WO2022026622A2 - Treatment of viral diseases - Google Patents

Abstract

The present invention relates to methods of preventing or treating in patients suffering from viral diseases such as Influenza A and novel coronavirus 2019 (COVID-19) comprising administering to a patient in need thereof a therapeutically effective amount of methionine enkephalin (MENK) alone or in combination with (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone or a pharmaceutically acceptable salt thereof.

Description

TREATMENT OF VIRAL DISEASES

CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. provisional application No.

63/057,641, which is titled TREATMENT OF VIRAL DISEASES, was filed July 28, 2020, and is incorporated herein by reference as if fully set forth.

SEQUENCE LISTING

[0002] The Sequence Listing filed herewith, titled “Sequence Listing,” and having a file size of 3,424 bytes is incorporated herein by reference as if fully set forth.

FIELD

[0003] The present invention relates to methods of treating patients suffering from diseases caused by or patients infected with viruses, Influenza A, novel coronavirus 2019 (COVID-19 caused by SARS-CoV-2 infection), or other coronaviruses. The present invention also relates to methods of decreasing the likelihood of infection or disease caused by said viruses. The present invention also relates to methods of increasing immune system factors above patient’s baseline. The methods comprise administering to a patient in need thereof a therapeutically effective amount of methionine enkephalin (MENK) alone or in combination with at least one of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone or a pharmaceutically acceptable salt thereof. The present invention also relates to compositions and dosage forms comprising methionine enkephalin (MENK) alone or in combination with at least one of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone or a pharmaceutically acceptable salt thereof.

BACKGROUND

[0004] The 2019-20 Wuhan coronavirus outbreak, formally the outbreak of novel coronavirus (COVID-19 or 2019-nCoV), is an ongoing viral epidemic primarily affecting Mainland China, along with isolated cases in 27 other countries and territories. [0005] In early December 2019 a new coronavirus, designated 2019-nCoV, was identified in Wuhan, the capital of China’s Hubei province, after 41 people developed pneumonia without a clear cause (2019-nCoV acute respiratory disease). The virus is capable of spreading from person to person and has been shown to spread from mother to child in the womb. The incubation period (time from exposure to onset of symptoms) ranges from 2 to 14 days, but it may be contagious during this period and after recovery. Symptoms include fever, coughing and breathing difficulties, and the virus can be fatal. Wuhan and Hubei Province have borne the brunt of the epidemic as the sudden shutdown of transportation links into and around the area slowed the shipping of vital medical supplies. The fatality rate in Wuhan was 4.1 percent and 2.8 percent in Hubei, compared to 0.17 percent elsewhere in mainland China.

[0006] A larger number of people may have been infected, but not detected (especially mild cases). According to official figures, As of 6 February 2020, there were 28,368 people infected of which 3,863 (14%) were in critical condition and 565 deaths were attributed to the virus since the first confirmed death on 9 January, with 1,387 recoveries.

[0007] Ultimately, the outbreak could be controlled with a protective vaccine to prevent COVID-19 infection. While vaccine are being distributed, and further vaccine research should be pursued intensely, the current vaccines need improvement. Further, the virus is mutating and immunity provided by current vaccines may not protect against future viral mutants. Still further, there exists today no generally accepted effective therapy to treat COVID-19 upon infection, despite an urgent need to find options to help these patients and preclude potential death.

SUMMARY

[0008] In an aspect, the invention relates to a method of addressing viral infection. The method comprises administering to a mammalian patient in need thereof at least one of (1) MENK or a pharmaceutically acceptable agent thereof or (2) a first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The method optionally comprises administering one or more antiviral agents or a pharmaceutically acceptable agent thereof. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus.

[0009] In an aspect, the invention relates to a pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The pharmaceutical composition also comprises at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof. The pharmaceutical composition may be for addressing a viral infection. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus.

[0010] In an aspect, the invention relates to a dosage form comprising a pharmaceutical composition. The pharmaceutical composition comprises at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The pharmaceutical composition also comprises at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof. The dosage form may be for addressing a viral infection. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus.

[0011] In an aspect, the invention relates to a use of a pharmaceutical composition. The pharmaceutical composition comprises at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The pharmaceutical composition also comprises at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof. The use may be for a method of addressing a viral infection. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus.

[0012] In an aspect, the invention relates to a use of a dosage form comprising a pharmaceutical composition. The pharmaceutical composition comprises at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The pharmaceutical composition also comprises at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof. The use may be for addressing a viral infection. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus.

[0013] In an aspect, the invention relates to a use of a pharmaceutical composition for preparing a medicament. The medicament may be for addressing viral infection. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus. The pharmaceutical composition comprises at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The pharmaceutical composition also comprises at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof.

[0014] In an aspect, the invention relates to a use of a dosage from comprising a pharmaceutical composition for preparing a medicament. The medicament may be for addressing viral infection. The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus. The pharmaceutical composition comprises at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof. The pharmaceutical composition also comprises at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings: [0016] FIG. 1 illustrates an experimental design. (A) RAW 264.7 cells were treated with MENK 24 h prior to influenza A/PR/8/34 H1N1 virus infection. (B) RAW 264.7 cells were treated with MENK 1 h after virus infection.

[0017] FIGS. 2A and 2B illustrate Influenza virus titer and TCID50. FIG. 2A shows Influenza virus titer of chicken embryo culture detected by Hemagglutination test (HA). FIG. 2B shows the dilution ratio of the virus TCID50 using Reed-Muench method.

[0018] FIGS. 3A and 3B illustrate the optimal concentration of MENK on RAW264.7 anti-influenza virus. FIG. 3A shows the effect of MENK on proliferation of RAW264.7 cells. FIG. 3B shows the effect of significant concentration of MENK on RAW264.7 anti-influenza virus. Data represent the mean ± SEM of three independent experiments. ★p<0.05, A Ap<0.01 versus the H1N1-C.

[0019] FIGS. 4A, 4B, and 4C illustrate morphology changes of RAW264.7 cells infected with H1N1 treated with MENK. FIG. 4A shows light microscope morphology of RAW264.7 cells at 24, 48, 72 hpi. Morphology of infected cells produced longer processus pseudopodia, changed to irregular polymorphism, and appeared vacuoles and granular substances in the cytoplasm. Pre-MENK treatment had a long fusiform shape, and with many pseudopods. Some cells occasionally appeared vacuoles and particulate substances in Post-MENK group. FIG. 4B shows Hoechst staining of RAW264.7 cells at 24, 48, 72 hpi. Infected cells had a few apoptotic cells with nucleus stained by dense or fragmented at 48 and 72 hpi, sporadic hyperpigmentation of nucleus in Pre-MENK group and Post- MENK group. FIG. 4C Panel represents apoptosis cells. Data represent the mean ± SEM of three independent experiments. ^<0.05, ★★p<0.01 versus H1N1-C group.

[0020] FIGS. 5A, 5B, 5C, and 5D illustrate that MENK inhibited the replication of influenza virus in RAW264.7 cells. FIG. 5 A, gene expression of virus was quantified at 48 hpi by quantitative PCR in RAW264.7 cells. FIG. 5B, FCM analyzed the expression of influenza NP in RAW264.7 cells infected with H1N1 at 24, 48,72 hpi. FIG. 5C represents the mean fluorescence intensity (MFI). FIG. 5D shows the localization and expression of influenza NP protein in RAW264.7 cells at 48 hpi. Influenza NP expressed on the cell nucleus was shown in green (DyLight 488), and in blue (DAPI) in nucleus. Data represent the mean ± SEM of three independent experiments. ^<0.05, ★★p<0.01 versus H1N1-C group.

[0021] FIGS. 6A and 6B illustrate MENK enhanced pro-inflammatory cytokine production by RAW264.7 cells infected with H1N1. FIG. 6A shows qPCR results to determine gene expressions of IL-6, TNF-a and IL-16 on RAW264.7 cells at 24, 48,72 hpi. FIG. 6B illustrates ELISA results to determine expressions of IL- 6, TNF-a and IL-16 in the cell supernatant. Results of qPCR presented as fold increase over the Normal-C group. Data represent the mean ± SEM of three independent experiments. ^<0.05, ★★p<0.01 versus H1N1-C group.

[0022] FIGS. 7A and 7B illustrate MENK upregulated MOR expression on RAW264.7 cells infected with H1N1. FIG. 7A shows gene expressions of MOR were quantified at 48 hpi by quantitative PCR in RAW264.7 cells. FIG. 7B shows localization and expression of MOR in RAW264.7 cells at 48 hpi. MOR expressed on the cell membrane was shown in green (DyLight 488), and nucleus in blue (DAPI). Results were presented as fold increase over the Normal-C group. Data represent the mean ± SEM of three independent experiments. ^<0.05, ★★p< 0.01 versus H1N1-C group.

[0023] FIGS. 8A, 8B, 8C, and 8D illustrate MENK inhibited RAW264.7 cells infected with H1N1 through up-regulating TLR4 and NF-KB. FIG. 8A shows gene expressions of TLR7, TLR4 and NF-KB p65 were quantified at 48 hpi by quantitative PCR in RAW264.7 cells. FIG. 8B shows FCM analysed the expression of TLR4 and NF-KB p65 in RAW264.7 cells infected with H1N1 at 24, 48,72 hpi. FIG. 8C represents mean fluorescence intensity (MFI). FIG. 8D shows the localization and expression of TLR4 and NF-KB p65 protein in RAW264.7 cells at 48 hpi. TLR4/ NF-KB p65 expressed on the cell membrane/nucleus was shown in green (DyLight 488), and nucleus in blue (DAPI). Data represent the mean ± SEM of three independent experiments. ^<0.05, ★★p<0.01 versus H1N1-C group. [0024] FIG. 9 illustrates in vitro antiviral results for Naltrexone-HCl.

[0025] FIG. 10 illustrates in vitro antiviral results for Remdesivir.

[0026] FIG. 11 illustrates in vitro antiviral results for Naloxone-HCl.

[0027] FIG. 12 illustrates in vitro antiviral results for 6-B-Naltresonxe Hydrate. [0028] FIG. 13 illustrates in vitro antiviral results for Remdesivir.

[0029] FIG. 14 illustrates the effect of pre- or post- MENK treatment.

DETAILED DESCRIPTION

[0030] Certain terminology is used in the following description for convenience only and is not limiting. The words “right,” “left,” “top,” and “bottom” designate directions in the drawings to which reference is made.

[0031] The words “a” and “one,” as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise. This terminology includes the words above specifically mentioned, derivatives thereof, and words of similar import. The phrase “at least one” followed by a list of two or more items, such as “A, B, or C” or “A, B, and C,” means any individual one of A, B or C as well as any combination thereof.

[0032] Abbreviations: MENK, methionine enkephalin; IAV, influenza A virus; MOR, p-opioid receptor; hpi, hour post infection; TLRs, Toll-like receptors; PRRs, pathogen- recognition receptors; NF-KB, nuclear factor KB; NP, nucleoprotein.

[0033] Numerical values or ranges preceded by “about” refer to the explicitly recited numbers, and the numbers within the experimental error of the measure contemplated. Embodiments described with the modifier “about” may be altered to remove “about” in order to form further embodiments herein. Likewise, embodiments described without the modifier “about” maybe altered to add “about” in order to form further embodiments herein.

[0034] A range expressed as being between two numerical values, one as a low endpoint and the other as a high endpoint, includes the values between the numerical values and the low and high endpoints. Embodiments herein include subranges of a range herein, where the subrange includes a low and high endpoint of the subrange selected from any increment within the range selected from each single increment of the smallest significant figure, with the condition that the high endpoint of the subrange is higher than the low endpoint of the subrange. [0035] Further embodiments herein include replacing one or more “including” or “comprising” in an embodiment with “consisting essentially of or “consisting of.” “Including” and “comprising,” as used herein, are open ended, include the elements recited, and do not exclude the addition of one or more other element. “Consisting essentially of means that addition of one or more element compared to what is recited is within the scope, but the addition does not materially affect the basic and novel characteristics of the combination of explicitly recited elements. “Consisting of refers to the recited elements, but excludes any element, step, or ingredient not specified.

[0036] The term “carrier” refers to a diluent, excipient, and/or vehicle with which an active compound is administered. The pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin, 18th Edition, which is incorporated by reference as if fully set forth.

[0037] A “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the present application includes both one and more than one such excipient.

[0038] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit/risk ratio. [0039] As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues, for example amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include, but are not limited to, salts from ammonia, L-arginine, betaine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine (2,2’-iminobis(ethanol)), diethylamine, 2-(diethylamino)-ethanol, 2-aminoethanol, ethylenediamine, N-ethyl-glucamine, hydrabamine, lH-imidazole, lysine, magnesium hydroxide, 4-(2-hydroxyethyl)- morpholine, piperazine, potassium hydroxide, l-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide, triethanolamine (2,2’,2”-nitrilotris(ethanol)), tromethamine, zinc hydroxide, acetic acid, 2,2-dichloro-acetic acid, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 2,5- dihydroxybenzoic acid, 4-acetamido-benzoic acid, (+)-camphoric acid, (+)-camphor- 10-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, decanoic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2- hydroxy-ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, D-glucoheptonic acid, D-gluconic acid, D-glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycine, glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, DL-lactic acid, lactobionic acid, lauric acid, lysine, maleic acid, (-)-L-malic acid, malonic acid, DL-mandelic acid, methanesulfonic acid, galactaric acid, naphthalene- 1,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, octanoic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid (embonic acid), phosphoric acid, propionic acid, (-)-L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid and undecylenic acid. A pharmaceutically acceptable salt can be, but is not limited to, salts formed with cations from metals. The metal may be aluminium, calcium, lithium, magnesium, potassium, sodium, or zinc, and the like (see Pharmaceutical salts, Berge, S. M. et al., J. Pharm. Sci, (1977), Vol.66, pp.1-19). A pharmaceutically acceptable salt herein may be any one of these. A pharmaceutically acceptable salt herein may be a hydrochloride salt.

[0040] Pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts may be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a mixture of two or more thereof. [0041] Salts of other acids than those mentioned above may also be useful for purifying or isolating the naltrexone ( e.g . trifluoro acetate salts), and also comprise an embodiment part of an embodiment of the invention.

[0042] A pharmaceutically acceptable salt of a compound of naltrexone may be readily prepared by using a desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as hydrochloric acid may be added to an aqueous suspension of a compound of naltrexone and the resulting mixture evaporated to dryness (e.g., by lyophilization) to obtain the acid addition salt as a solid. Alternatively, a compound of naltrexone may be dissolved in a suitable solvent, for example an alcohol. The alcohol may be isopropanol. The acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent. The less polar solvent may be diisopropyl ether or hexane. The precipitated acid addition salt may be isolated by filtration.

[0043] The acid addition salts of the compounds of naltrexone may be prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free base forms may differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents. The salts and/or free base forms may be part of embodiments herein. Some salts and free base forms may perform similarly with respect to methods of treatment herein.

[0044] Also included are both total and partial salts, that is to say salts with 1, 2 or 3, preferably 2, equivalents of base per mole of acid of formula I or salts with 1, 2 or 3 equivalents, preferably 1 equivalent, of acid per mole of base of formula I.

Formula I

[0045] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates.” For example, a complex with water is known as a “hydrate.” Solvates of a compound of the invention are within the scope and/or included in embodiments herein. The salts of naltrexone may form solvates ( e.g ., hydrates) and the embodiments herein may include one or more salt of naltrexone solvates. The meaning of the word “solvates” is well known to those skilled in the art as a compound formed by interaction of a solvent and a solute (i.e., solvation). Techniques for the preparation of solvates are well established in the art (see, for example, Brittain. Polymorphism in Pharmaceutical Solids. Marcel Decker, New York, 1999, which is incorporated herein by reference as if fully set forth).

[0046] As used herein, “pharmaceutically acceptable agent” means pharmaceutically acceptable salt and/or solvate. [0047] Embodiments herein provide potential options to treat COVID-19 in patients, with an emphasis on the necessity for speed and timeliness in developing new and effective therapies in this continuing pandemic. Options of drug repurposing, developing neutralizing monoclonal antibody therapy, and an oligonucleotide strategy targeting the virus may be at least part of embodiments herein. The fastest strategy to develop a treatment now, which could be resistant to any mutations the virus may have in the future, are contemplated herein. Embodiments herein may comprise use of or administering MENK alone or in combination with naltrexone. The use may be to prepare a medicament. The use or administering may be for treating. The use or administering may be for decreasing the likelihood of infection, or the likelihood of developing disease. The use or administering may be to modulate an immune response. The use or administering may be to prevent or treat viral infections, diseases, or disorders. The virus may be Influenza A, SARS-CoV-2, or other coronaviruses. The disease may be influenza or COVID-19. The immune modulation may be helping to recruit the immune system to build lasting immunity.

[0048] Embodiments also involve the use of naltrexone and methionine enkephalin (“MENK”). MEND is an endogenous neuropeptide, and is suggested to be involved in the regulatory loop between the immune and neuroendocrine systems, with modulation of various functions of cells related to both the innate and adaptive immune systems. The inventors’ present research findings show that MENK serves as an immune modulator to the pathway between DCs and CD4+T cells. Studies included changes of DCs in key surface molecules, the activity of acid phosphatases (ACPs), the production of IL-12, and the effects on murine CD4+T cell expansion and their cytokine production by MENK alone, and in combination with interkeukin-2 (IL-2) or interferon- (IFN- ). Surprisingly, the inventors found that MENK could markedly induce the maturation of DCs through the addition of surface molecules such as MHC class II, CD86, and CD40 on murine DCs, the production of IL-12, and the down-regulation of ACP inside DCs, which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation. The inventors also surprisingly found that MENK alone or in combination with IL-2 or IFN-, could markedly up-regulate both CD4+T cell expansion and the CD4 molecule expression in vivo and in vitro, and that MENK alone, or MENK+ IL-2, could enhance the production of interferon- from CD4+T cells. Moreover, MENK alone, or MENK+ IFN- , could enhance the production of IL-2 from CD4+T cells. It was therefore concluded that MENK can exert positive modulation to the pathway between dendtritic cells and CD4+T cells.

[0049] Embodiment include compositions comprising at least one of (1) one or more of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, or pharmaceutical agents thereof, and/or (2) antiviral agent, and/or (3) MENK.

[0050] Embodiments include methods of treating viral infection, methods of decreasing the likelihood of viral infection, methods of decreasing the likelihood of disease from viral infection, methods of treating a disease caused by viral infection, and methods of increasing immunes system factors in a patient. Collectively, these methods are referred to as methods of addressing viral infection. The methods of addressing viral infection may comprise administering at least one of (1) one or more of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, or pharmaceutical agents thereof, and/or (2) antiviral agent, and/or (3) MENK to a patient. The patient may be a mammal. The patient may be a human. The patient may be a rodent, a canine, a feline, a simian, or an ape.

[0051] An embodiment comprises a method for treating or preventing viral infections, or diseases caused by the infections, in a mammal comprising administering to a patient in need thereof a pharmaceutical composition comprising a compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and pharmaceutically acceptable salts thereof and mixtures thereof alone or in combination with one or more antiviral agents. The patient may be a mammal. The mammal may be a rodent, a canine, a feline, a simian, an ape, or a human. The patient may be a human. An embodiment of the method further comprises administration to the patient of a pharmaceutical composition comprising methionine enkephalin (MENK) or a pharmaceutically acceptable salt thereof. The (1) one or more of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, or pharmaceutical agents, and/or (2) antiviral agent, and/or (3) MENK may be separately administered, or administered combined in one formulation.

[0052] The antiviral agent may be chosen from the group consisting of an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal), or a combination of two or more thereof.

[0053] The viral infection may be by a virus chosen from the group consisting of influenza A, SARS-CoV-2 or novel coronavirus 2019 (COVID19) or a pathological strain thereof, 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV, SARS-CoV, molluscum contagiosum virus, HTLV, HTLV-1, hepatitis-A, HCV, HBV, HIV/AIDS, human papilloma virus, herpes virus, herpes simplex virus (which causes genital herpes), a virus that causes viral dysentery, measles virus, rubella virus, mumps virus, polio virus, rabies virus, Epstein-Barr virus (which causes mononucleosis), ebola virus, respiratory syncytial virus, dengue virus, yellow fever virus, lassa virus, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, a virus causing viral meningitis, west Nile virus, arbovirus, a parainfluenza virus, smallpox virus, dengue virus (which causes dengue hemorrhagic fever), cytomegalovirus, infant cytomegalic virus, JC virus (which causes progressive multifocal leukoencephalopathy), a virus that causes viral gastroenteritis, a hepatitis virus, herpes simplex virus (which causes cold sores or ocular herpes), a virus that causes encephalitis, varicella zoster virus (which causes chicken pox or shingles), viruses that cause California serogroup viral encephalitis, St. Louis encephalitis virus, rift valley fever virus, a virus that causes hand, foot, & mouth disease, hendra virus, an enterovirus, astrovirus, an adenovirus, Japanese encephalitis virus, lymphocytic choriomeningitis mammarenavirus (which causes lymphocytic choriomeningitis), human herpesvirus 6 or human herpesvirus 7 (which cause) roseola infantum, a virus that causes sandfly fever, papilloma virus, a human papilloma virus (which causes warts), cat scratch disease, parvovirus B19 (which causes slap-cheek syndrome), a parapox virus that causes orf, a virus that causes pityriasis rosea, and lyssavirus.

[0054] The viral infection may be influenza A infection.

[0055] The viral infection may be by SARS-CoV-2, also known as the novel coronavirus 2019 (COVID19), or a pathological strain thereof.

[0056] The anti-viral agent may be chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine, tocilizumab, EIDD-280 and etravirine. The anti-viral agent may be remdesivir. The anti-viral agent may be favipiravir.

[0057] The one or more anti-viral agent may be a combination of anti-viral agents, and the combination of anti-viral agents may be chosen from the group consisting of rilpivine, abacavir, and lamivudine; abacavir, dolutegravir, and lamivudine; abacavir, lamivudine, and zidovudine; atazanavir, and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine, and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumerate; emtricitabine, rilpivirine, and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; lopinavir, retinovir and interferon-beta lb; and lopinavir and ritonavir. The anti-viral agent may be chosen from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof.

[0058] The pharmaceutical compositions may further comprise one or more pharmaceutically acceptable excipients. The excipients may be charged aqueous species that have a net negative charge or a net positive charge.

[0059] A method herein may further comprise administration of a therapeutically effective amount of a compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin, and riboflavin or combinations thereof.

[0060] A method herein may further comprise administration of a therapeutically effective amount of a compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins or combinations thereof.

[0061] A method herein may further comprise administration of a therapeutically effective amount of an immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE or combinations thereof.

[0062] A method herein may further comprise administration of an antibacterial agent. The antibacterial agent may be Azithromycin.

[0063] The pharmaceutical composition comprising (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and pharmaceutically acceptable salts thereof and mixtures thereof may be in an immediate release form.

[0064] The amount of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and pharmaceutically acceptable salts thereof and mixtures thereof may between about 1.0 mg and about 300.0 mg. The amount may be between about 0.05 mg and about 6.0 mg. The amount may be between about 0.05 mg and about 4.5 mg. The amount may be the amount administered. The amount maybe the amount in a single dosage. The amount may be the amount in a single dosage form.

[0065] Embodiments of methods and compositions herein comprise an immediate release pharmaceutical composition. An immediate release pharmaceutical composition may be for administration once in a 24-hour period. The immediate release composition may comprise the pharmaceutically acceptable salt of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and release the pharmaceutically acceptable salt of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (- ) naloxone completely within about 60 minutes. [0066] The pharmaceutical composition may be in a dosage form for an administration route chosen from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration. A pharmaceutical composition herein may be in the form of a capsule or tablet. A pharmaceutical composition herein may be in the form of a dissolving film. A method herein may include administering by any one or more of these routes. A composition herein may comprise one or more of these dosage forms.

[0067] In the pharmaceutical composition comprising (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and pharmaceutically acceptable salts thereof, the ratio of (+) naltrexone and (-) naltrexone may be between about 1:99 and about 99:1. The ratio of (+) naltrexone and (-) naltrexone may be about 50:50. The ratio of (+) naloxone and (-) naloxone may be between about 1:99 and about 99:1. The ratio of (+) naloxone and (-) naloxone may be about 50:50. The ratio of (+) naltrexol and (-) naltrexol may be between about 1:99 and about 99:1. The ratio of (+) naltrexol and (-) naltrexol may be about 50:50.

[0068] Embodiments comprise prodrugs or pharmaceutically acceptable salts thereof of the compounds of formula I, i.e., compounds, which release an active parent drug (naltrexone) in, vivo when administered to a mammalian subject, in place of or in addition to the (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and pharmaceutically acceptable salts thereof. A prodrug is a pharmacologically active or more typically an inactive compound that is converted into a pharmacologically active agent by a metabolic transformation. Prodrugs of naltrexone are prepared by modifying functional groups present in naltrexone in such a way that the modifications may be cleaved in vivo to release the parent compound. In, vivo, a prodrug readily undergoes chemical changes under physiological conditions ( e.g ., are acted on by naturally occurring enzyme(s)) resulting in liberation of the pharmacologically active agent. Prodrugs of naltrexone wherein a hydroxyl or amino, of naltrexone is bonded to any group that may be cleaved in, vivo to regenerate the free hydroxyl, amino or carboxy group, respectively. Examples of prodrugs include esters (e.g., acetate, formate, and benzoate derivatives) of compounds of formula I or any other derivative, which upon being brought to the physiological pH or through enzyme action is converted to the active parent drug. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in the art (see, for example, Bundgaard. Design of Prodrugs. Elsevier, 1985, which is incorporated herein by reference as if fully set forth).

[0069] Prodrugs may be administered in the same manner as the active ingredient to which they convert or they may be delivered in a reservoir form, e.g., a transdermal patch or other reservoir which is adapted to permit (by provision of an enzyme or other appropriate reagent) conversion of a prodrug to the active ingredient slowly over time, and delivery of the active ingredient to the patient. [0070] Naltrexone may be formulated for administration in any convenient way for use in human or veterinary medicine and the invention therefore includes within its scope pharmaceutical compositions comprising a compound of the invention adapted for use in human or veterinary medicine. The compositions may be presented for use in a conventional manner with the aid of one or more suitable carriers. Acceptable carriers for therapeutic use are well-known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), which is incorporated herein by reference as if fully set forth. The choice of pharmaceutical carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, in addition to, the carrier any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s). [0071] Pharmaceutical Compositions Comprising Naltrexone or MENK [0072] Embodiments may comprise bulk naltrexone or MENK, administering them in bulk. Embodiments by comprise the active ingredient in a pharmaceutical formulation, e.g., wherein the agent is in admixture with a pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0073] Accordingly, the invention further provides a pharmaceutical composition comprising naltrexone or pharmaceutically acceptable salt thereof or MENK in admixture with a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, excipient, and/or vehicle with which an active compound is administered.

[0074] Naltrexone or MENK may be used in combination with other therapies and/or active agents. Accordingly, an embodiment provides a pharmaceutical composition comprising MENK or naltrexone or a solvate, hydrate, enantiomer, diastereomer, N-oxide or pharmaceutically acceptable salt thereof, a second active agent, and a pharmaceutically acceptable carrier. An embodiment provides a method herein comprising administering said pharmaceutical composition.

[0075] The pharmaceutical compositions may comprise as, in addition to, the carrier any suitable binder, lubricant, suspending agent, coating agent and/or solubilizing agent. Preservatives, stabilizers, dyes and flavoring agents also may be provided in the pharmaceutical composition. Antioxidants and suspending agents may be also present.

[0076] The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention may be prepared by processes known in the art, for example see W002/00196, which is incorporated herein by reference as if fully set forth.

[0077] The term “Immediate release” is defined as a release of compound from a dosage form in a relatively brief period of time, generally up to about 60 minutes.

[0078] Routes of Administration and Unit Dosage Forms

[0079] The routes for administration include oral ( e.g ., as a tablet, capsule, or as an ingestible solution), topical, mucosal {e.g., as a nasal spray or aerosol for inhalation), nasal, parenteral {e.g., by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, epidural and sublingual. The compositions of the invention may be especially formulated for any of those administration routes. In preferred embodiments, the pharmaceutical compositions of the invention are formulated in a form that is suitable for oral delivery.

[0080] There may be different composition/formulation requirements depending on the different delivery systems. It is to be understood that not all of the compounds need to be administered by the same route. When more than one active component is administered, then those components may be administered by different routes. By way of example, a pharmaceutical composition herein may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, formulations may be designed to be delivered by multiple routes.

[0081] Where an agent is to be delivered mucosally through the gastrointestinal mucosa, it could be able to remain stable during transit though the gastrointestinal tract; for example, it could be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile. For example, naltrexone may be coated with an enteric coating layer. The enteric coating layer material may be dispersed or dissolved in either water or in a suitable organic solvent. As enteric coating layer polymers, one or more, separately or in combination, of the following can be used; e.g., solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethylethylcellulose, shellac or other suitable enteric coating layer polymer(s). For environmental reasons, an aqueous coating process may be utilized. In such aqueous processes methacrylic acid copolymers are most preferred.

[0082] When appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges, which can be formulated in a conventional manner.

[0083] When the composition of the invention is to be administered parenterally, such administration includes one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.

[0084] Pharmaceutical compositions of the invention can be administered parenterally, e.g., by infusion or injection. Pharmaceutical compositions suitable for injection or infusion may be in the form of a sterile aqueous solution, a dispersion or a sterile powder that contains the active ingredient, adjusted, if necessary, for preparation of such a sterile solution or dispersion suitable for infusion or injection. This preparation may optionally be encapsulated into liposomes. The final preparation should be sterile, liquid, and stable under production and storage conditions. To improve storage stability, such preparations may also contain a preservative to prevent the growth of microorganisms. Prevention of the action of micro-organisms can be achieved by the addition of various antibacterial and antifungal agents, e.g., paraben, chlorobutanol, or ascorbic acid. In many cases isotonic substances are recommended, e.g., sugars, buffers and sodium chloride to assure osmotic pressure similar to those of body fluids, particularly blood. Prolonged absorption of such injectable mixtures can be achieved by introduction of absorption-delaying agents, such as aluminum monostearate or gelatin.

[0085] Dispersions can be prepared in a liquid carrier or intermediate, such as glycerin, liquid polyethylene glycols, triacetin oils, and mixtures thereof. The liquid carrier or intermediate can be a solvent or liquid dispersive medium that contains, for example, water, ethanol, a polyol {e.g., glycerol, propylene glycol or the like), vegetable oils, non-toxic glycerine esters and suitable mixtures thereof. Suitable flowability may be maintained, by generation of liposomes, administration of a suitable particle size in the case of dispersions, or by the addition of surfactants.

[0086] For parenteral administration, the compound is best used in the form of a sterile aqueous solution, which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions may be suitably buffered (for example, to a pH of from 3 to 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0087] Sterile injectable solutions can be prepared by mixing a compound of formula I or compounds or pharmaceutical compositions herein with an appropriate solvent and one or more of the aforementioned carriers, followed by sterile filtering. In the case of sterile powders suitable for use in the preparation of sterile injectable solutions, preparation methods may include drying in vacuum and lyophilization, which provides powdery mixtures of the active pharmaceutical ingredient(s) (API(s), which may be a compound(s) in a pharmaceutical composition herein), or a pharmaceutical composition herein, and desired excipients for subsequent preparation of sterile solutions.

[0088] Compounds of pharmaceutical compositions herein may be formulated for use in human or veterinary medicine by injection ( e.g ., by intravenous bolus injection or infusion or via intramuscular, subcutaneous or intrathecal routes) and may be presented in unit dose form, in ampoules, or other unit-dose containers, or in multi-dose containers, and may comprise an added preservative. The compositions for injection may be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, solubilizing and/or dispersing agents. The active ingredient may be in sterile powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0089] (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone or MENK can be administered (e.g., orally or topically) in the form of tablets, rapidly dissolving films, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

[0090] (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone or MENK may also be presented for human or veterinary use in a form suitable for oral or buccal administration, for example in the form of solutions, gels, syrups, mouth washes or suspensions, or a dry powder for constitution with water or other suitable vehicle before use, optionally with flavoring and coloring agents. Solid compositions such as tablets, rapidly dissolving films, capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules, bullets or premix preparations may also be used. Solid and liquid compositions for oral use may be prepared according to methods well-known in the art. Compositions herein may also comprise one or more pharmaceutically acceptable carriers and excipients which may be in solid or liquid form.

[0091] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.

[0092] A pharmaceutical composition herein may comprise one or more lubricating agent. The lubricating agent(s) may be selected from magnesium stearate, stearic acid, glyceryl behenate and talc.

[0093] The pharmaceutical compositions may be administered orally, in the form of rapid or controlled release tablets, microparticles, mini tablets, capsules, sachets, and oral solutions or suspensions, or powders for the preparation thereof. In addition to solid-state forms of an API(s) (which may be a compound(s) in a pharmaceutical composition herein)) or pharmaceutical composition herein in embodiments herein, oral preparations may be provided and include various standard pharmaceutical carriers and excipients. The pharmaceutical carriers and excipients may be selected from binders, fillers, buffers, lubricants, glidants, dyes, disintegrants, odourants, sweeteners, surfactants, mold release agents, antiadhesive agents, and coatings. Some excipients may have multiple roles in the compositions, e.g., act as both binders and disinte grants.

[0094] Examples of pharmaceutically acceptable disintegrants for oral pharmaceutical compositions herein include starch, pre-gelatinized starch, sodium starch glycolate, sodium carboxymethylcellulose, croscarmellose sodium, microcrystalline cellulose, alginates, resins, surfactants, effervescent compositions, aqueous aluminum silicates, and cross-linked polyvinylpyrrolidone. [0095] Examples of pharmaceutically acceptable binders for oral pharmaceutical compositions herein include acacia; cellulose derivatives, such as methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose, dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol, starch, pre-gelatinized starch, tragacanth, xanthane resin, alginates, magnesium-aluminum silicate, polyethylene glycol, or bentonite.

[0096] Examples of pharmaceutically acceptable fillers for oral pharmaceutical compositions herein include lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol, sorbitol, starch, cellulose (particularly microcrystalline cellulose), dihydro- or anhydro-calcium phosphate, calcium carbonate, and calcium sulphate.

[0097] Examples of pharmaceutically acceptable lubricants that may be in pharmaceutical compositions herein include magnesium stearate, talc, polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulphate, magnesium lauryl sulphate, sodium oleate, sodium stearyl fumarate, and colloidal silicon dioxide.

[0098] Examples of pharmaceutically acceptable odourants that may be included in oral pharmaceutical compositions herein include synthetic aromas and natural aromatic oils, including extracts of oils, flowers, fruits {e.g., banana, apple, sour cherry, peach) and combinations thereof, and similar aromas. Organoleptic acceptability for the population that will be taking the pharmaceutical compositions may be considered in choosing an odourant. [0099] Examples of pharmaceutically acceptable dyes that may be included in oral compositions herein include synthetic and natural dyes, titanium dioxide, beta-carotene, and extracts of grapefruit peel.

[00100] Examples of useful pharmaceutically acceptable coatings that may be included in oral compositions herein, which may facilitate swallowing, modify the release properties, improve the appearance, and/or mask the taste of the compositions, include hydroxypropylmethylcellulose, hydroxypropylcellulose, and acrylate -methacrylate cop olymers .

[00101] Examples of pharmaceutically acceptable sweeteners that may be included in oral pharmaceutical compositions herein include aspartame, saccharin, saccharin sodium, sodium cyclamate, xylitol, mannitol, sorbitol, lactose, and sucrose.

[00102] Examples of pharmaceutically acceptable buffers that may be included in pharmaceutical compositions herein include citric acid, sodium citrate, sodium bicarbonate, dibasic sodium phosphate, magnesium oxide, calcium carbonate, and magnesium hydroxide.

[00103] Examples of pharmaceutically acceptable surfactants that may be included in pharmaceutical compositions herein include sodium lauryl sulphate and polysorbates.

[00104] Pharmaceutical compositions herein may be in the form of solid compositions. Fillers in gelatin capsules may comprises similar components as above. Examples of excipients in this regard include lactose, starch, a cellulose, milk sugar, or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the active agent(s) may be combined with various sweetening or flavoring agents, coloring matter or dyes, emulsifying and/or suspending agents, and/or diluents. The diluents maybe water, ethanol, propylene glycol and glycerin, and combinations thereof.

[00105] (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and/or MENK may also, for example, be formulated in pharmaceutical compositions herein as suppositories or pessaries. The suppositories may include conventional suppository bases for use in human or veterinary medicine. The pessaries may include conventional pessary bases. [00106] (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and/or MENK may be formulated in pharmaceutical compositions herein for topical administration, for use in human and veterinary medicine, in the form of ointments, creams, gels, hydrogels, lotions, solutions, shampoos, powders (including spray or dusting powders), pessaries, tampons, sprays, dips, aerosols, drops ( e.g ., eye ear or nose drops), or pour-ons.

[00107] For application topically to the skin, the agent(s) of embodiments herein can be formulated as an ointment comprising the active compound(s) suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. These compositions may also contain other pharmaceutically acceptable excipients, such as polymers, oils, liquid carriers, surfactants, buffers, preservatives, stabilizers, antioxidants, moisturizers, emollients, colourants, and odourants.

[00108] Examples of pharmaceutically acceptable polymers that may be included in topical pharmaceutical compositions herein include acrylic polymers; cellulose derivatives (for example, carboxymethylcellulose sodium, methylcellulose or hydroxypropylcellulose); natural polymers (for example, alginates, tragacanth, pectin, xanthan and cytosan).

[00109] Examples of pharmaceutically acceptable oils that may be included in a pharmaceutical composition herein include mineral oils, silicone oils, fatty acids, alcohols, and glycols.

[00110] Examples of suitable pharmaceutically acceptable liquid carriers that may be in a pharmaceutical composition herein include water, alcohols or glycols. Examples of alcohols or glycols include ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and polyethylene glycol, or mixtures thereof. The API(s) (which may be a compound(s) in a pharmaceutical composition herein) or a pharmaceutical composition herein may be dissolved or dispersed in the liquid carrier. Non-toxic anionic, cationic or non-ionic surfactants, and inorganic or organic buffers may also be included. [00111] Examples of pharmaceutically acceptable preservatives that may be in a pharmaceutical composition herein include sodium benzoate, ascorbic acid, esters of p-hydroxybenzoic acid, antibacterial agents, and antifungal agents. For example, ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens, methyl paraben, ethyl paraben, and propyl paraben.

[00112] Examples of pharmaceutically acceptable stabilizers and antioxidants that may be in a pharmaceutical composition herein include ethylenediaminetetraacetic acid (EDTA), thiourea, tocopherol, and butyl hydroxyanisole.

[00113] Examples of pharmaceutically acceptable moisturizers that may be in a pharmaceutical composition herein include glycerine, sorbitol, urea, and polyethylene glycol.

[00114] Examples of pharmaceutically acceptable emollients that may be in a pharmaceutical composition herein include mineral oils, isopropyl myristate, and isopropyl palmitate.

[00115] A pharmaceutical composition herein may be in a form for dermal or transdermal administration, and dermally or transdermally administered, for example, via a skin patch.

[00116] For ophthalmic use, a pharmaceutical composition herein may be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.

[00117] As indicated, a pharmaceutical composition comprising naltrexone or MENK may be administered intranasally or by inhalation, and may be delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1, 1, 1,2-tetrafluoroethane (HFA 134AT) or 1, 1, 1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or other propellant gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound(s), e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate. [00118] Capsules and cartridges (made, for example, from gelatin) as an inhaler or insufflator herein may contain a powder mix of the compound and a suitable powder base such as lactose or starch.

[00119] For topical administration by inhalation, a pharmaceutical composition herein may be delivered for use in human or veterinary medicine via a nebulizer.

[00120] A pharmaceutical composition herein may comprise from 0.01 to 99% weight per volume of active material(s). For example, a pharmaceutical composition for topical administration may comprise from 0.01-10%, or 0.01-1% of the active material(s).

[00121] (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone and/or MENK can also be administered in the form of liposome delivery systems. A liposome delivery system herein may comprise small unilamellar vesicles, large unilamellar vesicles, and/or multilamellar vesicles. Liposomes may be formed from phospholipids, cholesterol, stearylamine or p hosp hatidylcholines .

[00122] A pharmaceutical composition or unit dosage form herein may be administered according to a dosage and administration regimen defined by routine testing in the light of the guidelines given above in order to obtain optimal activity while minimizing toxicity or side effects for a particular patient. Such fine tuning of the therapeutic regimen is routine in the light of the guidelines given herein. [00123] The dosage of the active agents herein may vary according to a variety of factors. The factors may include underlying disease conditions, the individual’s condition, weight, gender and age, and the mode of administration. An effective amount for treating a disorder can easily be determined by empirical methods known to those of ordinary skill in the art, for example by establishing a matrix of dosages and frequencies of administration and comparing a group of experimental units or subjects at each point in the matrix. The exact amount to be administered to a patient may vary depending on the state and severity of the disorder and the physical condition of the patient. A measurable amelioration of any symptom or parameter can be determined by a person skilled in the art or reported by the patient to the physician. A pharmaceutically acceptable amount may be an amount that leads to a measurable amelioration of any symptom or parameter.

[00124] The pharmaceutical composition or unit dosage form may be administered in a single daily dose, or the total daily dosage may be administered in divided doses. In addition, co-administration or sequential administration of another compound for the treatment of a disorder may be desirable. The combined active principles may be formulated into a simple dosage unit.

[00125] For combination treatment where the compounds are in separate dosage formulations, pharmaceutical compositions comprising compounds described herein may be administered concurrently, or each can be administered at staggered intervals. For example, a compound(s) herein may be administered in the morning and an additional antiviral compound may be administered in the evening, or vice versa. Additional compounds may be administered at specific intervals. The order of administration may depend upon a variety of factors including age, weight, gender and medical condition of the patient; the severity and aetiology of the disorders to be treated, the route of administration, the renal and hepatic function of the patient, the treatment history of the patient, and the responsiveness of the patient. Determination of the order of administration may be fine-tuned and such fine-tuning is routine in the light of the guidelines given herein.

[00126] Embodiments List

[00127] The following Embodiments List includes non-limiting embodiments.

The embodiments herein include but are not limited to those in the Embodiment List.

[00128] 1. A method of addressing viral infection comprising administering to a mammalian patient in need thereof at least one of (1) MENK or a pharmaceutically acceptable agent thereof or (2) a first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and optionally administering one or more antiviral agents or a pharmaceutically acceptable agent thereof.

[00129] The addressing may be one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient. The virus may be SARS-CoV-2. The virus may be the Influenza A virus.

[00130] 2. The method of embodiment 1, wherein the first pharmaceutical composition comprises the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and the method further comprises administering the MENK or a pharmaceutically acceptable agent thereof to the patient, wherein the MENK is either part of the first pharmaceutical composition or administered separately from the first pharmaceutical composition.

[00131] 3. The method of embodiment 1, wherein the first pharmaceutical composition comprises at least one of (a) the one or more antiviral agents or a pharmaceutically acceptable agent thereof, or (b) the MENK or a pharmaceutically acceptable agent thereof.

[00132] 4. The method of embodiment 1, wherein the administering further comprises administering a second pharmaceutical composition that comprises at least one of (a) the one or more antiviral agents or a pharmaceutically acceptable agent thereof, or (b) the MENK or a pharmaceutically acceptable agent thereof. [00133] 5. The method of any one or more of embodiments 1-4, wherein the patient is a human, an ape, a simian, a canine, a feline, or a rodent.

[00134] 6. The method of any one or more of embodiments 1-4, wherein the patient is a human.

[00135] 7. The method according to any one more of embodiments 1-6, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent is chosen from the group consisting of an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal), or a combination of two or more thereof.

[00136] 8. The method according to any one more of embodiments 1-7, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent comprises one chosen from the group consisting of hydroxychloroquine and chloroquine.

[00137] 9. The method according to any one more of embodiments 1-8, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent comprises remdesivir.

[00138] 10. The method according to any one more of embodiments 1-9, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent comprises favipiravir.

[00139] 11. The method according to any one more of embodiments 1-10, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent is chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine, tocilizumab, EIDD-280, and etravirine.

[00140] 12. The method according to any one more of embodiments 1-11, wherein the administering comprises administering two or more of the one or more antiviral agents, and the two or more antiviral agents are chosen from the group consisting of rilpivine, abacavir, and lamivudine; abacavir, dolutegravir, and lamivudine; abacavir, lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine, and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumerate; emtricitabine, rilpivirine, and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; lopinavir, retinovir and interferon-beta lb; and lopinavir and ritonavir. [00141] 13. The method according to any one more of embodiments 1-12, wherein said viral infection is from a virus chosen from the group consisting of influenza A, SARS-CoV-2, a pathological strain of SARS-CoV-2, 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV, SARS-CoV, molluscum contagiosum virus, HTLV, HTLV-1, hepatitis-A, HCV, HBV, HIV/AIDS, human papilloma virus, herpes virus, herpes simplex virus, a virus that causes viral dysentery, measles virus, rubella virus, mumps virus, polio virus, rabies virus, Epstein-Barr virus, ebola virus, respiratory syncytial virus, dengue virus, yellow fever virus, lassa virus, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, a virus causing viral meningitis, West Nile virus, arbovirus, a parainfluenza virus, smallpox virus, dengue virus, cytomegalovirus, infant cytomegalic virus, JC virus, a virus that causes viral gastroenteritis, a hepatitis virus, a virus that causes encephalitis, varicella zoster virus, viruses that cause California serogroup viral encephalitis, St. Louis encephalitis virus, rift valley fever virus, a virus that causes hand, foot, & mouth disease, hendra virus, an enterovirus, astrovirus, an adenovirus, Japanese encephalitis virus, lymphocytic choriomeningitis mammarenavirus, human herpesvirus 6, human herpesvirus 7, a virus that causes sandfly fever, a papilloma virus, a human papilloma virus, cat scratch disease, parvovirus B19, a parapox virus that causes orf, a virus that causes pityriasis rosea, and lyssavirus.

[00142] 14. The method according to embodiment 13, wherein the viral infection is by influenza A.

[00143] 15. The method according to embodiment 13, wherein the viral infection is by SARS-CoV-2 or a pathological strain thereof. [00144] 16. The method according to any one more of embodiments 1-15, wherein one or both of the first pharmaceutical composition or the second pharmaceutical composition further comprise one or more pharmaceutically acceptable excipients, optionally where the excipients are charged aqueous species that have a net negative charge or a net positive charge.

[00145] 17. The method according to any one more of embodiments 1-16 further comprising administration of a therapeutically effective amount of at least one compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin, and riboflavin.

[00146] 18. The method according to any one more of embodiments 1-17 further comprising administration of a therapeutically effective amount of at least one compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins.

[00147] 19. The method according to any one more of embodiments 1-18 further comprising administration of a therapeutically effective amount of at least one immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE.

[00148] 20. The method according to any one more of embodiments 1-19 further comprising the administration of an antibacterial agent.

[00149] 21. The method according to embodiment 20, wherein the antibacterial agent is Azithromycin.

[00150] 22. The method according to any one more of embodiments 1-21 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof are in immediate release form. [00151] 23. The method according to embodiment 22, wherein said immediate release pharmaceutical composition is for administration once in a 24 hour period, optionally within 60 minutes.

[00152] 24. The method according to embodiment 22 or 23 wherein said immediate release composition releases the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof completely within about 60 minutes.

[00153] 25. The method according to any one more of embodiments 1-24 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the amount of the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof is between about 1.0 mg and about 300.0 mg, 4.0, 5.0, between about 0.05 mg and about 6.0 mg, or between about 0.05 mg and about 4.5 mg. The amount may be in a range between and including any two amounts selected from 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, and 300 mg.

[00154] 26. The method according to any one more of embodiments 1-25, wherein the administrations for one or both of the first pharmaceutical composition or the second pharmaceutical composition is via a route chosen from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration.

[00155] 27. The method according to any one more of embodiments 1-26, wherein one or both of the first pharmaceutical composition or the second pharmaceutical composition are form of a capsule, a tablet, or a rapidly dissolving film.

[00156] 28. The method according to any one more of embodiments 1-27 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof comprises a mixture of (+) naltrexone and (-) naltrexone in ratio between about 1:99 and about 99:1, optionally 50:50. The ratio may be selected from 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 9:91, 8:92, 7:93, 6:94, 5:95, 4:96, 3:97, 2:98, and 1:99.

[00157] 29. The method according to any one more of embodiments 1-28 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof comprises a mixture of (+) naloxone and (-) naloxone in a ratio between about 1:99 and about 99:1, optionally 50:50. The ratio may be selected from 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 9:91, 8:92, 7:93, 6:94, 5:95, 4:96, 3:97, 2:98, and 1:99.

[00158] 30. The method according to any one of embodiments 1-29, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof comprises a mixture of (+) naltrexol and (-) naltrexol in a ratio between about 1:99 and about 99:1, preferably about 50:50. The ratio may be selected from 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 9:91, 8:92, 7:93, 6:94, 5:95, 4:96, 3:97, 2:98, and 1:99. [00159] 31. A pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof.

[00160] 32. The pharmaceutical composition of embodiment 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent is chosen from the group consisting of an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal) or combination thereof.

[00161] 33. The pharmaceutical composition of one or both of embodiments

31 or 32 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises at least one chosen from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof.

[00162] 34. The pharmaceutical composition of any one or more of embodiments 31-33 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises remdesivir.

[00163] 35. The pharmaceutical composition of any one or more of embodiments 31-34 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises favipiravir.

[00164] 36. The pharmaceutical composition of any one or more of embodiments 31-35 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises one chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine, tocilizumab, EIDD-280, and etravirine.

[00165] 37. The pharmaceutical composition of any one or more of embodiments 31-36 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises a combination of antiviral agents chosen from the group consisting of rilpivine, abacavir, and lamivudine; abacavir, dolutegravir, and lamivudine; abacavir, lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine, and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumerate; emtricitabine, rilpivirine, and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; lopinavir, retinovir and interferon-beta lb; and lopinavir and ritonavir. [00166] 38. The pharmaceutical composition of any one or more of embodiments 31-37 further comprising at least one compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin, and riboflavin.

[00167] 39. The pharmaceutical composition of any one or more of embodiments 31-38 further comprising at least one compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins.

[00168] 40. The pharmaceutical composition of any one or more of embodiments 31-39 further comprising at least one immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE.

[00169] 41. The pharmaceutical composition of any one or more of embodiments 31-40 further comprising an antibacterial agent. [00170] 42. The pharmaceutical composition of any one or more of embodiments 31-41, wherein the antibacterial agent is Azithromycin.

[00171] 43. A dosage form comprising the pharmaceutical composition of any one of claims 31-42.

[00172] 44. The dosage form of embodiment 43, wherein the dosage form is an immediate release dosage form.

[00173] 45. The dosage form of one or both of embodiments 43 and 44, wherein the dosage form is one of a capsule, a tablet, or a dissolving film.

[00174] 46. The dosage form of any one or more of embodiments 43-45, wherein the dosage form is for an administration route chosen from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration.

[00175] 47. The dosage form of any one or more of embodiments 43-46, wherein the dosage from comprises the concentration of the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and at least one of (1) one or more antiviral agents or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof suitable for a single day dose.

[00176] 48. A use of the pharmaceutical composition of any one of claim 31-

42 or the dosage form of any one of claims 43-47.

[00177] 49. The use of embodiment 48, wherein the use is for addressing a viral infection.

[00178] 50. The use of embodiment 49, wherein the addressing is one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient.

[00179] 51. The use of embodiment 50, wherein the virus is SARS-CoV-2 or

Influenza A virus.

[00180] 52. The use of any one or more of embodiments 49-51, wherein the addressing comprises the method of any one or more of embodiments 1-30. [00181] 53. The use of embodiment 48, wherein the use is preparing a medicament for addressing viral infection.

[00182] 54. The use of embodiment 53, wherein the addressing is one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient.

[00183] 55. The use of embodiment 54, wherein the virus is SARS-CoV-2 or

Influenza A virus.

[00184] 56. The use of any one or more of embodiments 53-55, wherein the medicament is for administering by the method of any one or more of embodiments 1-30.

[00185] 57. A method comprising administering to a patient the pharmaceutical composition of any one or more of embodiments 31-42 or the dosage form of any one or more of embodiments 43-47.

[00186] 58. The method of embodiment 57 comprising the method of any one or more of embodiments 1-30.

[00187] Examples

[00188] The following represents non-limiting examples. Embodiments herein may include one or more element from one or more example below.

[00189] MENK, as an immune adjuvant, has potential immune-regulatory activity on innate and adaptive immune cells. The inventors investigated the antiviral effect of MENK on influenza virus-infected murine macrophage cells (RAW264.7) and its underlying mechanisms. The results showed that MENK markedly inhibited influenza A virus (H1N1) replication in pre- and post-MENK treatment, especially in pre-MENK treatment. The mechanism exploration revealed that MENK (10 mg/mL) significantly inhibited the nucleoprotein (NP) of influenza virus and up-regulated levels of IL-6, TNF-a and IFN-b compared with those in H1N1 control group. Further experiments confirmed that antiviral effects of MENK were associated with promotion of opioid receptor (MOR) as well as activation of NF-KB p65 inducing cellular antiviral status. These data suggest that MENK should be potential candidate for prophylactic or therapeutic treatment against H1N1 influenza virus.

[00190] 1. Introduction

[00191] Influenza A virus is the most serious influenza type with high morbidity and mortality widespread, resulting in a mild to moderately severe respiratory disease, and even systemic complications, and be- come the first infectious disease with global disease surveillance [1-3]. In addition, mutant and re-emerging influenza strains might increase pathogenicity of the virus through altering viral receptor-binding specificity [4] and increasing viral polymerase activity [5], escape from immunity induced by prior infection and vaccination, and potentially develop into a rising global threat [6,7] Vaccines represent the most effective methods to prevent and control influenza virus infection, targeting antigenic drift in influenza virus HA protein [8] However, vaccines have drawbacks, including inadequate protection, high cost, difficulty in predicting representative strains, and time requirements for design and production [9-12] Therefore, novel anti-influenza strategies with high efficacy and low side effects are urgently in demand to prevent and control influenza epidemics.

[00192] As seen in the unfolding COVID-19 pandemic, similar difficulties present in combatting SARS-CoV-2.

[00193] Macrophages are antigen-presenting cells (APCs) and are known as innate immunity factor of defense against and eliminate infectious viruses. In the early stages of infection, rapid innate immune cells are effective in controlling respiratory epithelial cells infected with influenza virus and viral replication [13, 14] Macrophages, as important specialized phagocytic cells of the innate immune system, express pathogen-recognition receptors (PRRs), with the capacity of lysosomal degradation, presenting antigens and secreting antiviral cytokines and chemokines. Toll like receptors (TLRs) as a kind of PRRs, activate cellular signaling pathways that induce nuclear factor KB (NF-KB) transduction, with the function of regulating inflammation involving macrophage capacity [17,18] During influenza virus infection, macrophages produce proinflammatory cytokines and antiviral agent, in- duce acute-phase inflammation, enhance recruitment, activate other immune cells, and finally control early virus replication through establishing antiviral immunity [19,20]

[00194] MENK, an endogenous opioid peptide, composed of Tyr-Gly-Gly- Phe- Met, has potential to regulate both endocrine and immune systems via binding to opioid receptors (m,d,k) [21] MENK could trigger the second messengers Ca²⁺ and cAMP to modulate the phagocytic and boost pathogen elimination [22] Previous studies of in an inventor laboratory showed that MENK enhanced the production of cytokines, such as IL-1, IL-6, and exerted bidirectional modulation of cytotoxic activities by macrophage [23,24] Recently, published results indicated that MENK increased the level of macrophage surface marker CD64 and the ex pression of inflammatory cytokines TNF-a to exert an antitumor activity [25] Inventors demonstrated that MENK had anti-influenza virus activity by inhibiting inflammatory responses via binding to opioid receptors in vivo [26] However, the function of MENK in controlling influenza virus replication and related immunological regulation in macrophages remains obscured. Therefore, the following work was conducted with the intention to find related clues.

[00195] 2. Materials and methods

[00196] 2.1. Virus and cell culture

[00197] The influenza strain A/PR/8/34 (H1N1; PR8) was kindly provided by China Center for Disease Control and Prevention (Beijing, China). The virus was amplified in the allantoic cavities of lOd-old embryo chicken eggs [27] According to Reed and Muench methods, 50% tissue culture infective dose (TCID50) as a standardized indicator for viral titers was calculated by Hemagglutination test (HA). Murine macrophage cell line (RAW264.7) was purchased from the Cell Resource of Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 pg/mL streptomycin under a humidified atmosphere containing 5% C02 at 37 °C. When RAW264.7 cells were infected with influenza virus, the maintenance medium was changed to serum-free DMEM supplemented with 2 pg/mL TPCK-trypsin (Sigma, USA).

[00198] 2.2. Reagents [00199] MENK (>99% purity) was provided by America peptide Inc. RNeasy mini kit (74104) was purchased from Qiagen. One Step SYBR® Prime Script™ RT-PCR Kit (RR066A) was purchased from TaKaRa. The mAbs of Influenza A Virus Nucleoprotein (ab20343), NF- KB (ab 16502), TLR4 (ab22048) and MOR (abl0275) were purchased from Abeam. DyLight®488 IgG (H + L) was purchased from Earthox.

[00200] 2.3. Cell infection and treatment with MENK

[00201] The experiment was assigned to four groups: normal control group (Normal-C, cells were not infected and not treated with MENK), influenza virus infected control group (H1N1-C, cells were infected with H1N1 only), pre treatment of MENK group (pre-MENK, cells were treated with MENK 24 h prior to H1N1 infection), and post-treatment of MENK group (post-MENK, cells were treated with MENK 1 h post (infection) as shown in FIG. 1.

[00202] 2.4. Cell viability assay

[00203] The effect of MENK on RAW264.7 cells proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS). RAW264.7 cells (1 x 104 cells/ well) were seeded in 96-well plates and treated with MENK (20, 10, 1, 10- 1~ 10-12 mg/mL). After 24, 48, and 72 h cultivated, MTS reagent was added to each well, and incubated for additional 4 h. The absorbance value (OD) at 490 nm was measured using a microplate reader (Bio- Rad, USA), and data were analyzed by GraphPad Prism software.

[00204] 2.5. Antiviral effects of MENK

[00205] Pre-MENK treatment: RAW264.7 cells (2 x 104 cells/well) were seeded in 96 well plates for 24 h at 37 °C with 5% C02 and treated with MENK (10, 1, 10-1 and 10-2 mg/mL). After 24 h, MENK were removed and the cells were washed twice with 1 x PBS, inoculated 100 TCID50 H1N1 onto cells for 1 h, washed away unattached viruses, then added 100 pL DMEM to each well.

[00206] Post-MENK treatment: Virus inoculation was the same way as pre- MENK treatment. The virus-infected cells were treated with MENK (10, 1, 10-1 and 10-2 mg/mL) for 24 h. At 24 , 48, and 72 h pi, OD values were measured by MTS method. Data were analyzed to find out the optimal concentration of MENK. [00207] 2.6. Observation of cell morphology [00208] The RAW264.7 cells were infected with 100 TCID50 H1N1 and treated with optimal concentration of MENK (10 mg/mL) at pre-treatment and post-treatment model for 24 h. After 24, 48, and 72 h pi, morphology changes were observed under conventional light microscopy. The nuclei morphology of virus- infected cells was stained by Hoechst 33258. Images were captured using a fluorescence microscope (Olympus, Japan).

[00209] 2.7. Membranal and intracellular molecules analysis

[00210] For membranal molecules staining, the cells were washed and incubated with anti-TLR4 (1:100, 22048, Abeam) antibody for 1 h at room temperature, added secondary antibody DyLight®488 IgG (H + L) for 30 min. The cells were washed in cold PBS (l x ) and resuspended in 300 pL cold PBS (IX) containing 2% fetal calf serum (FCS). For intracellular molecules staining, the cells were washed and added 100 pL Fixation/ Permeabilization solution (554714, BD Biosciences), incubated at 4°C for 20 min, washed in BD Perm/WashTM Buffer (IX), added anti-influenza NP (1:100, 20343, Abeam), anti-NF-kB p65(l:100, 16502, Abeam) antibody, then washed and added DyLight®488 IgG (H + L) for 30 min. The cells were washed in BD Perm/WashTM Buffer (IX), resuspended in 300 pL BD Perm/WashTM Buffer (IX) and acquired by FACS Calibur (BD Biosciences).

[00211] 2.8. RNA extraction and qPCR analysis

[00212] RNA was extracted from RAW264.7 cells according to the specification of RNeasy (74104, Qiagen). qPCR was performed using One Step SYBR Prime Script RT-PCR Kit (RR066A, TaKaRa) with QuantStudio 6 Flex Real-time PCR system (ABI, USA). qPCR reactions completed as follow: 5 min at 42°C and 10s at 95°C, followed by 40 cycles-3s at 95°C, 30s at 60°C- and a melting curve step. Primer sequences are detailed in Table 1. Gene expression was calculated using the 2-DDOT method.

Table 1 PCR Primer Sequences.

Primer Direction Sequence SEQ ID NO

Virus Forward 5'-GACCGATCCTGTCACCTCTGAC-3' 1

Reverse 5'-AGGGCATTCTGGACAAAGCGTCTA-3' 2 IL-6 Forward 5'- ATGAAGTTCCTCTCTGCAAG-3' 3

Reverse 5'- GTGTAATTAAGCCTCCGACT-3' 4 TNF-a Forward 5'- CCAAAGGGATGAGAAGTTCC-3' 5

Reverse 5'- CTCCACTTGGTG GTTTGCTA-3' 6 IFN-b Forward 5'- AGCTCCAAGAAAGGACGAACAT-3' 7

Reverse 5'- GCCCTGTAGGTGAGGTTGATCT-3' 8 MOR Forward 5'-CCCAGTTCTTTATGCGTTCCT-3' 9

Reverse 5'-ATTAGCCGTGGAGGGGTGT-3' 10 TLR7 Forward 5'- GGTGGCAAAATTGGAAGATCC-3' 11

Reverse 5'- AGCTGTATGCTCTGGGAAAGGTT-3' 12 TLR4 Forward 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' 13

Reverse 5'-ACATTCGAGGCTCCAGTGAATTCGG-3' 14 NF-KB p65 Forward 5'-ATGTGCATCGGCAAGTGG-3' 15

Reverse 5'- C AGAAGTT GAGTTT C GGGT AG- 3' 16 GAPDH Forward 5'-ACCACCATGGAGAAGGCTGG-3' 17

Reverse 5'-CTCAGTGTAGCCCAGGATGC-3' 18

[00213] 2.9. ELISA assay

[00214] The cell culture supernatants were harvested at 24, 48, 72 h pi and stored at -20°C until analysis. The concentrations of IL-6, TNF-a and IFN-b were determined using BD ELISA kit (BD Biosciences) according to instructions included in ELISA kit.

[00215] 2.10. Immunofluorescence

[00216] The steps of RAW264.7 cells infected with 100TCID50 were as above.

The cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 (except MOR dection) and blocked with 2% BSA for 30 min at room temperature, incubated with anti-influenza NP (1:100, 20343, Abeam), anti-TLR4 (1:100, 22048, Abeam), anti-NF-kB p65 (1:100, 16502, Abeam), anti-MOR (1:100, ab 10275) antibody at 4°C overnight, followed by incubated for 1 h at room temperature with secondary antibody DyLight®488 IgG (H + L). Finally, added 25 pL DAPI-containing anti-fluorescence quencher to stain the cell nuclei. Images were acquired with a fluorescence microscope (Olympus, Japan).

[00217] 2.11. Statistical analyses [00218] All data were present as mean ± SEM and evaluated by one-way analysis of variance (ANOVA) or Student’s t-test. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, USA). Values of -*-p< 0.05 or ★★p<0.01 were considered statistically significant.

[00219] 3. Results

[00220] 3.1. Influenza virus titer and TCID50

[00221] The virus titer increased from 1:32 to 1:512 by chicken embryo culture (FIG. 2A). To determine the appropriate infection dose for application in cell experiments, the TCID50 was determined. The results showed that 100TCID50 was 1: 28.2 (Fig. 2B), equivalent to diluting the influenza A virus (H1N1) stock by 28.2 times to infect RAW264.7 cells.

[00222] 3.2. The effect of MENK on cell viability and antiviral ability in

RAW264.7 cells

[00223] To find an optimal concentration for subsequent experiment, the effects of MENK on cell viability and antiviral ability in RAW264.7 cells infected with influenza A virus (H1N1) were detected. The concentrations of MENK ranged from 20~10-7 mg/mL promoted cell proliferation, and 10, 1, 10-1, and 10-2 mg/mL were significant obviously in a dose-dependent manner (p < 0.01 or p < 0.05) (Fig. 3A). Based on the results, 10, 1, 10-1 and 10-2 mg/mL MENK were taken to investigate the optimal concentration of MENK against H1N1. As shown in FIG. 3B, pre-MENK and post-MENK treatment (10 and 1 mg/mL) statistically increased the proliferation rates of RAW264.7 cells at 24 h, 48 h, and 72 h pi compared with that in the H1N1-C group (p < 0.01 or p < 0.05), and 10 mg/mL MENK upregulated the higher proportion of cell proliferation than other concentration. Based on these results, the experiments were performed with MENK at optimal concentration of 10 mg/mL.

[00224] 3.3. MENK affected the morphological changes of RAW264.7 cells

[00225] The microscope images showed that RAW264.7 cells in the Normal- C group exhibited an initial state, mostly round and translucent. With lasting infection, RAW 264.7 cells displayed remarkable changes in cell morphology, typically produced longer protruding pseudopodia, changed to irregular polymorphism, and appeared to state of aging and death with vacuoles and granular substances in the cytoplasm. The cells in the pre-MENK group and post- MENK group had a long fusiform shape, and with many pseudopods, and occasional vacuoles and particulate substances appeared. See FIG. 4A.

[00226] Hoechst 33,258 staining showed that the nuclei of the Normal-C group were dark blue, while the H1N1-C group had some apoptotic cells with densely or fragmented stained nucleus. In contrast, the nucleus in pre-MENK group and post-MENK group showed sporadic hyperchromatism, and small amount of fragmented dense in FIG. 4B and FIG. 4C. These results indicated that MENK inhibited the apoptosis of macrophages caused by influenza virus.

[00227] 3.4. MENK inhibited influenza virus replication

[00228] The nucleoprotein (NP) of influenza virus encapsulated the negative strand of the viral RNA and was essential for replicative transcription. The relative viral amplification was markedly higher (112,760-fold) at 48 hpi in H1N1- C group, while 38,972-fold in pre-MENK group, and 60,534-fold in post-MENK group compared with that in Normal-C group (FIG. 5A).

[00229] The flow cytometry results showed that the expression of Influenza NP significantly increased in RAW264.7 cells infected with influenza virus, and the mean fluorescence intensity (MFI) decreased with last- ingness of infection. The difference between H1N1-C group and Normal-C group was significant at 24, 48, 72 h pi (p < 0.01). In comparison to the H1N1-C group, the level of Influenza NP decreased at each time point in pre-MENK group and post-MENK group (p < 0.01 or p < 0.05) as shown in FIGS. 5B and C.

[00230] The expression of influenza NP protein was localized by immuno fluorescence staining and the result showed that cells did not present NP protein in Normal-C group, but at a high level in RAW264.7 cells infected virus at 48 h. MENK treatment down-regulated the expression of influenza NP in pre-MENK group and post-MENK group (Fig. 5D). These findings illustrated that both pre- and post-administration of MENK could reduce influenza virus replication on RAW264.7 cells effectively, and pre-treatment was more effective.

[00231] 3.5. MENK promoted productions of inflammatory cytokines

[00232] As shown in FIG. 6A, the levels of inflammatory cytokines at mRNA levels increased on 24, 48, and 72 h post-infection with HlNl(IL-6, 4.67-fold/3.42- fold/ 2.71-fold; TNF-a, 6.02-fold/ 4.60-fold/ 3.25-fold; and IFN-b, 6.58-fold/ 4.65- fold/ 2.51-fold) (p < 0.01). In comparison, to H1N1-C group, the pre-treatment of MENK increased transcription of the above cytokines (IL-6, 8.02-fold/ 10.79-fold/ 5.43-fold; TNF-a, 12.45-fold/ 17.23-fold/ 9.98-fold; and IFN-B, 11.37- fold/ 15.54- fold/ 7.08-fold) (p < 0.01). Similarly, the post-treatment of MENK upregulated cytokines production (IL-6, 5.88-fold/ 7.79-fold/ 5.16-fold; TNF-a, 10.90-fold/ 14.07- fold/ 7.83-fold; and IFN-b, 9.67-fold/ 11.05- fold/ 5.64-fold) (p < 0.01 or p < 0.05). [00233] To further confirm changes of cytokines, the levels of IL-6, TNF-a and IFN-b in the cell supernatant were detected by ELISA. As shown in FIG. 6B, the secretions of IL-6, TNF-a and IFN-b in H1N1-C group were significantly higher than that in the Normal-C group on 24, 48, and 72 h pi (p < 0.01). Compared with H1N1-C group, pre-treatment of MENK significantly enhanced the level of IL-6, TNF-a, and IFN-b in the cell supernatant at 24, 48, and 72 h pi (p < 0.01). Those cytokines in post- MENK group to some extent had the same tendency as pre- MENK group (p < 0.01 or p < 0.05). All data suggested that MENK mediated the antiviral effects in RAW264.7 cells by regulating the pro-inflammatory cytokines (IL-6, TNF-a) and Type I IFN (IFN-B).

[00234] 3.6. MENK upregulated MOR expression

[00235] To correlate the observation above and assess the mechanisms basis for the effect of MENK, the mRNA expression of MOR in RAW264.7 cells was measured. There was no significant change of MOR between the Normal-C group and H1N1-C group. However, the expression of MOR obviously increased in pre- MENK group (4.10-fold) and post- MENK group (3.65-fold) as shown in FIG. 7A. [00236] Immunofluorescence staining showed opioid receptors were located on cell membrane. There were no significant changes of the fluorescence intensity in cells infected with H1N1, while the intensity re- markly enhanced in pre-MENK group and post-MENK group (FIG. 7B). These results further indicated that MENK exerted antiviral function by upregulating receptors as part of the mechanism of H INI therapy.

[00237] 3.7. MENK upregulated the level of TLR4 and NF-KB p65

[00238] There were no significant changes in the expression of TLR7 mRNA in four groups of RAW264.7 cells (p>0.05), but TLR4 mRNA changed to varying degrees. Thus, we detected the expressions of PRRs-related factors (TLR4 and NF- KB p65) by qPCR, FCM and immunofluorescence staining. The qPCR results showed that the mRNA levels of TLR4 were up-regulated in pre-MENK group (3.68-fold) and post-MENK group (3.31-fold) (p < 0.01). However, there was no significant difference in TLR4 mRNA between Normal-C group and H1N1-C group (p>0.05). Compare with H1N1-C group, the expressions of NF-KB p65 mRNA in creased in pre-MENK group (5.73-fold) (p < 0.01) and post-MENK group(4.69-fold) (p < 0.05) (FIG. 8A).

[00239] FCM analysis further confirmed changes of TLR4/NF-KB p65 in RAW264.7 cells infected with influenza virus and treated with MENK. As shown in FIGS. 8B and C, pre-MENK and post-MENK treatment significantly increased TLR4 and NF -KB p65 expressions at 24, 48, 72 h pi compared to those on untreated cells (p < 0.05 or p < 0.01).

[00240] However, there was no significant difference of levels of TLR4 between Normal-C group and H1N1-C group (p>0.05) As shown in FIG. 8D, the fluorescence microscopy images showed the subcellular localization of TLR4/NF- KB p65 in macrophages under different treatment conditions, expressed on cell membrane and nucleus respectively. The fluorescence intensity of TLR4 showed no significant change after cell infection, while pre- and post-MENK administration significantly enhanced the TLR4 expression. A small amount of NF-KB p65 expressed in Normal-C group, and significantly increased after cells infected. The expression of NF-KB p65 in pre-MENK group and post-MENK group were higher than that in H1N1-C group (p < 0.05 or p < 0.01). The results indicated that MENK could mediate the antiviral effects in RAW264.7 cells by modulating the key effectors of TLR4 and NF-KB p65 expressions, related to PRRs signaling pathway.

[00241] 3.8 Evaluation of Activity of Coronavirus in MRC5 Cells

[00242] MRC-5 cells (ATCC, CCL-171) were added to a 96-well flat-bottomed plate (Costar, 3595) at a density of 5 x 10³ cells/well in a volume of 100 mL in 10% complete DMEM [( DMEM (Lonza, 12-917F), 10% FBS (Gibco, 16140-089), 1% L- glutamine (Lonza, 17-605E), 100 U/mL penicillin and 100 mg/mL streptomycin (Lonza, 17-602E), 1% sodium pyruvate (Corning, 25-000-Cl) and 1% MEM non- essential amino acids (Corning, 25-025-Cl)]. Following a 24 hour incubation at 37°C/5% CO2 the media was removed and six concentrations of each compound, serially diluted half-logarithmically in assay media - 2% complete DMEM [(DMEM (Lonza, 12-917F), 2% FBS (Gibco, 16140-089), 1% L-glutamine (Lonza, 17-605E), and 100 U/mL penicillin and 100 mg/mL streptomycin (Lonza, 17-602E)] was added to triplicate wells in a volume of 100 mL. Coronavirus strain 229E (ATCC, VR-740) at a pre-determined titer was added to the experimental wells and virus controls in a volume of 100 mL. The cultures were incubated for 6 days at 37°C/5% CO2 at which time the cells were stained with a tetrazolium dye, XTT (2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide), solution. The XTT solution was prepared as a stock of 1 mg/mL in DMEM without additives. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS and stored in the dark at -20°C. The XTT/PMS solution was prepared immediately before use by adding 40 pL of PMS per mL of XTT solution. Fifty pL (50 mL) of XTT/PMS was added to each well of the plate and the plate incubated for 4 hours at 37°C. Following the incubation, the plates were sealed, and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 384 96 well plate format spectrophotometer. Microsoft Excel 2010 in combination with XLfit4 was used to analyze and graph the data. The EC25, EC50, and EC95 (25%, 50%, and 95% inhibition of virus replication), TC25, TC50, and TC95 (25%, 50%, and 95% reduction in cell viability) and a therapeutic index (TI, TC25/EC25, TC50/EC50, TC95/EC95) are provided. Raw data for both antiviral activity and toxicity with a graphic representation of the data are provided in a plate report summarizing the compound activity. Toxicity of the test compounds to target cells (compound plus cells in the absence of virus addition) at each concentration was evaluated in parallel. Remdesivir was evaluated in parallel as a positive control drug.

[00243] 3.9 Evaluation of Coronavirus Strain 229E Inhibition in a Cytopathic

Effect Assay in MRC5 Cells. MRC-5 cells were incubated at 37°C 15% CO2 with serially diluted compound prior to the addition of Cornavirus strain 229E. The cultures were then incubated for 6 days at 37°C/5% CO2. Following incubation, the cells were stained with the tetrazolium dye XTT and read at 450/650 nm on a spectrophotometer to evaluate cellular viability. Efficacy and toxicity values were calculated using linear regression analysis. The results are summarized in Table 2, below.

Table 2

Evaluation of Coronavirus Strain 229E Inhibition in a Cytopathic Effect Assay in

MRC5 Cells

[00244] The data for each compound is presented in the below tables. In addition, FIG. 9 illustrates in vitro antiviral results for Naltrexone-HCl, FIG. 10 illustrates in vitro antiviral results for Remdesivir, FIG. 11 illustrates in vitro antiviral results for Naloxone-HCl, FIG. 12 illustrates in vitro antiviral results for 6-B-Naltresonxe Hydrate, and FIG. 13 illustrates in vitro antiviral results for Remdesivir.

[00245] In Vitro Antiviral Results For Naltrexone-HCL. See FIG. 9. [00246] Assay: CoV Cytoprotection (XTT)

Concentration

Naltrexone-HCL mM

3.16

10

31.6

100

316

1000

[00247] Raw Data Naltrexone-HCL (pM)

Virus: CoV Strain: 229E Cells: MRC5

Reagent: 0.175

Virus Control: 0.007 Cell Control: 1.042 Differential: 1.049

[00248] In Vitro Antiviral Results For Remdesivir. See FIG. 10. [00249] Assay: CoV Cytoprotection (XTT)

Concentration

Remdesivir (mM) 0.003 0.01 0.03 0.1 0.32 1

[00250] Raw Data: Remdesivir (mM)

Virus: CoV Strain: 229E Cells: MRC5

Reagent: 0.175 Virus Control: -0.007 Cell Control: 1.042 Differential: 1.049

[00251] In Vitro Antiviral Results For Naloxone - HCL. See FIG. 11. [00252] Assay: CoV Cytoprotection (XTT)

Concentration Naloxone - HCL (mM)

3.16

10

31.6 100

316

1000

[00253] Raw Data: Naloxone - HCL (mM)

Virus: CoV Strain: 229E x=1.5

Cells: MRC5

Reagent: 0.156 Virus Control: 0.072 Cell Control: 1.502

Differential: 1.430

[00254] In Vitro Antiviral Results For 6-B-Naltrexone Hydrate. See FIG. 12. [00255] Assay: CoV Cytoprotection (XTT)

Concentration 6-B-Naltrexone Hydrate (mM) 3.16 10 31.6 100 316 1000

[00256] Raw Data 6-B-Naltrexone Hydrate (mM)

Virus: CoV Strain: 229E x=1.5 Cells: MRC5

[00257] In Vitro Antiviral Results For Remdesivir. See FIG. 13. [00258] Assay: CoV Cytoprotection (XTT)

Concentration Remdesivir (mM) 0.003 0.01 0.03 0.1 0.32 1

[00259] Raw Data: Remdesivir (mM)

Virus: CoV Strain: 229E x=1.5

Cells: MRC5

Reagent: 0.153 Virus Control: 0.079 Cell Control: 1.435 Differential: 1.356

[00260] 4. Discussion

[00261] Macrophages are an efficient phagocytic component of innate immune response to infection and play a critical role in the clearance of pathogenic molecules and apoptotic cells, then activate adaptive immune response [28-30] Several studies have shown that clodronate liposome-mediated depletion of macrophages led to virus multiply replication and systemic spread of virus, exacerbated the progress of disease [31,32] MENK, as an immune regulating factor, can regulate NK cells, DC, macrophages, CD4+T cells, and CD8+T cells via binding to opioid receptor expression on immune cells [33-38]

[00262] The data herein demonstrate that the optimal concentration of MENK treatment increased the proliferation rate of RAW264.7 cells infected with virus. Moreover, pre- and post-treatment of MENK decreased influenza virus replication in RAW264.7 cells, and pre-treatment was more effective. See FIG. 14. After influenza virus infection, the macrophages rapidly produced type I IFN, cytokines and chemokines, which would regulate inflammatory response and immune response. TNF-a, a major pro-inflammatory cytokine, is produced by macrophages infected with influenza, could amplify production of other pro- inflammatory cytokines and chemokines, and accelerate the recruitment of neutrophils and monocytes to the site of infection [39] IL-6 seems to play protective role in the model of IAV infection, and accelerates viral clearance and limit inflammatory response [40], and blocking IL-6 might induce in- adequate for inflammation control. Type I IFN (IFN-a/b) is expressed by virtually all cells, and is known as important mediator of virus elimination and activated antiviral state for protecting against acute influenza virus infection [41] Results herein showed that MENK administration significantly enhanced the level of IL-6, TNF-a and IFN-b, and suggested that MENK upregulated the non-specific immune response and enhanced the level of inflammatory cytokines to accelerate viral clearance in RAW264.7 cells infected with H1N1, which may be one aspect of its antiviral efficacy through reverse the immunophenotype of macrophages. Previously, inventors reported that MENK exerted an antitumor activity by inducing tumor- associated macrophages (TAMs) polarization from M2 to Ml type [25,42]

[00263] Previous experiments confirmed that MENK showed no direct killing effect on influenza virus. When MENK was co-cultured with virus for 24 h, 48 h, and 72 h, there was no change in virus titer. So, the consideration was what factors make MENK regulate RAW264.7 cells against influenza virus infection. Inventor data demonstrated that the MOR expressed on cell membrane was upregulated by MENK, while it did not change in H1N1-C group. This indicated that MENK played antiviral effects via binding to MOR. Inventors have reported that knockdown opioid receptor significantly cancelled anti-tumor function of MENK [25] Therefore, data herein furthered the understanding that MENKs antiviral effects were achieved by binding to opioid receptors. Inventors also found the degree of upregulation of receptors and inflammatory cytokines in pre-MENK administration was higher than post MENK. This suggested that MENK in prophylactic administration, up-regulated the status of macrophages before infection. Once the virus invaded, macrophages rapidly exerted immunobiological effects to recognize, phagocytose and eliminate virus, which may be the main reason for better preventive effect than that of therapeutic administration. Previous experiments of MENK inhibiting influenza virus infection in vivo by inventors also supported this view [26] Furthermore, to explore the underlying antiviral mechanisms of MENK, we followed the search in pattern-recognition receptors (PRRs)-related factors of TLR and NF-KB, and p65. TLRs are important receptors of PRRs, and have emerged as key sensors of innate immunity to viruses and highly expressed on immune cells. TLR7 is the major PRRs for the recognition of ssRNA viruses [43,44], but inventor findings showed that there was no regulatory effects on TLR7 by MENK, and an interesting finding was that MENK could significantly upregulate the expression of TLR4 in RAW264.7 cells. However, inventors previously reported that activation of the TLR7 pathway by influenza virus infection was inhibited by MENK treatment in mice. The reason may be that MENK performed different functions in vivo and in vitro. In vivo, MENK bound to opioid receptors on the surface of various immune cells and stromal cells, such as DC, macrophages, NK, T cells, epithelial cells, endothelial cells, etc. These various cells exerted multiple effects to eliminate infection. Therefore, the TLR7 pathway and downstream cytokines induced by influenza virus were not excessively activated. In vitro, MENK acted as an immunomodulator and had a positive immunoregulatory function, which promoted the conversion of macrophage types into classical Ml type with proinflammatory activity, upregulated the TLR4 pathway and induced the release of inflammatory cytokines. The previous findings further confirmed this point. MENK triggered activation of BMDCs via upregulating TLR4 through MyD88/NF-KB signaling pathway in vitro [45] According to documented reports, activation of TLR4-signaling during influenza infection induced an exaggerated inflammatory response in vivo [46-48] Inventor results indicated that MENK treatment significantly increased TLR4 and NF-KB p65 expression in RAW264.7 cells infected with influenza virus, while there was no significant difference in level of TLR4 between Normal-C group and H1N1-C group. Therefore, inventor data indicated that MENK maybe activate TLR4-NF-KB p65 signaling, increased inflammatory cytokines and typellFNs, induced celluar antiviral state. An inventor team recently found MENK regulated cytokines production by downregulating TLR-MyD88-TRAF6-NF-KB p65 signaling pathway as a treatment for type 2 diabetes mellitus (T2DM) [49] Through comprehensive analysis, MENK tended to inhibit the TLR pathway in vivo, to reduce the expression of inflammatory factors and to upregulate TLR pathway and its downstream cytokines, then stimulate innate immune cells to exert anti-effects in vitro. However, the detailed mechanisms for this phenomenon need further study in depth. Taken together, these studies indicate that MENK up -regulates macrophage opioid receptor, activates macrophages and positively regulates macrophage function to augment immune inflammatory response inducing celluar antiviral state, resulted in inhibition of influenza virus invasion and intracellular replication. Thus, the innate cellular immune regulation of MENK can provides inspired the prevention and treatment of influenza virus. Moreover, MENK acts on innate immune cells by binding to opioid receptors, rather than the virus itself, supporting its use as an adjuvant of influenza vaccine. Therefore, inventor results illustrate that MENK is unrpedictably effective as a nonspecific agent or vaccine adjuvant in preparation of prophylactic or therapeutic vaccines.

[00264] Prophetic Examples

[00265] Tables below show prophetic data.

[00266] 1. Validating a method of addressing SARS-CoV-2 infection, in vitro.

[00267] In the case of SARS-CoV-2, a novel coronavirus, the following method could be used. Vero cells stably expressing human TMPRSS2 would be generated by retroviral transduction and blasticidin-based selection. All media would be supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech), lx non-essential amino acid solution (lOx stock, PAA) and 10 mM sodium pyruvate (ThermoFisher Scientific). For seeding and sub-cultivation, cells would be first washed with phosphate buffered saline (PBS) and then incubated in the presence of trypsin/EDTA solution (PAN- Biotech) until cells detached. [00268] Primary lung tissue samples would be obtained and for the preparation of human airway epithelial cells, bronchus tissue would be derived from human donors. Primary human airway epithelial cells would be harvested from the mucosal surface using a scalpel and resuspended in growth medium. After incubation at 37°C, 5% CO2 for 2 h to remove adherent fibroblast cells, non adherent cells would be seeded on a collagen I coated flask and maintained at 37°C, 5% CO2 until used as infection target cells.

[00269] Target cell cultures (Vero cells, above) would be transfected with ACE2 and others with an irrelevant receptor ( e.g ., DsRed) as a negative control. Transfection would be carried out by calcium-phosphate precipitation. Primary cells would be used after preparation as described above. Target cells would be washed with PBS and supernatant inoculated with a fixed amount {e.g., 8xl0⁷genome equivalents) of a known SARS-CoV-2 isolate for a fixed amount of time {e.g., 1 h). After infection, cells would be washed and after a culture period (eg 24 h), a fixed amount of culture supernatant would be subjected to viral RNA extraction using a viral RNA kit {e.g., Macherey-Nagel) according to the manufacturer’s instructions. Virus genome templates would be detected by real time RT-PCR using a standard SARS-CoV-2 specific amplification and detection protocol.

[00270] Validation would be assessed by comparing the relative quantity of virus genome templates that are present as measured by RT-PCR in cultures that were treated with the API(s) in PBS solution or mock treated with PBS solution alone. A positive validation would be exemplified by prophetic results similar to those shown below, which illustrate that treatment with the API but not control could prevent increasing infection of cells by virus over time. The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00271] 2. Validating a method of addressing a viral infection, in vitro.

[00272] In the case of viruses that are not SARS-CoV-2, commercially available kits would be generally available and could be employed as directed by the manufacturer.

[00273] Validation could be assessed by comparing the relative quantity of virus genome templates that are present as measured by RT-PCR in cultures that were treated with the API(s) in PBS solution or mock treated with PBS solution alone. A positive validation could be exemplified by prophetic results as shown above, which illustrate that treatment with the API but not control prevents increasing infection of target cells by virus over time. The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00274] 3. Validating a method of addressing a viral infection, in vivo: small animal models

[00275] An ever-expanding variety of small animal models continue to be developed and could be used to validate a given API, however, the Syrian Hamster model has an especially diverse range in terms of viral strains (Miao et al. 2019). The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00276] Male and female Syrian hamsters, aged 6-10 weeks old, could be obtained and kept in Biosafety Level-2 housing and would be given access to standard pellet feed and water ad libitum until virus challenge. Phosphate- buffered saline (PBS) would be used to dilute virus stocks to a fixed concentration, and inocula would be verified for proper dosing prior to inoculation. PBS containing a fixed amount of virus stock ( e.g ., 10⁵ plaque-forming units) or PBS alone for controls, in an appropriate volume (e.g., IOOmI) would be introduced intranasally while animals are under anesthetic. Animals would be monitored for clinical signs of disease. Validation of an API treatment (“API” in the table below) could be made by measuring one or more of the following and making comparisons versus those same measurements in control (“Control” below) treatment: Change in body weight ( e.g ., treatment could maintain body weight post infection, whereas control subjects would have decreased body weight post infection), decreased activity in the running wheel (e.g., decreased total rotations in control versus unchanged rotations in treatment), total viral load in throat swabs by RT-PCR as described for in vitro validation herein, lung and nasal tissue pathology that corresponds to the particular human pathologies of the viral disease (in the example of SARS-CoV-2 (Gruber et al. 2020). The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00277] 4. Validating a method of addressing a viral infection, in vivo: Human

Clinical Trial

[00278] For validation of an API individually or selected from any compound(s) or any pharmaceutical composition herein, a human clinical study could be performed that would include participants that have been clinically tested and diagnosed with an active viral infection. A study of these participants could be conducted to measure the symptom severity of disease (including one or more of the following: fatigue, cough, headache, improvement in cognition), a reduction of inflammatory markers (including one or more of the following: IL-1, IL-6, IL-18, D-Dimer, and/or CRP), and an improvement in quality of life.

[00279] A valid API could demonstrate the following self-reported primary outcome:

• A reduction in symptoms of fatigue as measured by the Chadler Fatigue Scale (11 items with 2 subscales, 1 for physical fatigue (items 1-7) and 1 for mental fatigue (items 8-11))

[00280] A valid API could demonstrate one or more of the following self- reported secondary outcomes:

• A reduction in cough in patients that experienced cough post infection

• A reduction in severity and frequency of headache in patients that experienced headaches post infection (NCS and Frequency Scales)

• An improvement in Montreal Cognitive Assessment test (MoCA) in patients that experienced a decline in MoCA post infection

• An improvement in quality of life as measure by the EQ-5i

[00281] A valid API could result in the restoration to baseline of one or more of the following blood-based biomarkers associated with viral infection and post infection as measured by commercially available blood protein and cytokine/chemokine assays that measure: IL-1, IL-6, IL-18, D-Dimer, CRP. [00282] A valid API could result in a decrease in the relative quantity of virus genome templates that are present as measured by RT-PCR in blood samples from patients that were treated with the API(s) or mock treated with PBS solution alone.

[00283] The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00284] 5. Validating a method of treating a viral infection, in vivo: small animal models

[00285] An ever-expanding variety of small animal models continue to be developed and could be used to validate a given API, however, the Syrian Hamster model has an especially diverse range in terms of viral strains (Miao et al. 2019). The API(s) could be selected from any compound(s) or any pharmaceutical composition herein. [00286] Male and female Syrian hamsters, aged 6-10 weeks old, could be obtained and kept in Biosafety Level-2 housing and would be given access to standard pellet feed and water ad libitum until virus challenge. Phosphate- buffered saline (PBS) would be used to dilute virus stocks to a fixed concentration, and inocula would be verified for proper dosing prior to inoculation. PBS containing a fixed amount of virus stock ( e.g ., 10⁵ plaque-forming units) or PBS alone for controls, in an appropriate volume (e.g., IOOmI) would be introduced intranasally while animals are under anesthetic. Animals would be monitored for clinical signs of disease. Validation of an API treatment (“API” in figure below) would be made by measuring one or more of the following and making comparisons versus those same measurements in control (“Control” below) treatment: Change in body weight (e.g., treatment could maintain body weight post infection, whereas control subjects may have decreased body weight post infection), decreased activity in the running wheel (e.g., decreased total rotations in control versus unchanged rotations in treatment), total viral load in throat swabs by RT-PCR as described for in vitro validation herein, lung and nasal tissue pathology that corresponds to the particular human pathologies of the viral disease (in the example of SARS-CoV-2 (Gruber et al. 2020). The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00287] 6. Validating a method of treating a viral infection, in vivo: Human

Clinical Trial

[00288] For validation of an API individually or selected from any compound(s) or any pharmaceutical composition herein, a human clinical study would be performed that would include participants that have been clinically tested and diagnosed with an active viral infection. A study of these participants would be conducted to measure the symptom severity of disease (including one or more of the following: fatigue, cough, headache, improvement in cognition), a reduction of inflammatory markers (including one or more of the following: IL-1, IL-6, IL-18, D-Dimer, and/or CRP), and an improvement in quality of life.

[00289] A valid API could demonstrate the following self-reported primary outcome:

• A reduction in symptoms of fatigue as measured by the Chadler Fatigue Scale (11 items with 2 subscales, 1 for physical fatigue (items 1-7) and 1 for mental fatigue (items 8-11))

[00290] A valid API could demonstrate one or more of the following self- reported secondary outcomes:

• A reduction in cough in patients that experienced cough post infection

• A reduction in severity and frequency of headache in patients that experienced headaches post infection (NCS and Frequency Scales)

• An improvement in Montreal Cognitive Assessment test (MoCA) in patients that experienced a decline in MoCA post infection

• An improvement in quality of life as measure by the EQ-5ii

[00291] A valid API could result in the restoration to baseline of one or more of the following blood-based biomarkers associated with viral infection and post infection as measured by commercially available blood protein and cytokine/chemokine assays that measure: IL-1, IL-6, IL-18, D-Dimer, CRP [00292] A valid API could result in a decrease in the relative quantity of virus genome templates that are present as measured by RT-PCR in blood samples from patients that were treated with the API(s) or mock treated with PBS solution alone.

[00293] The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00294] 7. Validating a method of decreasing the likelihood of a viral infection, in vivo: small animal models

[00295] Incorporating the steps needed to infect a small animal model suited to validate addressing a viral infection (infection of Syrian Hamsters, above), to validate a method of decreasing the likelihood of a viral infection, one or more experimentally inoculated hamsters could act as transmission vectors and be co housed with API pre-treated hamsters, or hamsters that received only a sham treatment until such time that all hamsters that were untreated became observably symptomatic.

[00296] Validation of an API treatment could be made by comparing the percent of treated versus non-treated co-housed hamsters which became infected. The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00297] 8. Validating a method of decreasing the likelihood of a viral infection, in vivo: Human Clinical Trial

[00298] For validation of an API individually or selected from any compound(s) or any pharmaceutical composition herein, a human clinical study would be performed that would include participants that have been clinically tested and are confirmed to not yet have an active viral infection. Participants would be enrolled and those in the treatment group would be provided a maintenance dose of the API(s), and blood samples would be obtained on a regular basis (eg every 72 hours) until such time that all participants in the control cohort were diagnosed with a viral infection.

[00299] A valid API could demonstrate the following primary outcomes:

• Detection of viral genome template by RT-PCR in a greater percentage of participants that were not treated than those who were treated. • A decrease in the relative quantity of virus genome templates that are present as measured by RT-PCR in blood samples from patients that were treated with the API(s) or untreated.

[00300] The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00301] 9. Validating a method of treating a disease caused by viral infection, in vitro could performed as described for an above described in vitro experiment. A decreased viral load, increased cell survival, or any of the measures mentioned above could be the basis to deduce success at treating a disease caused be viral infection.

[00302] 10. Validating a method of treating a disease cause by viral infection, in vivo: small animal models

[00303] Incorporating the steps needed to infect a small animal model suited to validate addressing a viral infection (infection of Syrian Hamsters, above), to validate a method of treating a disease caused by viral infection, two cohorts of hamsters could be inoculated hamsters, and after an incubation period (eg 24-72 hours post inoculation), one cohort could be treated with API(s) whereas a second cohort would receive only a sham treatment until such time that all hamsters that were untreated became observably symptomatic.

[00304] Validation of an API treatment could be made by measuring one or more of the following and making comparisons versus those same measurements in control (“Control” below) treatment: Change in body weight ( e.g ., treatment maintained body weight post infection, whereas control subjects had decreased body weight post infection), decreased activity in the running wheel (e.g., decreased total rotations in control versus unchanged rotations in treatment), total viral load in throat swabs by RT-PCR as described for in vitro validation herein, lung and nasal tissue pathology that corresponds to the particular human pathologies of the viral disease (in the example of SARS-CoV-2 (Gruber et al. 2020). The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00305] 11. Validating a method of treating a disease caused by viral infection, in vivo: Human Clinical Trial [00306] For validation of an API individually or selected from any compound(s) or any pharmaceutical composition herein, a human clinical study could be performed that would include participants that have been clinically tested and diagnosed with an active viral infection. Once confirmed to be actively infected and exhibiting symptoms of disease, one cohort could be treated with the API and a second cohort would be studied but not treated. A study of these participants could be conducted that regularly measures the symptom severity of disease (including one or more of the following: fatigue, cough, headache, improvement in cognition), a reduction of inflammatory markers (including one or more of the following: IL-1, IL-6, IL-18, D-Dimer, and/or CRP), and an improvement in quality of life.

[00307] A valid API could demonstrate the following self-reported primary outcome:

• A reduction in symptoms of fatigue as measured by the Chadler Fatigue Scale (11 items with 2 subscales, 1 for physical fatigue (items 1-7) and 1 for mental fatigue (items 8-11))

[00308] A valid API could demonstrate one or more of the following self- reported secondary outcomes:

• A reduction in cough in patients that experienced cough post infection

• A reduction in severity and frequency of headache in patients that experienced headaches post infection (NCS and Frequency Scales)

• An improvement in Montreal Cognitive Assessment test (MoCA) in patients that experienced a decline in MoCA post infection

• An improvement in quality of life as measure by the EQ-5iii

[00309] A valid API could result in a decrease in the relative length of time before restoration to baseline of one or more of the following blood-based biomarkers associated with viral infection and post-infection as measured by commercially available blood protein and cytokine/chemokine assays that measure: IL-1, IL-6, IL-18, D-Dimer, CRP.

[00310] A valid API could result in a decrease in the relative length of time before there was a decrease in the relative quantity of virus genome templates that are present as measured by RT-PCR in blood samples from patients that were treated with the API(s) or mock treated with PBS solution alone.

[00311] A valid API could result in a decrease in the relative length of time that clinically observable symptoms persist in participants that are treated versus those that are not treated with the API.

[00312] The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00313] 12. Validating a method of increasing immune system factors, in vivo: small animal models

[00314] Incorporating the steps needed to infect a small animal model suited to validate addressing a viral infection (infection of Syrian Hamsters, above), to validate a method of increasing immune system factors, one cohorts of hamsters could be pre-treated with the API, and a second would be untreated. Subsequent to pre-treatment (or control), hamsters in both cohort could be inoculated with an infectious dose of virus, and at the same time (time = 0) blood samples would be obtained from both cohorts. The study could continue throughout the period when all hamsters that were untreated became observably symptomatic, until the completion of the study ( e.g ., when all hamsters have resolved infection or been euthanized). During the study, blood samples would be collected on a regular basis (e.g., every 24 hours).

[00315] Validation of an API treatment would be made by measuring one or more of the following and making comparisons versus those same measurements in control (“Control” below) treatment: Change in the phenotypes of effector and regulatory leukocytes, inflammatory cytokines, inflammatory chemokines, and the level of neutralizing antibodies.

[00316] A validated API would exhibit one or more of the following trends over the course of the study:

• An increased frequency of neutralizing antibodies in the blood

• An increased frequency of virus-specific effector cells

• A decreased frequency of virus-specific regulatory cells

• A level of inflammatory cytokines and chemokines that return to baseline coincident with resolution of disease symptoms [00317] The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

[00318] 13. Validating a method of increasing immune system factors, in vivo :

Human Clinical Trial

[00319] For validation of an API individually or selected from any compound(s) or any pharmaceutical composition herein, a human clinical study could be performed that would include participants that have been clinically tested and are confirmed to not yet have an active viral infection. Participants would be enrolled and those in the treatment group would be provided a maintenance dose of the API(s), and blood samples would be obtained on a regular basis (eg every 72 hours) until such time that all participants in the control cohort were diagnosed with a viral infection.

[00320] A validated API would exhibit one or more of the following trends over the course of the study:

• An increased frequency of neutralizing antibodies in the blood

• An increased frequency of virus-specific effector cells

• A decreased frequency of virus-specific regulatory cells

• A level of inflammatory cytokines and chemokines that return to baseline coincident with resolution of disease symptoms

[00321] The API(s) could be selected from any compound(s) or any pharmaceutical composition herein.

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[00323] In addition to the work above MENK, as an immune adjuvant, has potential immune -regulatory activity on innate and adaptive immune cells. An aim of the examples herein was to investigate the antiviral effect of MENK on influenza virus-infected murine macrophage cells (RAW264.7) and its underlying mechanisms. The results showed that MENK markedly inhibited influenza A virus (H1N1) replication in pre- and post-MENK treatment, especially in pre- MENK treatment.

[00324] The mechanismic exploration revealed that MENK (10 mg/mL) significantly inhibited the nucleoprotein (NP) of influenza virus and up-regulated levels of IL-6, TNF-a and IFN-b compared with those in H1N1 control group. Further experiments confirmed that antiviral effects of MENK was associated with promotion of opioid receptor (MOR) as well as activation of NF-KB p65 inducing cellular antiviral status. The data shows that MENK was an unpredictable and surprisingly successful candidate for prophylactic or therapeutic treatment against H1N1 influenza virus. By extension, the data also show that MENK could be an unpredictable and surprisingly successful candidate for prophylactic or therapeutic treatment against other viruses, including but not limited to SARS-CoV-2 and others described herein.

[00325] The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[00326] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

Claims

CLAIMS What is claimed is:

1. A method of addressing viral infection comprising administering to a mammalian patient in need thereof at least one of (1) MENK or a pharmaceutically acceptable agent thereof or (2) a first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and optionally administering one or more antiviral agents or a pharmaceutically acceptable agent thereof.

2. The method of claim 1, wherein the first pharmaceutical composition comprises the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and the method further comprises administering the MENK or a pharmaceutically acceptable agent thereof to the patient, wherein the MENK is either part of the first pharmaceutical composition or administered separately from the first pharmaceutical composition.

3. The method of claim 1, wherein the first pharmaceutical composition comprises at least one of (a) the one or more antiviral agents or a pharmaceutically acceptable agent thereof, or (b) the MENK or a pharmaceutically acceptable agent thereof.

4. The method of claim 1, wherein the administering further comprises administering a second pharmaceutical composition that comprises at least one of (a) the one or more antiviral agents or a pharmaceutically acceptable agent thereof, or (b) the MENK or a pharmaceutically acceptable agent thereof.

5. The method of claim 1, wherein the patient is a human, an ape, a simian, a canine, a feline, or a rodent.

6. The method of claim 1, wherein the patient is a human.

7. The method according to any one of claims 1-6, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent is chosen from the group consisting of an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal), or a combination of two or more thereof.

8. The method according to any one of claims 1-6, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent comprises one chosen from the group consisting of hydroxychloroquine and chloroquine.

9. The method according to any one of claims 1-6, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent comprises remdesivir.

10. The method according to any one of claims 1-6, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent comprises favipiravir.

11. The method according any one of claims 1-6, wherein the administering comprises administering the one or more antiviral agent or a pharmaceutically acceptable agent thereof, and the one or more antiviral agent is chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine, tocilizumab, EIDD-280, and etravirine.

12. The method according any one of claims 1-6, wherein the administering comprises administering two or more of the one or more antiviral agents, and the two or more antiviral agents are chosen from the group consisting of rilpivine, abacavir, and lamivudine; abacavir, dolutegravir, and lamivudine; abacavir, lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine, and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumerate; emtricitabine, rilpivirine, and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; lopinavir, retinovir and interferon-beta lb; and lopinavir and ritonavir.

13. The method according to any one of claims 1-6, wherein said viral infection is from a virus chosen from the group consisting of influenza A, SARS- CoV-2, a pathological strain of SARS-CoV-2, 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS- CoV, SARS-CoV, molluscum contagiosum virus, HTLV, HTLV-1, hepatitis-A, HCV, HBV, HIV/AIDS, human papilloma virus, herpes virus, herpes simplex virus, a virus that causes viral dysentery, measles virus, rubella virus, mumps virus, polio virus, rabies virus, Epstein-Barr virus, ebola virus, respiratory syncytial virus, dengue virus, yellow fever virus, lassa virus, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, a virus causing viral meningitis, West Nile virus, arbovirus, a parainfluenza virus, smallpox virus, dengue virus, cytomegalovirus, infant cytomegalic virus, JC virus, a virus that causes viral gastroenteritis, a hepatitis virus, a virus that causes encephalitis, varicella zoster virus, viruses that cause California serogroup viral encephalitis, St. Louis encephalitis virus, rift valley fever virus, a virus that causes hand, foot, & mouth disease, hendra virus, an enterovirus, astrovirus, an adenovirus, Japanese encephalitis virus, lymphocytic choriomeningitis mammarenavirus, human herpesvirus 6, human herpesvirus 7, a virus that causes sandfly fever, a papilloma virus, a human papilloma virus, cat scratch disease, parvovirus B19, a parapox virus that causes orf, a virus that causes pityriasis rosea, and lyssavirus.

14. The method according to claim 13, wherein the viral infection is by influenza A.

15. The method according to claim 13 wherein the viral infection is by SARS-CoV-2 or a pathological strain thereof.

16. The method according any one of claims 1-6, wherein one or both of the first pharmaceutical composition or the second pharmaceutical composition further comprise one or more pharmaceutically acceptable excipients, optionally where the excipients are charged aqueous species that have a net negative charge or a net positive charge.

17. The method according to any one of claims 1-6 further comprising administration of a therapeutically effective amount of at least one compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin, and riboflavin.

18. The method according to any one of claims 1-6 further comprising administration of a therapeutically effective amount of at least one compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins.

19. The method according to any one of claims 1-6 further comprising administration of a therapeutically effective amount of at least one immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE.

20. The method according to any one of claims 1-6 further comprising the administration of an antibacterial agent.

21. The method according to claim 20, wherein the antibacterial agent is Azithromycin.

22. The method according to any one of claims 1-6 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof are in immediate release form.

23. The method according to claim 22, wherein said immediate release pharmaceutical composition is for administration once in a 24-hour period, optionally within 60 minutes.

24. The method according to claim 22 wherein said immediate release composition releases the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof completely within about 60 minutes.

25. The method according to any one of claims 1-6 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the amount of the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof is between about 1.0 mg and about 300.0 mg, between about .05 mg and about 6.0 mg, or between about 0.05 mg and about 4.5 mg.

26. The method according to any one of claims 1-6, wherein the administrations for one or both of the first pharmaceutical composition or the second pharmaceutical composition is via a route chosen from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration.

27. The method according to any one of claims 1-6, wherein one or both of the first pharmaceutical composition or the second pharmaceutical composition are form of a capsule, a tablet, or a rapidly dissolving film.

28. The method according to any one of claims 1-6 comprising the administering of the first pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof comprises a mixture of (+) naltrexone and (-) naltrexone in ratio between about 1:99 and about 99:1, optionally 50:50.

29. The method according to any one of claims 1-6, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof comprises a mixture of (+) naloxone and (-) naloxone in a ratio between about 1:99 and about 99:1, optionally 50:50.

30. The method according to any one of claims 1-6, wherein the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof comprises a mixture of (+) naltrexol and (-) naltrexol in a ratio between about 1:99 and about 99:1, preferably about 50:50.

31. A pharmaceutical composition comprising at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and at least one of (1) one or more antiviral agent or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof.

32. The pharmaceutical composition of claim 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent is chosen from the group consisting of an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal) or combination thereof.

33. The pharmaceutical composition of claim 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises at least one chosen from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof.

34. The pharmaceutical composition of claim 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises remdesivir.

35. The pharmaceutical composition of claim 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises favipiravir.

36. The pharmaceutical composition of claim 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises one chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine, tocilizumab, EIDD-280, and etravirine.

37. The pharmaceutical composition of claim 31 comprising the one or more antiviral agent or a pharmaceutically acceptable agent thereof, wherein the one or more antiviral agent comprises a combination of antiviral agents chosen from the group consisting of rilpivine, abacavir, and lamivudine; abacavir, dolutegravir, and lamivudine; abacavir, lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine, and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumerate; emtricitabine, rilpivirine, and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; lopinavir, retinovir and interferon-beta lb; and lopinavir and ritonavir.

38. The pharmaceutical composition of claim 31 further comprising at least one compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin, and riboflavin.

39. The pharmaceutical composition of claim 31 further comprising at least one compound selected from the group consisting of L-arginine, L- homoarginine, homocysteine, L-glutamine and immunoglobins.

40. The pharmaceutical composition of claim 31 further comprising at least one immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE.

41. The pharmaceutical composition of claim 31 further comprising an antibacterial agent.

42. The pharmaceutical composition of claim 41, wherein the antibacterial agent is Azithromycin.

43. A dosage form comprising the pharmaceutical composition of any one of claims 31-42.

44. The dosage form of claim 43, wherein the dosage form is an immediate release dosage form.

45. The dosage form of claim 43, wherein the dosage form is one of a capsule, a tablet, or a dissolving film.

46. The dosage form of claim 43, wherein the dosage form is for an administration route chosen from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration.

47. The dosage form of claim 43, wherein the dosage from comprises the concentration of the at least one compound selected from the group consisting of (+) 6B naltrexol, (-) 6B naltrexol, (+) naltrexone, (-) naltrexone, (+) naloxone and (-) naloxone, and pharmaceutically acceptable agents thereof, and at least one of (1) one or more antiviral agents or a pharmaceutically acceptable agent thereof or (2) MENK or a pharmaceutically acceptable agent thereof suitable for a single day dose.

48. A use of the pharmaceutical composition of any one of claim 31-42 or the dosage form of any one of claims 43-47.

49. The use of claim 48, wherein the use is for addressing a viral infection.

50. The use of claim 49, wherein the addressing is one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient.

51. The use of claim 50, wherein the virus is SARS-CoV-2 or Influenza A virus.

52. The use of claim 48, wherein the use is preparing a medicament for addressing viral infection.

53. The use of claim 52, wherein the addressing is one of a method of treating viral infection, a method of decreasing the likelihood of viral infection, a method of decreasing the likelihood of disease from viral infection, a method of treating a disease caused by viral infection, or a method of increasing immune system factors in a patient.

54. The use of claim 53, wherein the virus is SARS-CoV-2 or Influenza A virus.