WO2022103565A1 - Myeloperoxidase-containing compositions and uses thereof - Google Patents

Abstract

Compositions comprising myeloperoxidase and methods of using said combinations to treat viral pathogens are provided, A viral pathogen that may be treatable with compositions and methods disclosed herein include Sars-CoV-2, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and human influenza A virus (including H1N1).

Description

MYELOPEROXIDASE-CONTAINING COMPOSITIONS AND USES THEREOF

BACKGROUND

[0001] The present disclosure generally relates to myeloperoxidase-containing compositions for treating viral pathogens on mucosal and epithelial surfaces.

[0002] Infectious diseases represent a continuous and increasing threat to human health and mortality. Emerging diseases, increasing resistances to treatments, and increased disease spread fueled by cross-border travelling and transportation concern the whole world.

[0003] Non-specific hygiene agents, such as sanitizers, detergents or other chemical disinfectants can be useful for infection management. However, many cause irritation and may result in mild through to debilitating symptoms such as including dryness, irritation, itching, and even cracking and bleeding¹. Further, non-specific hygiene agents can non- specifically react with and inhibit normal immunophysiologic defense mechanisms. For example, they may inhibit the antimicrobial function of phagocytic leukocytes.

[0004] An ideal hygiene agent would exert potent reactivity against a broad range of pathogens with minimum toxicity to host cells, preferably aiding and/or augmenting the natural protective agencies of the body against infection. To a limited extent, these requirements are met by certain antibiotics. The selective bactericidal action of antibiotics is based on differences between prokaryotic and eukaryotic cells with regard to protein synthesis, nucleic acid replication, and the presence or composition of the cell wall. However, the emergence of antibiotic resistance has resulted in the inability to treat previously treatable bacterial infections, and/or the reservation of health professionals to administer antibiotics considered the last line of defense due to the risk of the possible emergence of resistance. Notably, viruses, yeasts and fungi are unaffected by antibiotics, and treatment with antibiotics may make a patient susceptible to non-bacterial infection (e.g. resulting in yeast/fungi overgrowth).

[0005] Compositions comprising myeloperoxidase are an example of an alternative targeted hygiene agent that are known to be useful with the respect to the control of bacterial, yeast and fungal infectious agents. As disclosed in U.S. Patent Nos. 5,888,505, 6,294,168, and 8,945,540, myeloperoxidase selectively binds to and kills target microorganisms (in the presence of peroxide and halide) without significantly damaging host cells and normal flora. In this regard, myeloperoxidase-containing compositions demonstrate a high degree of selectivity and can therefore be employed as targeted hygiene agent in the therapeutic or prophylactic treatment of bacterial, yeast and fungal infectious agents in human or animal subjects. Further, myeloperoxidase has recently been shown to detoxify bacterial lipopolysaccharide in the absence of components/triggers normally required for catalytic activity². How the enzyme actually functions as targeted hygiene agent against bacteria has yet to be fully elucidated.

[0006] Viruses also present a significant problem with respect to human health and mortality. A case in point is the emergence of coronavirus SARS-CoV-2, the causative agent of the COVID-19 pandemic. Coronaviruses belong to the family of Coronaviridae, and are the largest group of viruses causing respiratory and gastrointestinal infections. COVID-19 can present as a mild to acute severe infection of the respiratory tract³, and there is currently no preventative vaccine or specific treatment available for the disease⁴. [0007] Other viruses, such as herpes simplex virus (HSV), human immunodeficiency virus (HIV) and human influenza A virus (including H1 N1 which is the causative agent of Swine flu), equally present as human health risks, particularly in hospital and healthcare settings.

[0008] For infection management during the administration of healthcare, and minimizing exposure risk to caregivers (particularly doctors and other first responders) and patients, there is a need for treatment options that minimize or eliminate the risk of virus transmission.

SUMMARY

[0009] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0010] E-101 solution is a first-in-class myeloperoxidase-containing composition developed for topical application⁵. Once activated, the reactive species hydrogen peroxide, hypochlorous acid, and singlet oxygen are generated. E-101 solution has been shown to exhibit broad antimicrobial activity in vitro and in vivo against bacteria (including antibiotic resistant and sensitive species), fungi, and yeast, and maintains its activity in the presence of serum and blood^6-9. In a phase three clinical trial for preventing surgical site infections after colorectal surgery, E-101 tested at high concentrations (300 GU/ml) was found to be as safe as saline when applied to open wounds (ClinicalTrials.gov NCT01297959).

[00 ] The myeloperoxidase component of E-101 solution selectively binds to the surface of target bacteria, yeast and fungal pathogens optimizing direct oxidative damage while sparing normal flora^{10 11}. Further, singlet oxygen is produced with a restricted kill radius and exhibits a short half-life, thus precluding collateral damage to surrounding host cells and tissue^{12 13}.

[0012] The present disclosure is predicated on the surprising and unexpected finding that myeloperoxidase-containing compositions, exemplified by the E-101 solution, exhibit antiviral properties.

[0013] According to one aspect, the disclosure provides a method of treating a virus infection in a human or animal subject, said method comprising administering to said subject an effective amount of a myeloperoxidase. In some embodiments, the myeloperoxidase catalyzes halide oxidation and disproportionation of peroxide to singlet molecular oxygen thereby inhibiting and/or killing said virus.

[0014] In other embodiments, the method further comprises administering an effective amount of peroxide or a peroxide-producing oxidase. A substrate for the oxidase may be optionally administered. Preferably, the peroxide-producing oxidase is glucose oxidase and the substrate is glucose.

[0015] In further embodiments, the method further comprises administering the myeloperoxidase with a halide, preferably a chloride or bromide.

[0016] In yet other embodiments, the myeloperoxidase is administered in a first composition together with at least one further composition comprising one or more of: a halide, peroxide or a peroxide-producing oxidase, and a substrate for the peroxide- producing oxidase. Alternatively, the myeloperoxidase may be formulated in a composition which also comprises one or more of a halide, peroxide or a peroxide- producing oxidase, and a substrate for the peroxide-producing oxidase.

[0017] In yet other embodiments, the method may exclude administration of one or more of: a halide, a peroxide or peroxide producing oxidase, or a substrate for said oxidase.

[0018] In further embodiments, the myeloperoxidase is administered topically to a mucosal or epithelial surface. Further, the method may be effective in treating a virus selected from the group consisting of: Sars-CoV-2, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and human influenza A virus (including H1 N1).

[0019] In yet another aspect of the disclosure, there is provided a combination for treating a virus infection in a human or animal subject, said combination comprising: a myeloperoxidase, and at least one of a halide, and peroxide or a peroxide producing oxidase. The combination may be formulated in one or more compositions. Preferably, where formulated in two or more compositions, said compositions may be premixed for administration, or administered concurrently or sequentially. [00 0] In some embodiments, the combination excludes at least one of: a halide, or peroxide or peroxide producing oxidase.

[0021] In yet another aspect of the disclosure, there is provided a composition for treating a virus infection in a human or animal subject, said compositions comprising: a myeloperoxidase, and optionally one or more of: a halide, peroxide or a peroxide producing oxidase, a substrate for said oxidase, and a pharmaceutically acceptable carrier.

[0022] In some embodiments, the composition may comprise a myeloperoxidase, but exclude one or more of: a halide, peroxide or a peroxide producing oxidase, a substrate for said oxidase, and a pharmaceutically acceptable carrier.

[0023] Other embodiments will be evident from the following detailed description.

DETAILED DESCRIPTION

Definitions

[0024] The terms “a,” “an,” “the” and similar referents used in the context of describing inventive concepts (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0025] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0026] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to provide better illumination and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element is essential.

[0027] Except where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term ‘about’. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding conventions. The term "about" may be understood to refer to a range of +/- 10%, such as +/- 5% or +/- 1% or, +/- 0.1 %. [00 8] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1 , 7, 34, 46.1 , 23.7, or any other value or range within the range.

[0029] The terms “protein” and “polypeptide” are used interchangeability herein. The 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature is used throughout this disclosure. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0030] Reference is made herein to “enzymes", such as myeloperoxidase or glucose oxidase. In the present context, an enzyme is a protein/polypeptide which acts as a catalyst to bring about a specific biochemical reaction. Included within the scope of enzymes of the present disclosure include those isolated from a natural source having the unmodified amino acid sequence identical to that found in nature, as well as "functional derivatives" thereof.

[0031] A “derivative” of an enzyme of the disclosure generally retains the characteristic enzymatic activity observed in the wild-type, native or parent form to the extent that the derivative is effective for similar purposes as the wild-type, native or parent form.

[0032] The term “functional derivative” when used in the contact of enzymes of the disclosure encompasses naturally occurring, synthetically or recombinantly produced nucleic acids or fragments and encode enzymes having the functional characteristics of the native, unmodified parent enzyme present disclosure. A “functional derivative” may include a "substituted variant" which is a variant in which at least one amino acid residue in a native sequence has been removed and inserted into the same position by a different amino acid. The substitution may be single, wherein only one amino acid in the molecule is substituted; or there may be multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be located at successive sites. Likewise, an amino acid can be substituted with multiple residues, including substitutions and insertions. An "insertion variant" is a variant in which one or more amino acids are inserted into an amino acid immediately adjacent to a particular position in a native sequence. Immediately adjacent to the amino acid means attached via an alpha-carboxy or alphaamino functional group of the amino acid. A "deleted variant" is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deleted variant has one or two amino acids deleted in a particular region of its molecule. [0033] The term isolated or purified refers to a material that is removed from its original environment (e.g. the natural environment, if it is naturally occurring). For example, the material is said to be “purified” when it is present in a particular composition in a higher concentration than exists in a naturally occurring or wild type organism or in combination with components not normally present upon expression from a naturally occurring or wild type organism. For example, a naturally-occurring protein/polypeptide present in a living organism is not isolated, but the same protein/polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such proteins/polypeptides could, for example, be part of a composition, and still be isolated in that such a composition is not part of the natural environment of the proteins/polypeptides. [0034] The term "pharmaceutically acceptable" as used herein refers to substances that do not cause substantial adverse allergic or immunological reactions when administered to a subject. A "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, coatings, dispersion agents, wetting agents, isotonic and absorption delaying agents and disintegrants.

[0035] As used herein, "treat", "treating" or "treatment" of a disease, condition or disorder means accomplishing one or more of the following: (a) reducing the severity and/or duration; (b) limiting or preventing development of characteristic symptoms; (c) inhibiting worsening of symptoms; (d) limiting or preventing recurrence; and (e) limiting or preventing recurrence of symptoms. That is, the terms include both prophylactic or preventive treatment (that prevent and/or slow the development of a targeted pathologic disease, condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a disease, condition or disorder; and treatment of a subject at risk of contracting a disease or suspected to have contracted a disease, as well as a subject who is ill or has been diagnosed as suffering from a disease, condition or disorder. The terms do not necessarily imply that a subject is treated until total recovery. The terms may also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms may also include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As non-limiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional. [0036] As used herein, "prevent", "preventing", "prevention", or "prophylaxis" of a disease or disorder means preventing that a disorder occurs in subject. “Prevention” includes reduction of risk, incidence and/or severity of a disease, condition or disorder. [0037] As used herein, the expressions is for administration and is to be administered" have the same meaning as "is prepared to be administered". In other words, the statement that an active compound "is for administration" has to be understood in that said active compound has been formulated and made up into doses so that said active compound is in a state capable of exerting its therapeutic activity.

[0038] The terms "effective amount" or "therapeutic amount" are intended to mean that amount of a substance that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. The term "prophylactically effective amount" is intended to mean that amount of a pharmaceutical drug that will prevent or reduce the risk of occurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician.

[0039] The terms "comprise", "comprises", "comprised" or "comprising", "including" or "having" and the like in the present specification and claims are used in an inclusive sense, that is to specify the presence of the stated features but not preclude the presence of additional or further features.

[0040] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of’ excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.

Myeloperoxidase-Containing Compositions

[0041] The present disclosure is directed to compositions and methods for inhibiting and/or killing viral infections by contacting the site of infection with a composition comprising myeloperoxidase. In the practice, viruses may be inhibited and/or killed through contact with a myeloperoxidase in the presence of a peroxide and bromide or chloride.

[0042] In one aspect, the compositions and methods of the present disclosure exhibit myeloperoxidase-mediated antiviral activity against a broad range of enveloped viral pathogens. Enveloped viruses utilize a pathogenic process that involves human mucosa as the infection entry gate followed by different pathways of virus dissemination into susceptible organs. The envelopes are typically derived from portions of the host cell membranes (phospholipids and proteins), but include some viral glycoproteins. Without being bound by theory, the inventors postulate that the compositions and methods of the present disclosure disrupt the viral envelope, slowing viral spread within the host cell resulting in decrease severity of disease in an individual and/or a reduction in virus transmissibility. Preferably, enveloped viruses which may be inhibited and/or killed by compositions of the present disclosure include, but are not limited to, SARS-CoV-2, HIV, human influenza A virus, and H1 N1 .

[0043] The compositions and methods of the present disclosure are based on the use of dioxygenating myeloperoxidase which, as previously shown, exhibits selective affinity for pathogenic microorganisms (bacteria and fungi specifically). As such, high potency microbicidal action can be directed to the target microorganisms without associated host tissue destruction or disruption of normal flora. As shown herein, compositions and methods of the present disclosure have been surprisingly and unexpectedly found to exhibit anti-viral activity.

[0044] In some embodiments, the present disclosure provides compositions for inhibiting and/or killing the virus growth, said compositions comprising myeloperoxidase. The compositions may optionally comprise or exclude: hydrogen peroxide or a source of hydrogen peroxide, and/or a halide (such as chloride or bromide).

[0045] In related embodiments, the present disclosure provides methods of treating a human or animal subject in need of such treatment comprising administering to a site of infection in the subject a composition comprising myeloperoxidase. Again, the composition may optionally comprise or exclude: hydrogen peroxide or a source of hydrogen peroxide, and a halide (such as chloride or bromide).

[0046] In some embodiments, myeloperoxidase effective in the present disclosure is a halide:hydrogen peroxide oxidoreductase (e.g., EC No. 1.11.1.7 and EC No. 1.11.1.10 under the International Union of Biochemistry) for which halide, e.g., chloride or bromide, is the electron donor or reductant and peroxide is the electron receiver or oxidant. However, any haloperoxidase which catalyzes the halide dependent generation of singlet molecular oxygen from hydrogen peroxide may be used in the present disclosure. Suitable haloperoxidases, include myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), chloroperoxidase (CPO), and functional derivatives thereof. Preferably, the myeloperoxidase used in the present disclosure is porcine-derived myeloperoxidase.

[0047] The enzymatic activity of a myeloperoxidase solution can be determined by reaction with guaiacol in the presence of hydrogen peroxide in sodium phosphate buffer. The reaction generates a product with strong absorbance at 470 nm. The activity is determined from the kinetics of the increase in absorbance compared to a reference standard. Myeloperoxidase activity is commonly expressed in Guaiacol units/mL (GU/mL), and is also expressed as micrograms of MPO per milliliter (pg/mL). The conversion of pg to GU of MPO is based on 0.375 GU per pg of MPO. The specific activity is calculated from its activity and the total protein concentration and expressed in GU/mg protein.

[0048] Effective amounts of myeloperoxidase employed in the compositions of the disclosure may vary widely depending on conditions under which the compositions are employed, the environment of use and the desired result. For most purposes, the compositions of the disclosure will generally comprise at least about 0.05 pg/ml (0.01875 GU/ml) of myeloperoxidase. In some embodiments, the compositions of the disclosure will comprise from about 1 to about 50,000 pg/ml of myeloperoxidase (i.e., from about 0.375 to about 18,750 GU/ml), more preferably from about 5 to about 10,000 pg/ml of myeloperoxidase (i.e., from about 1 .875 to about 3,750 GU/ml), and even more preferably from about 10 to about 5,000 pg/ml of myeloperoxidase (i.e., from about 3.75 to about 1 ,875 GU/ml).

[0049] Peroxide-producing oxidases effective in the present disclosure include, for example, oxidases, such as glucose oxidase, cholesterol oxidase and galactose oxidase. As a representative example, when the oxidase is glucose oxidase and its substrate is glucose, the compositions of the present disclosure may comprise from about 0.05 to about 3,000 U/ml, more preferably from about 0.1 to about 1 ,000 U/ml, and even more preferably from about 1 to about 500 U/ml of glucose oxidase, and from about 0.1 to about 100 mM, more preferably from about 0.5 to about 80 mM, and even more preferably from about 1 to about 50 mM glucose. Preferably, the glucose oxidase as used in compositions of the present disclosure is derived from Aspergillus Niger.

[0050] In some embodiments, the myeloperoxidase/oxidase may optionally be supplied to a site of infection with at least two amino acids, preferably at least three amino acids, selected from the group consisting of glycine, L-alanine, D-alanine, L-alanine anhydride, L-glutamine, L-glutamic acid, glycine anhydride, hippuric acid, L-histidine, L- leucine, D-leucine, L-isoleucine, D-isoleucine, L-lysine, L-ornithine, D-phenylalanine, L- phenylalanine, L-proline, L-hydroxyproline, L-serine, taurine, L-threonine, D-threonine, L- tyrosine, L-valine, D-valine, beta amino acids, such as beta alanine, L-beta- homoleucine, D-beta-homoleucine, 3-aminobutanoic acid, L-2,3-diaminopropionic acid monohydrochloride, D-2,3-diaminopropionic acid monohydrochloride, L-3- aminoisobutyric acid, D-3-aminoisobutyric acid, ethyl 3-aminobutyrate, sarcosine methyl ester hydrochloride and nipecotic acid, or an alkyl ester or pharmaceutically acceptable salt thereof. In a particular embodiment, the myeloperoxidase/oxidase may be formulated together with the amino acids, or alternatively the ammo acids may be supplied as a separate composition for premixing before administration, or simultaneous or concurrent administration.

[0051] Effective amounts of the amino acids which may be optionally employed in the compositions of the disclosure will vary depending on the amount of myeloperoxidase/oxidase in the compositions and conditions present in the environment of use. In an example, the compositions may generally comprise from about 0.1 to about 500 mM, more preferably from about 0.2 to about 100 mM, and even more preferably from about 0.3 to about 50 mM of each of the amino acids of the disclosure.

[0052j The compositions of the present disclosure may optionally comprise a halide. When the halide is chloride, the amount of chloride used in the compositions of the present disclosure will preferably fall in the range of about 10 pmol chloride to about 200 pmol per ml of solution (i.e., 10 to 200 mEq chloride/L) chloride. The physiologic concentration of chloride in plasma is about 105 mEq/L. When included, the compositions of the present disclosure may comprise from about 0.5 pmol bromide to about 20 pmol bromide per ml (i.e., 0.5 to 20 mEq bromide/L) of liquid composition, more preferably from about 1 pmol bromide to about 10 pmol bromide per ml (i.e., 1 to 10 mEq bromide/L) of liquid composition, and most preferably from about 100 nmol bromide to about 1 pmol bromide per ml of liquid composition.

[0053j The compositions may optionally comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions may be conveniently provided in a liquid carrier. Any liquid carrier may be generally used for this purpose, provided that the carrier does not significantly interfere with the selective binding capabilities of the myeloperoxidase or with enzyme activity. Alternatively, the compositions may be provided in solid form with activation on solubilization in liquid.

[0054j In embodiments that include a substrate for the peroxide-producing oxidase, the myeloperoxidase/oxidase system lends itself to construction as a binary formulation in which the composition's active agents are formulated in two separate parts for consolidation at the time of use. For example, the first composition of the binary formulation may comprise a solution containing both the myeloperoxidase and the oxidase. In some embodiments, the first composition may optionally comprise two orthree amino acids. In some embodiments, the three amino acids are glycine, l-alanine and l-proline. The second composition of the binary formulation may comprise a substrate for the oxidase, e.g., glucose (i.e., dextrose) in the case of glucose oxidase. The substrate may be provided, for example, in the form of a solid wafer. In some embodiments, the myeloperoxidase composition may additionally comprise alcohol in order to facilitate oxidase substrate solubilization and utilization by the oxidase.

[0055j In one embodiment, the methods of the present disclosure comprise administering to a site, prophylactically or therapeutically, a combination of compositions. For example, a first composition comprising myeloperoxidase and a peroxide-producing oxidase may be administered (optionally comprising at least two amino acids). A second composition comprising a substrate for the oxidase may be separate. In some embodiments, the first composition and the second composition are mixed before administration to the site of infection. In some embodiments the first composition and the second composition are administered concurrently to the site. In some embodiments the first composition and the second composition are administered sequentially to the site. The first composition and the second composition may be administered in any order.

[0056] For topical applications, the anti-viral compositions can be administered in any effective pharmaceutically acceptable form to warm blooded animals, including human and animal subjects, at any mucosal or epithelial surface. For example, the compositions of the disclosure may be administered in topical, lavage, oral, vaginal or rectal suppository dosage forms, as a topical, buccal, nasal spray, aerosol for inhalation or in any other manner effective to deliver active myeloperoxidase to a site of infection. The route of administration will preferably be designed to obtain direct contact of the compositions with viral pathogens. In one aspect of the present disclosure, the compositions of the present disclosure are delivered or administered topically to areas of a human or animal subject that are infected or susceptible to infection, such as, for example, to the gums, eyes, ears, skin, wounds, vaginal areas, groin areas, bed sores, burns, areas under medical dressings, diapers or other coverings which are likely to be moist, and the like.

[0057] For topical applications, the pharmaceutically acceptable carrier may take the form of liquids, creams, foams, lotions, ointments, suspensions, suppositories or gels, and may additionally comprise aqueous or organic solvents, buffering agents, emulsifiers, gelling agents, moisturizers, stabilizers, surfactants, wetting agents, preservatives, time release agents, and minor amounts of humectants, sequestering agents, dyes, perfumes, and other components commonly employed in pharmaceutical compositions for topical administration. In addition, the compositions of the present disclosure may be impregnated in dressings or coverings for application to a subject.

[0058] As an illustrative example, a composition suitable for use as an antimicrobial (or anti-infective) solution may comprise from about 1 to 50,000 pg/ml (i.e., from about 0.375 to about 18,750 GU/ml) of myeloperoxidase, from 0.01 to 500 units of glucose oxidase, and optionally: from 0.1 to 500 pmol/mL (i.e., from 0.1 to 500 mM) of glycine, from 0.1 to 500 pmol/mL (i.e., from 0.1 to 500 mM) of D-isoleucine, from O to 100 pmol/mL (i.e., from 0 to 100 mM) of L-alanine, and from 50 to 500 mEq/L of chloride. The above composition may be combined with from 1 to 500 pmol/mL (i.e., from 1 to 500 mM) of glucose or dextrose and used as a liquid disinfectant or sterilizing solution.

[0059] Further examples of the present disclosure are described below. However, it should be noted that the present disclosure should not be limited to these examples, and that the present disclosure is susceptible to variations, modifications and/or additions other than those specifically described, and it is to be understood that the present disclosure includes all such variations, modifications and/or additions which fall within the scope of the claims.

EXAMPLES

Example 1 - Materials

[0060] Glucose oxidase (GO) from Aspergillus Niger was purchased from Biozyme, Inc., UK, Cat #GO3A, 270 U/mg). Porcine Myeloperoxidase (p-MPO) (Exoxemis, Inc., Little Rock, Ark. U.S.A., 375 U/mg). Sterile stock solutions of D-glucose (dextrose) and sodium chloride were prepared and used at a final concentration of 150 mM each, [from US8945540B2 - appropriate here? Not discussed in BSL Report or draft paper]

[0061] Stock solutions of E-101 enzyme solution containing pMPO and GO and substrate solution containing glucose (dextrose) were prepared at Exoxemis, Inc. (Omaha, NE). The final concentrations of enzyme solutions were 100 GU/mL (0.248 mg pMPO/mL) and 50 GU/mL (0.124 mg pMPO/mL), prepared in saline just prior to use. The substrate solution (comprising dextrose) was prepared separately, and the enzyme and substrate solutions were mixed together to activate the system. The activated formulations were held at room temperature for about 20 +/- 5 minutes for oxidant generation before the addition of mucin and target viruses. Mucin (Sigma) was prepared in PBS to achieve a final concentration of 0%, 0.5%, and 1%.

[0062] Catalase (Sigma, Cat #C-40) was prepared as a 1% stock solution in sterile 0.9% normal saline. Dey/Engley (D/E) Neutralizing Broth was obtained from Sigma Aldrich (Cat # D3435).

[0063] Virus strains tested were Human Coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), Herpes Simplex Virus type 1 (ATCC #VR-260), Human Influenza A/WS/33, H1 N1 (ATCC #VR-1520), and Human Immunodeficiency Virus (HIV-1 , strain Mn; ZeptoMetrix Part #0810027CF). [0064] The host cells HCT-8, ATCC #CCI_-244; human colon adenocarcinoma, epithelial; Vero (African green monkey kidney cells [ATCC #CCL:-81]), Madin Darby Canis Kidney (MDCK [ATCC #CCL-34]), and H9 (human T-cell leukemia) cells (ATCC #HTB- 176) were used for the Virucidal Suspension Tests of Coronavirus, Herpes Simplex Virus type 1 , Influenza A/WS/33 H1 N1 and Human immunodeficiency Virus (HIV-1), respectively. Cells were maintained as monolayers in disposable cell culture labware. Prior to testing, host cell cultures were seeded onto 24- well cell culture plates. Cell monolayers were sufficiently confluent (80%) and less than 48 hours old before inoculation with virus. The growth medium (GM; RPMI with 10% FBS and 1% antibiotic and L- glutamine) was replaced by maintenance medium (MM; RPMI with 2% FBS and 1% antibiotic and L-glutamine) to support virus propagation.

[0065] The mucin preparation for 1% final mucin concentration was prepared as follows: 2.0 grams of mucin (Sigma) was added to 10 ml of DPBS (Gibco) and mixed well. 1 mL of the 20% mucin concentration was added to 1 mL of the virus suspension to achieve a concentration of 10% mucin. 0.5 mL aliquot of virus/10% mucin suspension was added to 4.5mL of the designated test concentration to achieve a final concentration of 1% mucin.

[0066] The mucin preparation for 0.5% final mucin concentration was prepared as follows: A 2.0mL aliquot of the 20% mucin solution was added to 2.0mL of DPBS and mixed well. 1 mL of the 10% mucin concentration was added to 1 mL of the virus suspension to achieve a concentration of 5% mucin. 0.5 mL aliquot of virus/10% mucin suspension was added to 4.5 mL of the designated test concentration to achieve a final concentration of 0.5% mucin.

[0067] A 1.0mL aliquot of maintenance medium was added to a 10mL aliquot of the virus suspension for the preparation of the neat virus.

Example 2 - Testing the virucidal properties of E-101 solution

[0068] The virucidal properties of E-101 solution at two concentrations (100 and 50 GU/mL) was challenged with Human Coronavirus, strain OC43 in presence of 0%, 0.5%, and 1% mucin. Additional challenge enveloped viruses tested at one concentration (100 GU/mL) without mucin added included Herpes Simplex Virus type 1 (ATCC #VR-260), Human Influenza A/WS/33, H1 N1 (ATCC #VR- 1520), and Human Immunodeficiency Virus (HIV-1 , strain Mn; ZeptoMetrix Part #0810027CF).

[0069] A Virucidal Suspension test in vitro time-kill method) based on the ASTM E1052-11 , Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension was used¹⁰. The Virucidal Suspension Test includes the following parameters: Virucidal suspension test, virus control, cytotoxicity control, neutralization control, neutralizer toxicity control, and cell culture control. In the virucidal suspension test, a 0.5 ml aliquot of test virus was added to 4.5 ml E-101 solution at a 90% (v/v) concentration to achieve a 100 or 50 GU/ml pMPO final E-101 Solution concentration/virus mixture. The mixtures were exposed for 15 and 45 minutes. Immediately after each 60 exposure time, the mixture was neutralized in Dey/Engley (D/E) Neutralizing Broth plus 1% catalase to stop virucidal activity and serially diluted in maintenance medium (MM). Each dilution was plated in four replicates.

[0070] A virus control consisted of a 0.5 ml aliquot of test virus added to 4.5 ml MM and exposed for 15 and 45 minutes at ambient temperature. Subsequent test virus dilutions were made in MM and serially diluted in MM. Each dilution was plated in four replicates. A cytotoxicity control consisted of a 0.5 ml aliquot of MM added to 4.5 ml of the highest concentration of E-101 solution. The MM/E-101 mixture was neutralized in D/E Neutralizing Broth plus catalase, mixed and serially diluted in MM. Each dilution was plated in four replicates.

[0071] A neutralization control consisted of a 0.5 mL aliquot of MM added to 4.5 mL of the highest concentration of E-101 solution. The MM/E-101 mixture was diluted 1 :10 in D/E Neutralizing Broth plus catalase. An aliquot of virus was added to the neutralized E- 101 solution, mixed and exposed to neutralize E-101 for 10 to 20 minutes. The effect of neutralizer on the virus infectivity was also assessed by adding neutralizer (D/E Neutralizing Broth plus catalase) alone followed by exposure for 10 to 20 minutes. Subsequent 10-fold serial dilutions of neutralized E- 101/virus suspension were made in MM. Each dilution was plated in four replicates. Intact cell culture replaced by MM in all cell control wells served as the control of cell culture viability. The plates were incubated in CO₂ for 7 to 21 days at 33°C + 2°C and monitored for cytopathic/cytotoxic effect.

[0072] Viral and toxicity titers were expressed -logw as 50% titration end point for infectivity. To calculate the viral titer, a 50% tissue culture infectious dose (TCIDso) was calculated by the Spearman-Karber method¹⁴. The logw reduction [(log TCIDso virus control) - (logwTCIDso Virucidal Suspension Test)] and percent reduction [(1 -TCIDso test/TCIDso virus control) x 100] in viral infectivity resulting from treatment were calculated. [0073] The results of the time-kill suspension test performed for Coronavirus OC43 when tested at a final E-101 Solution concentration of 100 GU/mL pMPO and 50 GU/mL in the presence of mucin are shown in Tables 1 and 2. No difference in results were observed for both concentrations of E-101 solution concentrations. E-101 Solution reduced the infectivity of coronavirus by >4.25 logw (>99.99%) at 15 minutes and 4.00 logw (>99.99%) at 45 minutes in the absence of mucin. E-101 Solution reduced the infectivity of coronavirus by >4.50 logw (>99.99%) at 15 minutes and 4.00 logw (>99.99%) at 45 minutes in the presence of 1% mucin. E-101 Solution reduced the infectivity of coronavirus by >4.50 logw (>99.99%) at 15 minutes and 4.25 logw (>99.99%) at 45 minutes in the presence of 0.5% mucin.

Table 1. Time-kill suspension test results for Coronavirus strain OC43 to E-101 solution at 100 GU/mL

GU = guaiacol unit. A GU is the amount of MPO enzyme that catalyzes the conversion of 1 micromole of hydrogen peroxide per minute at 25°C.

Table 2. Time-kill suspension test results for Coronavirus strain OC43 to E-101 solution at 50 GU/mL

[0074] The results of the time-kill suspension test performed for HSV-1 , HIV-1 , and

HIA/WS/33/H1 N1 viruses when tested at a final E-101 solution concentration of 100 GU/mL pMPO is shown in Table 3. E-101 solution reduced the infectivity of HSV-1 and HIV-1 by 5.50 logw (>99.99%) and 4.25 logw (>99.99%), respectively, following 15 and 45 minutes of exposure. E-101 solution reduced the infectivity of HIA virus, 2.00 logw (>99.00%) at 15 minutes and 4.25 logw (>99.99%) following 45 minutes of exposure.

Table 3. Time-kill suspension test results for HSV-1 , HIV-1 , and HIA/WS/33/H1 N1 virus to E-101 solution at 100 GU/mL

[0075] Discussion or mention of any piece of prior art in this specification is not to be taken as an admission that the prior art is part of the common general knowledge of the skilled addressee of the specification.

[0076] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the present disclosure.

[0077] The contents of all references, and published patents and patent applications cited throughout the application are hereby incorporated by reference. Those skilled in the art will recognize that the disclosure may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the disclosure.

BIBLIOGRAPHY

1. WHO Guidelines on Hand Hygiene in Health Care: First Global Patient Safety Challenge Clean Care Is Safer Care. Geneva: World Health Organization; 2009. Chapter

14, Skin reactions related to hand hygiene. https://www.ncbi.nlm.nih.gov/books/NBK144008/ 2. Myeloperoxidase and Eosinophil Peroxidase Inhibit Endotoxin Activity and Increase Mouse Survival in a Lipopolysaccharide Lethal Dose 90% Model, Robert C. Allen, Mary L. Henery, John C. Allen, Roger J. Hawks, and Jackson T. Stephens. J Immunol Res. 2019(3):1-10.

3. CDC, Interim Clinical Guidelines for Management of Patients with Confirmed Coronavirus Disease (COVID-19) https://www.cdc.gov/coronavirus/2019nCoV/hcp/clinical-guidance-137 mamagementpatient. html.

4. Cevik M, Bamford CGG, HO A. 2020. COVID-19 pandemic-a focused review for clinicians. Clin Microbiol Infect 26:842-847.

5. Mechanism of Microbicidal Action of E-101 Solution, a Myeloperoxidase-Mediated Antimicrobial, and Its Oxidative Products Gerald A. Denys, Neil C. Devoe, Polyxeni Gudis, Meghan May, Robert C. Allen, Jackson T. Stephens Jr. Infection and Immunity Jun 2019, 87 (7) e00261-19.

6. Denys GA, Grover P, O'Hanley P, Stephens JT, Jr. 2011. In vitro antibacterial activity of E-101 Solution, a novel myeloperoxidase-mediated antimicrobial, against grampositive and gram-negative pathogens. J Antimicrob Chemother 66:335-342.

7 Denys GA, Davis JC, O’Hanley PD, Stephens JT, Jr. 2011. In vitro and in vivo activities of E-101 solution against Acinetobacter baumannii isolates from U.S. military personnel. Antimicrob Agents Chemother 55:3603-3608.

8. Denys GA, Davis JC, O’Hanley PD, Stephens JT, Jr. 2014. E-101 solution, a novel myeloperoxidase mediated topical antimicrobial demonstrates in vivo efficacy in whole animal models of surgical infection prevention. J Antimicrob Photon 129:278-287.

9. Denys GA, Pillar CM, Sahm DF, O’Hanley P, Stephens JT, Jr. 2014. Five-year longitudinal 154 assessment (2008 to 2012) of E-101 solution activity against clinical target and antimicrobial-resistant pathogens. Antimicrob Agents Chemother 58:4911-4914.

10. Allen RC, Stephens JT, Jr. 2011. Myeloperoxidase selectively binds and selectively kills microbes. Infect Immun 79:474-485. 11. Allen RC, Stephens JT, Jr. 2011. Reduced-oxidized difference spectral analysis and chemiluminescence-based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes. Luminescence 26:208-213.

12. Ameta, SC, Punjabi PB, Chobisa CS, Mangal N, Bhardwaj R. 1990. Singlet molecular oxygen. Asian J Chem Rev 1 :106-124.

13. Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension ASTM International, West Conshohocken, PA 2011 , www.astm.org.

14 Schmidt N, Lennette DA, Lennette ET, Lennette EH, Emmons RW (ed). 1995. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, 7th ed. American Public Health Association, Washington, DC.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a virus infection in a human or animal subject, said method comprising administering to said subject an effective amount of a myeloperoxidase.

2. The method of claim 1 , wherein the myeloperoxidase catalyzes halide oxidation and disproportionation of peroxide to singlet molecular oxygen thereby inhibiting and/or killing said virus.

3. The method of claim 1 , further comprising administering an effective amount of peroxide or a peroxide-producing oxidase.

4. The method of claim 3, further comprising administering a substrate for the oxidase.

5. The method of claim 4, wherein the peroxide-producing oxidase is glucose oxidase and the substrate is glucose.

6. The method of claim 1 , wherein the myeloperoxidase is administered with a halide.

7. The method of claim 6, wherein the halide is a chloride or bromide.

8. The method of claim 1 , wherein the myeloperoxidase is administered in a first composition together with at least one further composition comprising one or more of: a halide, peroxide or a peroxide-producing oxidase, and a substrate for the peroxide-producing oxidase.

9. The method of claim 8, wherein said compositions are: premixed before administration, or administered concurrently or sequentially.

10. The method of claim 1 , wherein the myeloperoxidase is administered topically to a mucosal or epithelial surface.

11 . The method of claim 1 , wherein the virus is selected from the group consisting of: Sars-CoV-2, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and human influenza A virus (including H1 N1).

12. The method of claim 1 , which excludes administration of one or more of: a halide, a peroxide or peroxide producing oxidase, or a substrate for said oxidase.

13. A method of treating a virus infection in a human or animal subject, said method consisting of administering to said subject an effective amount of: a myeloperoxidase, and optionally one or more of: a halide, a peroxide or peroxide producing oxidase, a substrate for said oxidase, and a a pharmaceutically acceptable carrier.

14. A combination for treating a virus infection in a human or animal subject, said combination comprising: a myeloperoxidase, and at least one of a halide, and peroxide or a peroxide producing oxidase.

15. The combination of claim 14, wherein the myeloperoxidase catalyzes halide oxidation and disproportionation of peroxide to singlet molecular oxygen inhibiting and/or killing said virus.

16. The combination of claim 14, which is formulated in a composition that is for administration to said human or animal subject.

17. The combination of claim 14, which is formulated in at least two compositions, and wherein said compositions are: premixed for administration to said human or animal subject, or for administration concurrently or sequentially to said human or animal subject.

18. The combination of claim 14, wherein the peroxide-producing oxidase is glucose oxidase.

19. The combination of claim 14, wherein the halide is a chloride or bromide.

20. The combination of claim 14, comprising from about 1 to about 50,000 pg/ml of myeloperoxidase.

21. The combination of claim 14, wherein the peroxide-producing oxidase generates from 100 pmol to 50 pmol peroxide per ml per minute when in the presence of a substrate for the oxidase.

22. The combination of claim 14, which is formulated for topical administration to a mucosal or epithelial surface of said subject.

23. The combination of claim 14, wherein the virus is selected from the group consisting of: Sars-CoV-2, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and human influenza A virus (including H1 N1).

24. A combination for treating a virus infection in a human or animal subject, said combination consisting of: a myeloperoxidase, a halide, and peroxide or a peroxide producing oxidase, and optionally a substrate for said oxidase, and a pharmaceutically acceptable carrier.

25. The combination of claim 14, wherein excludes at least one of: a halide, or peroxide or peroxide producing oxidase.

26. A composition for treating a virus infection in a human or animal subject, said compositions comprising: a myeloperoxidase, and optionally one or more of: a halide, peroxide or a peroxide producing oxidase, a substrate for said oxidase, and a pharmaceutically acceptable carrier.

27. The composition of claim 26, wherein the myeloperoxidase catalyzes halide oxidation and disproportionation of peroxide to singlet molecular oxygen inhibiting and/or killing said virus.

28. The composition of claim 26, in which is the myeloperoxidase is formulated together with a peroxide producing oxidase.

29. The composition of claim 26, wherein the peroxide-producing oxidase is glucose oxidase.

30. The composition of claim 26, comprising from about 1 to about 50,000 pg/ml of myeloperoxidase.

31. The composition of claim 26, wherein the peroxide-producing oxidase generates from 100 pmol to 50 pmol peroxide per ml per minute when in the presence of a substrate for the oxidase.

32. The composition of claim 28, wherein said combination comprises about 10 to about 5,000 pg/ml of myeloperoxidase, and from about 1 to about 500 U/ml of glucose oxidase.

33. The composition of claim 26, which is formulated for topical administration to a mucosal or epithelial surface of said subject.

34. The composition of claim 26, wherein the virus is selected from the group consisting of: Sars-CoV-2, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and human influenza A virus (including H1 N1).

35. A composition for treating a virus infection in a human or animal subject, said composition consisting of: a myeloperoxidase, and optionally one or more of a halide, peroxide or a peroxide producing oxidase, a substrate for said oxidase, and a pharmaceutically acceptable carrier.