阅读说明：本技术 用于治疗或预防病毒感染的组合物和方法 (Compositions and methods for treating or preventing viral infections ) 是由 C·扎维拉 林明盛 于 2021-04-23 设计创作，主要内容包括：本文公开的组合物和方法可用于治疗或预防病毒感染。组合物可口服给予需要预防性或治疗性治疗的受试对象和/或组合物可用于表面消毒以防止或限制疾病通过表面接触传播。(The compositions and methods disclosed herein are useful for treating or preventing viral infections. The compositions can be administered orally to a subject in need of prophylactic or therapeutic treatment and/or the compositions can be used for surface disinfection to prevent or limit the spread of disease through surface contact.)

1. A composition for treating or preventing a viral infection, comprising:

humic acid or derivatives thereof; and

a peptide comprising the sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Lys (SEQ ID NO:1) (CZV 2.14).

2. The composition of claim 1, wherein the peptide consists essentially of the amino acid sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Lys (SEQ ID NO: 1).

3. The composition of claim 1, wherein the peptide consists of the amino acid sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Lys (SEQ ID NO: 1).

4. The composition of claim 1, wherein the humic acid and the peptide are covalently bound to each other, or wherein the humic acid and the peptide are ionically bound to each other, or wherein the humic acid and the peptide are electrostatically attracted to each other.

5. The composition according to claim 1, characterized in that the weight ratio of humic acid to peptide is between 500 and 100000.

6. The composition of claim 1, further comprising one or more pharmaceutical carriers, excipients, preservatives, colorants and/or diluents.

7. A method of treating or preventing a viral infection in a subject, comprising administering to the subject a therapeutically effective amount of the composition of claim 1.

8. The method of claim 7, wherein the peptide consists essentially of the amino acid sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Lys (SEQ ID NO: 1).

9. The method of claim 7, wherein the peptide consists of the amino acid sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Lys (SEQ ID NO: 1).

10. The method according to claim 7, wherein the humic acid and the peptide are covalently bound to each other, or the humic acid and the peptide are ionically bound to each other, or the humic acid and the peptide are electrostatically attracted to each other.

11. The method of claim 7, wherein the composition further comprises one or more pharmaceutical carriers, excipients, preservatives, colorants and/or diluents.

12. The method of claim 7, wherein the therapeutically effective amount is from 20 mg/kg/day to 50 mg/kg/day.

13. The method of claim 7, wherein the therapeutically effective amount is administered once daily, twice daily, or three times daily.

14. The method of claim 7, wherein the composition is administered orally.

15. The method of claim 14, wherein the composition is formulated as a capsule, tablet, emulsion, tincture, syrup, or food additive.

16. The method of claim 7, wherein the viral infection is selected from the group consisting of severe acute respiratory syndrome, middle east respiratory syndrome, human immunodeficiency virus, herpes simplex virus, cytomegalovirus, varicella zoster virus, epstein barr virus, influenza, hemorrhagic fever, respiratory syncytial virus, and combinations thereof.

17. The method of claim 7, wherein the viral infection is selected from the group consisting of SARS-CoV-2, HIV-1, HIV-2, HSV-1, HSV-2, H1N1, H3N2, RSV-2, and combinations thereof.

18. A method of disinfecting a surface, comprising:

dispersing the composition of claim 1 in a solvent to produce a mixture; and

contacting the composition with the mixture surface.

19. The method according to claim 18, characterized in that, the solvent has a neutral pH, an acidic pH or a basic pH.

20. The method of claim 18, wherein the solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, butanol, octanol, acetonitrile, benzyl alcohol, ethylene glycol, propylene glycol, dioxane, tetrahydrofuran, methyl acetate, ethyl acetate, acetone, potassium hydroxide, amines, aminoalcohols, phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, sulfonic acid, acetic acid, tartaric acid, lactic acid, citric acid, salicylic acid, C₅-C₂₀Carboxylic acids or combinations thereof.

Background

We are in epidemic disease caused by a highly contagious and fatal coronavirus (SARS-CoV-2, COVID-19) that infects the original population lacking natural immunity. Large-scale research efforts to develop vaccines are underway, but the only current method to slow the spread of disease is to have people around the world avoid contact with others as much as possible and put on protective masks when entering public places. These limitations have had an impact on people's livelihood, mental state, interpersonal relationships, and broader health. Therefore, there is an urgent need to find a composition having preventive and/or therapeutic effects on a novel coronavirus. Also, to ensure that we are not without much iron in the face of the next new virus, it would be beneficial to develop a composition that is effective against multiple viruses.

Humic Acid (HA) refers to a mixture of acids formed after decomposition/humification of organic matter in soil, water, peat and sediment. Stable compounds resistant to further decomposition and soluble in alkaline media in lignite or leonardite deposits are called humic acids. As early as 1992, researchers discovered the efficacy of humic acid on viruses. Neyts et al, "poly (hydroxy) Carboxylates as Selective Inhibitors of Cytomegalovirus and Herpes Simplex Virus Replication (Poly (hydroxy) Carboxylates as Selective Inhibitors of Cytomegalovirus and Herpes Simplex Virus Replication)," 3(4), 8.1.1992, page 215-222.) they hypothesize that the polyanionic form of HA present in the basal medium interacts with the positively charged domains of the viral envelope glycoprotein to block Virus attachment to the cell surface. Since then, more studies have shown that administration of HA prior to or simultaneously with exposure to viruses can not only prevent infection of HIV-1, HSV-2, VZV, EBV, H1N1 and H3N2 in a variety of cell lines, but can also serve as a therapeutic effect after the cells have been infected. (broad spectrum antiviral efficacy of natural and synthetic humates, virology subcontracts, antiviral research and antibacterial chemistry programs, microbiology and infectious disease departments, screening and testing programs for antiviral, immunomodulatory, antitumor and/or drug delivery activities, national allergy and infectious disease institute, national institutes of health, 2002, 9 days 8/2002.) the same studies show that HA is not cytotoxic in vitro at levels at least up to 100 μ g/mL, and another study finds that HA is non-toxic in vivo at concentrations up to 50 mg/kg. (Schiller, F. et al, Results of directed clinical trials of ammonium humate for the topical treatment of human Herpes Virus (HVH) infections (Results of a directed clinical trial of an oriented clinical trial of ammonium humate for the local treatment of human Herpes Virus (HVH) infections), "dermatology (Dermatols. Monatsschr.)1979 month 7; 165 (7): 505-9.) thus, humic acids are promising candidates for further study of compositions with antiviral prophylactic and/or therapeutic efficacy.

Disclosure of Invention

The compositions and methods disclosed herein are useful for treating or preventing viral infections. The compositions may be administered orally to a subject in need of prophylactic or therapeutic treatment and/or the compositions may be used for surface disinfection to prevent or limit the spread of disease by surface contact.

In one aspect, a composition for treating or preventing viral infection comprises humic acid and a peptide (CZ V2.14) comprising the sequence of Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Ly s (GEPPPGKPAKDAGK) (SEQ ID NO: 1).

In one embodiment, the peptide consists of the amino acid sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-As p-Ala-Gly-Lys (SEQ ID NO: 1).

In one embodiment, the peptide consists of the amino acid sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-As p-Ala-Gly-Lys (SEQ ID NO: 1).

In one embodiment, the humic acid and the peptide are covalently bound to each other, or the humic acid and the peptide are ionically bound to each other, or the humic acid and the peptide are electrostatically attracted to each other.

In one embodiment, the weight ratio of humic acid to peptide is between 500 and 100000, or between 5000 and 80000, or between 10000 and 70000, or between 25000 and 60000, or between 40000 and 50000.

In one embodiment, the weight ratio of humic acid to peptide is about 50000 (e.g., about 250mg humic acid to 5 μ g peptide per tablet).

In one embodiment, the composition for treating or preventing a viral infection further comprises one or more pharmaceutical carriers (carriers), excipients, preservatives, colorants and/or diluents.

In one aspect, a method of treating or preventing a viral infection in vivo comprises administering to a subject a therapeutically effective amount of a humic acid and peptide composition disclosed herein.

In one embodiment, the therapeutically effective amount is from 20 mg/kg/day to 50 mg/kg/day, or from 25 mg/kg/day to 40 mg/kg/day, or about 30 mg/kg/day. In one embodiment, the therapeutically effective amount is administered in portions once a day, twice a day, or three times a day.

In one embodiment, the composition is administered orally. For example, the composition may be formulated as a capsule, tablet, emulsion, tincture, syrup, or wet or dry food additive.

In one embodiment, the viral infection treated or prevented by the disclosed compositions is selected from the group consisting of Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), Varicella Zoster Virus (VZV), Epstein Barr Virus (EBV), influenza, hemorrhagic fever, Respiratory Syncytial Virus (RSV), or a combination thereof.

In one embodiment, the viral infection is selected from SARS-CoV-2, HIV-1, HIV-2, HSV-1, HSV-2, H1N1, H3N2, RSV-2, or a combination thereof.

In one aspect, a method of disinfecting a surface comprises dispersing a humic acid and peptide composition disclosed herein in a solvent to produce a mixture and contacting the composition with the mixture surface.

In one embodiment, dispersing includes dissolving, and in this case, the mixture is synonymous with the solution.

In one embodiment, the contacting step comprises wiping, dipping, spraying, dipping, or a combination thereof.

In one embodiment, the solvent has a neutral pH, an acidic pH, or a basic pH. Suitable solvents may for example be selected from water, methanol, ethanol, n-propanol, isopropanol, butanol, octanol, acetonitrile, benzyl alcohol, ethylene glycol, propylene glycol, dioxane, tetrahydrofuran, methyl acetate, ethyl acetate, acetone, potassium hydroxide, amines, amino alcohols, phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, sulfonic acid, acetic acid, tartaric acid, lactic acid, citric acid, salicylic acid, C₅-C₂₀Carboxylic acids or combinations thereof.

Detailed Description

Generally, the terms and phrases used herein have their art-recognized meanings as may be found by reference to standard texts, journal references, and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of this specification.

An "amino acid" is a molecular building block of a protein. An "amino acid residue" is the simplest discrete unit or monomer of a protein chain or peptide.

In the context of the present specification, the term "substantially purified" refers to a state of purity of at least 50%, preferably at least 70%, more preferably at least 85%, still more preferably at least 95%, and wherein the physiologically active peptide is present in the substantial absence of other physiologically active peptides or proteins.

Peptides with conservative amino acid substitutions are within the scope of the invention. A recognized principle of protein and peptide chemistry is that certain amino acid substitutions, known as "conservative" amino acid substitutions, can often be made in proteins or peptides without altering the conformation or function of the protein or peptide. These alterations comprise the substitution of any of these amino acids with any of isoleucine (L), valine (V), leucine (L); aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) in place of asparagine (N), and vice versa; serine (S) for threonine (T) and vice versa. Such substitutions are not the only amino acid substitutions that may be considered "conservative". Other substitutions may also be considered conservative, depending on the context of the particular amino acid. For example, glycine (G) and alanine (A) are generally interchangeable, as are alanine (A) and valine (V). Methionine (M) is relatively hydrophobic and is often interchangeable with leucine (L) and isoleucine (I), and sometimes with valine (V). The remarkable features of lysine (K) and arginine (R) in their amino acid residues are that the different pK values of the two amino acid residues are not significant, and their charge positions are often interchangeable. Cysteine (C) may typically be replaced by serine (S) when the ability of cysteine to form disulfide bonds is undesirable or not required. Other changes may be considered "conservative" in certain circumstances.

Nucleic acids encoding peptides

The peptides disclosed herein may be encoded by isolated nucleic acids. As used herein, the term "nucleic acid" encompasses both DNA and RNA as well as both single-stranded and double-stranded forms; if double-stranded, also includes DNA-RNA hybrids. According to the generally accepted base-pairing Watson-Crick rules, the listing of single-stranded nucleic acid sequences also includes the complement thereof. The nucleic acids encoding these peptides may be DNA or RNA; however, in many applications, DNA is preferred.

The term "isolated" is used herein to indicate that the nucleic acid is present in a form substantially separated from nucleic acid molecules that do not encode the peptides disclosed herein. In the context of the present specification, the term "isolated" refers to a state of purity of at least 50%, preferably at least 70%, more preferably at least 85%, and still more preferably at least 95%.

However, nucleic acids may be incorporated into larger nucleic acid molecules, such as vectors (vectors) used to transfect suitable host cells and produce peptides, and the term "isolated" should not be construed to exclude such incorporation into larger, genetically engineered molecules that do not naturally occur.

The nucleic acid sequence is selected to encode the amino acid sequence of a particular peptide according to the conventional triple genetic code. Since the genetic code for specifying an amino acid by a triplet codon in a nucleic acid sequence is degenerate and many amino acids are specified by more than one codon, all possible substitutions of codons may be used. However, in some cases, the efficiency of transcription and/or translation of a nucleic acid sequence may be affected by codon usage. In this case, it is preferred to use codons which increase the efficiency of transcription and/or translation of the nucleic acid sequence.

Vectors and host cells

Vectors comprising the DNA operably linked to at least one control element that affects the expression of the DNA are also contemplated. These control elements may be promoters, operators, enhancers or other nucleic acid sequences that affect the expression of the DNA. The vector may be derived from prokaryotic or eukaryotic sources. The vector may comprise a sequence of chromosomal, nonchromosomal or synthetic DNA sequences. Typically, these vectors contain one or more cloning sites containing restriction endonuclease sequences that are readily cleaved by specific restriction endonucleases. It is generally preferred that these restriction endonucleases produce sticky or "sticky" ends to more efficiently clone the desired sequence. Some suitable prokaryotic cloning vectors comprise plasmids from E.coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, or RP 4. Prokaryotic vectors also contain derivatives of phage DNA such as M13 and other filamentous single stranded DNA phages. Other vectors, such as baculovirus vectors, may be used.

Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of lambda phage, the control regions of fd coat protein, the glycolytic promoters of yeast (e.g., the promoter of 3-phosphoglycerate kinase), the promoters of yeast acid phosphatase (e.g., Pho5), the promoters of yeast alpha mating factor, and promoters from polyoma, adenovirus, retrovirus, and simian virus (e.g., the early and late promoters of SV 40), as well as other sequences known to control gene expression in prokaryotic or eukaryotic cells and viruses or combinations thereof. Vectors for yeast are available. A suitable example is the 2. mu. plasmid, which is a good example. Vectors for use in animal cells are also known. These vectors comprise derivatives of SV40, adenovirus, retrovirus-derived DNA sequences, and shuttle vectors that are a combination of functional mammalian vectors as described above (such as the vectors described above), as well as functional plasmid and phage DNA. Another suitable vector is a baculovirus vector. However, vectors suitable for expression in E.coli (E. coli) are generally preferred.

The vector is inserted into a host cell for expression. Typically, such vectors are inserted into host cells by methods well known in the art, such as transfection, transformation, electroporation, direct injection of DNA, lipofection, and other well known methods. The method used may be selected according to the host cell chosen and the size and conformation of the DNA. Some useful expression host cells include well-known prokaryotic and eukaryotic cells. Some suitable prokaryotic hosts include, for example, E.coli, e.g., E.coli SG-936, E.coli HB101, E.coli W3110, E.coli.1776, E.coli.2282, E.coli DHI, and E.coli MRCI. Other bacterial and fungal host cells may be used, such as Pseudomonas, Bacillus species such as Bacillus subtilis and Streptomyces. Other host cells which may be used are eukaryotic cells such as yeast and other fungi, insect cells, animal cells such as COS cells and CHO cells, human cells and plant cells in tissue culture.

Method for producing peptide

Solid state peptide synthesis

Peptides can be synthesized by standard solid-state Peptide Synthesis methods, such as those described in m.bodanszky, "(Principles of Peptide Synthesis)" (schpringer-Verlag, berlin, second edition, 1993). This involves synthesis on insoluble polymers such as derivatized styrene-divinylbenzene copolymers. The reaction sequence used is standard.

Genetic engineering

Peptides can be prepared by genetic engineering. Generally, a method of producing a substantially purified peptide having physiological activity comprises the steps of: (1) culturing a host cell transfected with a vector comprising DNA encoding the peptide, the DNA operably linked to at least one control element that affects expression of the DNA; and (2) isolating the peptide produced by the host cell to produce a substantially purified peptide.

Methods of Expression are described, for example, in d.v. goeddel, "" Gene Expression Technology "(Gene Expression Technology)" (Academic Press, san diego, 1991). Generally, such methods are well known in the art.

Once expressed, the peptides can be isolated by standard protein separation techniques including ion exchange chromatography on resins such as diethylaminoethyl cellulose or carboxymethyl cellulose, chromatography on size exclusion media (gel filtration), isoelectric focusing, chromatofocusing and other standard methods, for example r.k. scopes, "protein purification: principles and practices (Protein Purification: Principles and Practice) "(3 rd edition, (Springer-Verlag, New York, 1994).

If polyclonal or monoclonal antibodies are prepared against these peptides, these antibodies can be used for affinity chromatography by standard methods such as those described in the Scopes book above. Such methods for preparing polyclonal or monoclonal antibodies are well known in the art and need not be described in further detail herein. Typically, polyclonal antibodies are generated by injecting the peptide into an antibody-producing mammal, such as a rat, rabbit, sheep or goat, with or without a suitable adjuvant, such as Freund's complete adjuvant. The peptide may be conjugated to a carrier (carrier) protein such as keyhole limpet hemocyanin. Once a polyclonal antibody is produced, cells producing such polyclonal antibody can be fused with appropriate fusion partners by standard techniques to obtain hybridomas producing monoclonal antibodies of defined specificity.

Preparation method of humic acid

Humic acid can be extracted from any substance containing well-decomposed organic matter by treating the substance with a sodium hydroxide solution to dissolve the organic matter. Acid is then added dropwise to lower the pH to about 2, and the organic material flocculated to the top can be mechanically separated from the liquid portion. The flocculated material is humic acid, which when dried, and optionally crushed and sieved, forms a black solid called humate.

Humic acid is also commercially available from commercial suppliers.

Application method

The humic acid and peptide compositions disclosed herein can be used in a variety of ways. When used as a medicament, the compositions are usually administered orally in the form of capsules, tablets, emulsions, tinctures, syrups or food additives. When used as a surface disinfectant, the composition is typically dissolved or dispersed in a solvent to produce a solution or mixture for contacting (e.g., wiping, spraying, or otherwise immersing) a surface.

The preferred dosage of the drug is about 30mg/kg humic acid and polypeptide per day, with a weight ratio of about 50000. The dose to be administered may be determined by one of ordinary skill in the art based on the clinical severity of the problem, the age and weight of the patient, the exposure of the patient to conditions that may affect the chances of infection, the presence or absence of potential systemic problems (e.g., diabetes, circulatory disorders, and immune compromised states), and other pharmacokinetic factors generally understood in the art (e.g., liver and kidney metabolism). Freirich et al "Quantitative Comparison of Anticancer drug Toxicity in mice, rats, hamsters, dogs, monkeys and humans" (Quantitative companion of sensitivity of Anticancer Agents in mice, rats, Hamster, Dog, Monkey and Man) "," Cancer chemotherapeutic Agents "(Cancer chemi. Rep.), 50: animal-to-human dose correlations based on mg/kg of various sizes and species are described in 219-244 (1966). Dosage regimens may be adjusted to optimize prophylactic and/or therapeutic response. The dosage may be divided and administered daily or the dosage may be reduced proportionally according to the treatment.

The active ingredient is usually mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, and the like or combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers or pH buffers, and the like. For a more detailed description of the foregoing, reference is made to standard Pharmaceutical texts, such as Remington's Pharmaceutical Sciences, Mack publishing Co. easton, Pa. (1970).

The method according to the invention can be used for the treatment of humans or animal species of social or economic importance, such as dogs, cats, horses, sheep, cattle, goats or pigs. The method according to the invention is not limited to use in humans.

Pharmaceutical composition

In general, the pharmaceutical compositions disclosed herein comprise: (1) humic acid; (2) SEQ ID NO: 1; (3) optionally a pharmaceutically acceptable carrier.

Physiologically effective amounts can be determined by one of ordinary skill in the art with reference to the dosages described above.

Conventional pharmaceutically acceptable carriers (carriers) known in the art may comprise alcohols such as ethanol, serum protein, cholesterol, human serum albumin, liposomes, buffers such as phosphate, water, sterile saline or other salts, electrolytes, glycerol, hydroxymethyl cellulose, propylene glycol, polyethylene glycol, polyoxyethylene sorbitan, other surfactants, vegetable oils, and conventional antibacterial or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Pharmaceutically acceptable carriers (carriers) meet industry standards for sterility, isotonicity, stability and non-pyrogenicity.

The invention is illustrated by the following examples. These examples are for illustrative purposes only and are not intended to limit the present invention.

Examples

Example 1: therapeutic effect of Humic Acid (HA) and peptide (CZV2.14) composition on flu symptoms

This example compares the efficacy of a composition comprising Humic Acid + peptides on Influenza Symptoms to the results of Humic Acid alone using the protocol described in Amar, s., Ecovar, a Randomized, Double-Blind, Placebo-Controlled, Parallel Group Pilot Study on day 9, month 2, 2018, investigating the effect of Humic Acid on Influenza Symptoms (a Randomized, Double-blade, Placebo-Controlled, Parallel-Group pillow Study to investigatate the Effects of Humic Acid on Symptoms of Influenza).

The 20 participants were divided into treatment and placebo groups of 10 people each. Tables 1-4 show the results of the humic acid study versus the humic acid + peptide study.

Table 1. influenza symptom score.

TABLE 2 The percentage changes of TNF-. alpha.and IL-8 markers at week 2.

TABLE 3 weekly Visual Analog Scale (VAS).

Psychometric response scale for the study: wewers et al, 1994.

An increase in VAS score was observed in the HA + peptide compared to HA alone. In the HA group alone, symptoms were observed to be more severe at baseline and a lower VAS score.

Statistically significant increases in VAS scores (p 0.001) were observed from baseline to weeks 1 and 2 in the HA + peptide group and placebo, with greater increases in subjects from screening to weeks 1 and 2 in the HA + peptide group compared to placebo.

Table 4% change in CD4+ and CD8+ markers at week 2.

CD4+ and CD8+ T cells are useful biomarkers of impaired immune activity and identification of insufficient antibody responses in vivo. Researchers have found that many individuals have heterosubtype-specific CD4+ and CD8+ T cells that help recognize conserved internal epitopes common to different serotypes; also, in the presence of such heterosubtypic T cells, influenza individuals have reduced immunity, disease severity, and duration of infection. In this study, the cell count of T lymphocytes in CD4+ and CD8+ in serum was measured two weeks after treatment in subjects treated with humic acid or placebo. Although not statistically significantly different, the subjects who received humic acid had a 3% increase in the absolute number of CD4+ cells from screening to week 2, while the subjects who received placebo had a 3% decrease in the CD4+ cell count. It was found in this study that humic acid treatment actually increased CD4+ and CD8+ cells, suggesting that humic acid treatment may play a role in modulating human immune system responses.

Example 2: evaluation of the in vitro antiviral Properties of humic acid and polypeptide (CZV2.14)

A peptide (CZV2.14) comprising the sequence Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Lys-Asp-Ala-Gly-Lys (SEQ ID NO:1) is mixed with humic acid. The evaluation of the in vitro antiviral properties of the humic acid and the polypeptide shows that the virus killing activity of the humic acid and the polypeptide on HSV-1 at 3 days is 100 percent and the virus killing activity of the humic acid and the polypeptide on HSV-2 at 3 days is 60 percent, which is determined by a plaque test. In addition, there was complete viral attachment inhibition (100%) on day 3 for CMV and RSV-2 as measured by the virus yield assay. Without being bound by theory, it is believed that the polypeptide portion of the HA + CZV2.14 mixture binds to the spike protein (S-glycoprotein) of the virus, and HA associates with the polypeptide (e.g., by electrostatic attraction) forming a humic acid "shell" around the virus, thereby chemically and physically blocking the virus from entering the cell and preventing infection.

Example 3: in vivo evaluation of compositions comprising humic acid and peptide (CZV2.14)

Case #1

Singapore, 2 months in 2020: a6-mouth ambulance is sent to hospital for hospitalization, and the age is 20-54 years. COVID-19 was detected as positive.

Assay-24 hour treatment Window (PCR)

Treatment: comprising humic acid and comprising SEQ ID NO:1, 2 tablets or 2 capsules of 3 times daily for 3 days.

Emission Standard-negative assay X2, with 24 hours intervals

First assay, 24 hours-negative after initial treatment

Second assay, 48 hours after initial treatment-negative

All family members are discharged from hospital, #3 no complication occurs

Case #2

Hong kong, 3/4 months 2020:

background: central manager 1(CM1) contacts a dealer's business point to a customer known to be infected with COVID-19. CM1 was taking humic acid and CZV2.14 Product (Product) without illness, but a colleague (CM2) known to have no unadministered Product (CM2) was mixed with CM1 and the client did develop symptoms of fever, chills and vomiting. She was finally detected as positive for the COVID-19 test and was admitted to the hospital for hospitalization.

The hospitalization process is as follows: CM2 refused to take humic acid and CZV2.14 products in hospitals because her primary treatment provider is receiving another medication, she was concerned about accidental interactions; however, the symptoms worsen. The distributor received CM2 consent and started to take therapeutic doses of humic acid and CZV2.14 product three times a day, 3 tablets each, on 2 months 4 of 2020. Fever disappeared 24 hours after completion of the initial treatment dose. By 4 months and 4 days 2020, CM2 was free of any symptoms.

Note: the common practice in hong Kong is to publish the name of the COVID-19 infected person in a public record. The information of CM2 was published in 2020, 4 months and 2 days.

Employer responses: the dealer was aware of the CM2 infection on 31/3/2020 and turned off the local service for 4 days. At this time, all staff were instructed to take a therapeutic dose of humic acid and CZV2.14 product three times a day, 3 tablets each time. The dealer performs a PCR check on all employees before they return to work. All employees also participated in a nightly web workshop where they could learn the COVID-19 and ask questions about humic acid and CZV2.14 products to obtain compliance with the prophylactic dose of humic acid and CZV2.14 products.

Description of the products

Prevention guidance: 1 tablet/1 capsule is taken twice a day

The method for preventing the disease attack comprises the following steps: the capsule is administered 3 times daily in a dosage of 1 tablet/1 granule

Chronic diseases-diabetes and heart disease

Autoimmune diseases-rheumatoid arthritis, IBD, lupus

Excessive obesity

Genome inducers (NLRP3, CCL2, IL-1A)

Treatment, COVID-19 (+): 2 tablets/2 capsules are orally taken for 3 times a day

Treatment, COVID-19(+) onset: orally taking 3 tablets/3 capsules 3 times daily

Example 4: in vitro measurement of viral fusion inhibition and killing activity of CZV2.14 and CZV2.14+ humic acid on SARS-CoV-2 based on IC50 and IC90

20 SARS-CoV2 positive patients were treated with a composition comprising varying doses of Humic Acid (HA) and polypeptide (CZV 2.14). The results of the study are shown below.

¹Peak heat-day 1; AF is a non-exothermic temperature of 98.6 DEG F (37 ℃) or less

²tid is three times a day

³Gradually reducing to 750mg tid X1-4 days, 500mg bid X1-3 days, 250mg tid X1-2 days

⁴COVID-19Ab inception date + status ═ day 0

⁵S is symptomatic and AS is asymptomatic

¹Chang D, et al, Time Kinetics of Viral Clearance and symptom Resolution in Novel Coronavirus infections (Time Kinetics of Viral Clearance and Resolution of Symptoms in Novel Coronavir Infection) American respiratory and Severe Care medicine (Am J Respir Crit Care Med.) for 2020 years; 201(9):1150-1152。

doi:10.1164/rccm.202003-0524LE

Observations and conclusions

Chang reports patient population admission. The HA + peptide group received treatment in an outpatient setting. The standard of practice in china when conducting this study is to isolate infected persons by hospitalization. In any event, symptoms between the two studies indicate that they are a reasonable match (e.g., fever, cough, and dyspnea); however, the age range of the HA + peptide group (19-72 years) was broader than that of the conventional group (24-43 years).

High fever (greater than or equal to 102 ° f) was recorded in 7 individuals in the HA + peptide group. Of which 5 were over 50 years of age. Residual symptoms were observed in 1 patient (67 years) of the HA + peptide group, while 8 of 16 reported by Chang.

In Chang's data, 50% of the population develop residual symptoms after reaching the COVID negative state. In the HA + peptide group, 7.1 patients exhibited residual symptoms after adjustment to 6 patients with asymptomatic clinical course.

Observation # 1: 9 patients in the HA + peptide group were over 50 years old. The reduction of TNF-alpha (and IL-6) in the HA + peptide group may provide a more rapid clinical degradation of the viral inflammatory response.

Observation # 2: considering the variation of physiological adaptability with age, 9 patients aged over 50 years in the HA + peptide group had more residual symptoms. The anti-inflammatory activity of HA + peptides, in addition to their ability to enhance the immune system, may contribute to this improved outcome.

In Chang's data, people using HA + peptide between 24-43 years of age reached virus negativity 1.5 days earlier than their age matched cohort. The entire HA + peptide group, aged between 19-72 years, achieved virus negativity approximately 1 day before the data reported by Chang.

The average number of days of fever for the HA + peptide group was 1.7 days less than in the Chang report. Similar findings were also observed in the HA + peptide and influenza populations. Inhibition of TNF- α and enhancement of the immune system by HA + peptides may be beneficial to the recovery of an individual.

Statement regarding incorporation by reference and alteration

All references, such as patent documents, cited in this application include issued or granted patents or equivalents; patent application publications; and non-patent literature or other source materials; which is incorporated by reference herein in its entirety as if individually incorporated by reference.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the invention, and it will be apparent to those of skill in the art that the invention may be carried out using a wide variety of apparatus, apparatus components and method steps set forth in the present specification. As will be apparent to those skilled in the art, the methods and devices useful in the present methods and devices may comprise a wide variety of optional compositions and processing elements and steps.

When a group of substituents is disclosed herein, it is understood that all individual members and all subgroups of the group are disclosed separately. When a markush group or other group is used herein, all individual members of the group, as well as all possible combinations and subcombinations of the group, are intended to be individually included in the disclosure.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes a plurality of such peptides and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a", "an", "one or more" and "at least one" are used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably. The statement "according to any of claims XX-YY" (where XX and YY refer to claim numbering) is intended to provide multiple dependent claims in alternative forms and in some embodiments is interchangeable with the statement "as in any of claims XX-YY".

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such invention by virtue of prior disclosure.

Whenever a range is given in the specification, for example, an integer range, a temperature range, a time range, a composition range, or a concentration range, all intermediate ranges and subranges, as well as all individual values included in the given range, are intended to be included in the present disclosure. As used herein, a range specifically includes the values provided as the endpoints of the range. As used herein, a range specifically includes all integer values of the range. For example, a range of 1 to 100 specifically includes the endpoints 1 and 100. It should be understood that any subrange or individual value within a range or subrange included in the description herein may be excluded from the claims herein.

As used herein, "comprising" is synonymous with "including" or "containing" or "characterized by" and is used interchangeably, and is inclusive or open-ended and does not exclude additional, non-recited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of … does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. In each instance herein, any of the terms "comprising," "consisting essentially of …," and "consisting of …" can be substituted with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

All functional equivalents of the materials and methods known in the art are intended to be included in this disclosure. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

SEQUENCE LISTING

<110> C zavila

Lin Mingsheng

<120> compositions and methods for treating or preventing viral infections

<130> 9-20 US

<150> US 63/015,376

<151> 2020-04-24

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic peptide

<400> 1

Gly Glu Pro Pro Pro Gly Lys Pro Ala Lys Asp Ala Gly Lys

1 5 10