiment, the compounds are used as the sole active ingredient. In another particular embodiment, the compounds are used in combination with other active ingredients.

[00164] When the combination is in the form of a single composition, the ingredients present in the combination are always administered simultaneously. [00165] When the combination is in the form of several compositions (or units), each of them having at least one of the ingredients of the combination, the compositions or (units) may be administered simultaneously, sequentially or separately.

[00166] Simultaneous administration means that the ingredients or compositions (or units) are administered at the same time.

[00167] Sequential administration means that the ingredients or compositions (or units) are administered at different time points in a chronologically staggered manner.

[00168] Separate administration means that the ingredients or compositions (or units) are administered at different time points independently of each other.

[00169] While the disclosure is described in conjunction with the enumerated aspects, it will be understood that they are not intended to limit the invention to those aspects. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The nomenclature used in this application is based on IUPAC systematic nomenclature, unless indicated otherwise.