TW202017939A - Antiviral use of mussel adhesive proteins - Google Patents

Abstract

There is provided a mussel adhesive protein or a derivative thereof for use in the treatment of a viral infection and/or as an antiviral agent. Viruses that may be mentioned include herpes simplex, type 1 virus, herpes simplex, type 2 virus, human papillomavirus, influenza virus and parainfluenza virus.

Description

Antiviral use of mussel adhesion protein

The present invention relates to new uses of known compounds.

Viruses are very small organisms that contain genetic material (DNA or RNA) that can infect organisms. The virus invades the living cell and adsorbs itself to the living cell, after which it multiplies to produce more virus particles (viral particles), which are adsorbed to and enter the susceptible cells.

Viruses may kill cells or change cell functions, and their by-products cause infection of other cells. This then usually results in what is called a viral disease (or viral infection).

Generally, viruses infect only one type of cell, but can spread in many ways, including contact with infected individuals or their body secretions, animals (such as arthropods), or inanimate objects. The virus can also be spread by inhalation or swallowing.

After the virus infection, the biological immune defense system is triggered. Lymphocytes and monocytes try to attack and destroy aggressive viruses. This is called the body's innate or natural immunity. Innate immune responses can often cause patients to feel unwell or tired. If the patient's immune system is damaged, or is not effective enough to prevent the spread of the virus, this can cause severe discomfort, and in some cases, morbidity and/or death.

Antiviral drugs usually work by interfering with viral replication, such as slowing the rate of replication to increase the likelihood of innate immune response interference. There is clearly a clinical need for drugs that exhibit better and/or different antiviral properties.

Mussel adhesion proteins are also known as Mytilus edulis foot protein (mefp), which are proteins secreted by marine shellfish species such as edible mussels ( Mytilus edulis ), thick-shelled mussels ( Mytilus ccruscus ), and jade Mussels ( Perna viridis ). Adhesion proteins are secreted from the mycelium by mussels, where they are produced and stored in the mycelium. When secreted on the surface of a solid (such as rock and other solid objects, such as metal, wood, glass, etc.), a water-proof bond is formed, which fixes the mussel to the solid object. Mussels are usually attached to coastal reefs in groups or attached to the bottom of boats. The bond is extremely strong and has the ability to resist wave impacts in coastal waters.

Studies on edible mussels, Mediterranean mussels ( Mytilus galloprovincialis ), California mussels ( Mytilus californias ) and emerald mussels ( Perna viridis ) have so far identified 11 separate subtypes of adhesion proteins derived from mussel foot silk: mfp-1 (with It is called "mefp-1" and can be used interchangeably hereinafter), mfp-2/mefp-2, mfp-3/mefp-3, mfp-4/mefp-4, mfp-5/mefp-5, mfp-6 /mefp-6, collagen, pre-COL-P, pre-COL-D, pre-COL-NG, PTMP (proximal thread matrix protein) and DTMP (distal proximal silk matrix protein ( distal proximal thread matrix protein)). See, for example, Zhu et al., Advances in Marine Science , 32 , 560 (2014) and Gao et al., Journal of Anhui Agr. Sci. , 39 , 19860 (2011).

All mussel adhesion proteins (including their subtypes) have two structural features, including the following: (1) Lysine, so that the protein carries a high positive charge (due to the NH ₂ terminal); (2) 3, 4-Dihydroxyphenylalanine (DOPA, dopamine), whose catechol moiety is responsible for the formation of strong covalent bonds, thereby conferring the ability to bind mussel adhesion proteins to solid surfaces.

Products based on mussel adhesive protein products are currently used in a limited number of areas (including as tissue adhesives for microporous bonding, and for treating wounds and burns). Commercial products are either used directly as a solution for mussel adhesion proteins or stored as lyophilized powder to dissolve before use.

As far as the applicant is aware, the prior art does not specifically disclose the use of mussel adhesion proteins as antiviral agents to prevent the onset or spread of viral diseases.

According to the present invention, there is provided at least one mussel adhesion protein or derivative thereof, such as a pharmaceutically acceptable derivative, for use in the treatment of viral infections.

In the context of the present invention, the term "mussel adhesive protein" includes any adhesive protein that can be derived (isolated or extracted) from the silk of the mussel species, including those mentioned in this application and preferably edible mussels (blue mussels).

The term therefore includes full-length proteins, including all subtypes, which are or can be derived from mussels, such as collagen pre-COL-P, pre-COL-D and pre-COL-NG, mussel foot silk matrix proteins PTMP and DTMP , And more preferably mfp or mefp, such as mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, and especially mefp-1, and includes any mixture or combination of such proteins, such as mefps . Although according to the present invention, mixtures/combinations of the above-mentioned MAP subtypes can be provided as MAP "components", it is preferred that the purity of the main MAP subtype (e.g. mefp-1) is at least 25% by weight of the total amount of any such mixture %.

Therefore, at least one mussel adhesive protein, which is an essential element of the present invention, is hereinafter referred to as at least one "MAP". Mussel adhesion proteins are collectively or individually referred to as "MAP" below.

Known methods for extraction, preparation, separation and purification of naturally occurring MAP can be used, such as mixed adsorption chromatography (see Chinese Patent No. ZL200710179491.0), carboxymethyl ion exchange chromatography (see Chinese Patent No. ZL200710179492.5) and/or Salting out and dialysis (Chinese Patent No. ZL200910087567.6). Commercial sources of MAP include USUN Bio Co. (China; sold as MAP Medical Device®), BD Biosciences (USA), Kollodis (Korea), and Biopolymer (Sweden). Alternatively, MAP can be prepared using known recombinant DNA methods.

Derivatives of MAP include pharmaceutically acceptable derivatives, such as isolated lower molecular weight products (eg, molecular weights ranging from about 500 Da to about 2,000 (eg, about 1,200, such as about 800) Da, which may allow easier penetration through biofilms , Such as skin barriers or mucosal surfaces. Such derivatives may also include other compounds that contain the same sequence as the sequence already identified in the naturally occurring MAP or that are (eg, minor) variant amino acid sequences, and that the compound can be chemically And/or biological methods (such as chemical modification of naturally occurring MAP, or direct synthesis). I use "the (eg minor) variant of the amino acid sequence determined in naturally occurring MAP" to indicate that these sequences will not Changes that negatively affect the necessary properties of the relevant naturally occurring MAP to a measurable degree.

Derivatives of MAP include "MAP peptides" and their salts (eg cationic salts), which are decapeptides with the following sequence: Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (see Waite, Int. J. Adhesion and Adhesives , 7 , 9 (1987)). The MAP peptide can be derived and/or isolated as a low molecular weight derivative of naturally occurring MAP, or can be synthesized , for example, as described by Yamamoto in J. Chem. Soc., Perkin Trans. 1 , 613 (1987). See also Dalsin et al., J. Am. Chem. Soc. , 125 , 4253 (2003). Other derivatives include peptides of the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, their regioisomers, stereoisomers or salts, which can be prepared by similar techniques (see, For example, Kanyalkar et al. in Biomaterials , 389 (2002) and Belli et al. in Dental Materials , 26 , e125 (2010)).

Such derivatives of MAP can be used alone or in combination with one or more other such derivatives and/or one or more of the aforementioned full-length MAPs according to the invention.

As described below, the applicant has unexpectedly discovered that MAP and its derivatives have antiviral properties that can allow the treatment of viral infections themselves (ie treatment of viral infections) or viral diseases by interfering with the replication of the virus in the host, This is different from treating any symptoms of any viral infection or disease, such as pain and/or inflammation. Such antiviral properties may also allow the prevention of such infections or disease outbreaks, protect the host's cells from (eg, further) viral infections, prevent or prevent viral infections Or the spread of the disease (within a single host, or from one host to a new host), or to prevent reactivation of the virus after latent in the host.

According to yet another aspect of the present invention, there is provided a method of treating a viral infection, the method comprising administering at least one MAP or derivative thereof to a patient in need of such treatment.

"Patient" includes amphibian and preferably mammal (especially human) patients.

Viral infections that may be mentioned include those caused by viruses in the following families: adenoviridae (eg adenovirus), papillomavirus (eg human papillomavirus), polyomavirus (eg BK virus; JC virus), Herpesviridae (eg herpes simplex virus type 1; herpes simplex virus type 2; varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpes virus type 8), pox Viridae (e.g. variola virus), Hepatoviridae (e.g. hepatitis B virus), Parvoviridae (e.g. Parvovirus B19), Astroviridae (e.g. human astrovirus), Calicivirus (e.g. Noro Virus; Norwalk virus), MicroRNA virus family (eg Coxsackie virus, Hepatitis A virus; Poliovirus; Rhinovirus), Coronaviridae (eg severe acute respiratory syndrome virus), Flavivirus family (eg C Hepatitis virus; yellow fever virus; dengue virus; West Nile virus; tick-borne encephalitis virus), retroviridae (eg, human immunodeficiency virus; HIV), togaviridae (eg, rubella virus), sand Viridae (e.g. Reisa virus), Bunyavirudae (e.g. Hantavirus; Crimea-Congo hemorrhagic fever virus; Hantavirus), Filaviridae (e.g. Ebola virus; Marburg Virus; Ravn virus), Orthomyxoviridae (eg influenza viruses, including influenza A viruses (eg H1N1 and H3N2 viruses), influenza B virus or influenza C virus), paramyxoviridae (eg measles virus) Mumps virus; parainfluenza virus, respiratory fusion cell virus), rhabdoviridae (e.g. rabies virus), hepatitis virus family (e.g. hepatitis E virus), reoviridae (e.g. rotavirus; circovirus) ; Coltivirus; Banna virus), and viruses that do not belong to the family, such as hepatitis D virus.

Viruses that may be more specifically mentioned include herpes simplex virus type 1 and herpes simplex type 2 Herpes virus, human papilloma virus, influenza virus and parainfluenza virus.

According to the present invention, MAP/derivatives can be administered locally or systemically, such as oral administration, intravenous or intraarterial administration (including through intravascular and other perivascular devices/dosage forms (eg stents)), intramuscular administration , Skin administration, subcutaneous administration, transmucosal administration (e.g. sublingual or buccal administration), rectal administration, intravaginal administration, transdermal administration, nasal administration, lung administration (e.g. tracheal administration Or bronchial administration), topical administration or administration via any other parenteral route in the form of a pharmaceutical preparation containing the compound in a pharmaceutically acceptable dosage form. The topical administration form can be enhanced by creating a spray containing the active ingredient, for example, by using powder aerosols or by means of water mist using appropriate atomization techniques or equipment, such as sprayers.

Preferred modes of delivery of MAP and its derivatives include local delivery (e.g. to the skin or mucosal surface, including lung mucosa, in suitable (e.g., pharmaceutically acceptable) vehicles and/or commercially available formulations, or Preferably nasal or vaginal mucosa), but can also include oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular or intraperitoneal delivery.

The MAP/derivative will usually be administered in the form of one or more (eg, pharmaceutical) formulations mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier, which can be appropriately scheduled for administration The choice is based on the route and standard pharmaceutical or other (e.g. cosmetic) practices. Acceptable carriers can be chemically inert to the active compound and can have no deleterious side effects or toxicity under the conditions of use. Such carriers can also confer immediate release or modified release of the active ingredient.

Suitable pharmaceutical formulations may be based on commercially available or can be otherwise in accordance with the literature (e.g., Remington The Science and Practice of Pharmacy , 22 nd edition, Pharmaceutical Press (2012) , and Martindale-The Complete Drug Reference, 38 th Edition, Pharmaceutical Press (2014) and the documents mentioned therein, all relevant disclosures in all documents are incorporated by reference to the technique described in (). Otherwise, the preparation of suitable formulations containing MAP and its derivatives can be achieved by those with ordinary knowledge in the technical field using conventional techniques without creativity.

MAP (eg Mefp-1) and its derivatives may be in the form of aqueous formulations, such as emulsions, suspensions and/or solutions (eg (optional) buffered aqueous formulations (eg solutions), such as physiological saline-containing formulations (Eg solution), phosphate-containing formulation (eg solution), acetate-containing formulation (eg solution) or borate-containing formulation (eg solution)), or lyophilized powder.

In an alternative embodiment, the active ingredient can be combined with appropriate excipients to prepare: ● Gel formulations (gel matrix materials suitable for it include cellulose derivatives, carbomers and alginates, Xanthan gum, gelatin, pectin, carrageenan, gellan gum, starch, xanthan gum, cationic guar gum, agar, non-cellulosic polysaccharide, vinyl polymer, acrylic resin, polyvinyl alcohol, carboxyvinyl polymerization Substances, and especially hyaluronic acid); ● lotion (concentrate; suitable matrix materials for it include cellulose derivatives, glycerin, non-cellulose polysaccharides, polyethylene glycol and propylene glycol with different molecular weights); ● paste Or ointment (paste base materials suitable for it include glycerin, petrolatum, paraffin, polyethylene glycols with different molecular weights, etc.); creams or foams (excipients suitable for them (such as foaming agents) Including hydroxypropyl methylcellulose, gelatin, polyethylene glycol with different molecular weight, sodium lauryl sulfate, fatty alcohol sodium polyoxyethylene ether sulfonate, corn gluten powder and acrylamide); powder gas Sol (suitable excipients include mannitol, glycine, dextrin, dextrose, sucrose, lactose, sorbitol and polysorbate); and/or liquid (aerosol) spray for oral use or use For inhalation (suitable excipients include viscosity modifiers such as hyaluronic acid, emulsifiers, buffers, alcohols, water, preservatives, sweeteners, flavors, etc.).

Depending on the situation, the following substances may also be included in such formulations: humectants such as glycerin, glycerin, polyethylene glycol, trehalose, glycerin, petrolatum, paraffin oil, hyaluronic acid and their Salts (e.g. sodium and potassium salts), caprylic/capric acid triglycerides, and the like; and/or antioxidants such as vitamins and glutathione; and/or pH adjusting agents such as acids, bases and pH Buffer. In addition, the following may be included: surfactants/emulsifiers such as cetyl alcohol (cetyl alcohol), fatty acids (eg stearic acid), sodium lauryl sulfate (sodium lauryl sulfate), sorbitan esters (E.g. sorbitan stearate, sorbitan oleate, etc.), monoglycerides (such as glyceryl monostearate) polyethoxylated alcohols, polyvinyl alcohol, polyol esters, polyoxy Ethylene alkyl ethers (eg polyoxyethylene sorbitan monooleate), polyoxyethylene castor oil derivatives, ethoxylated fatty acid esters, polyoxyglycerides, lauryl dimethylamine oxide, bile salts (E.g. sodium deoxycholate, sodium cholate), phospholipid, N,N-dimethyldodecylamine-N-oxide, cetyltrimethylammonium bromide, poloxamer, lecithin , Sterols (such as cholesterol), sugar esters, polysorbates, and the like; preservatives, such as phenoxyethanol, ethylhexylglycerol, and the like; and thickeners, such as taurine propionyl diacetyl Methyl ester/VP copolymer. In particular, it may include stearic acid, glyceryl monostearate, cetyl alcohol, sorbitan stearate, cetyl alcohol, caprylic/capric acid glycerides, etc., especially in cream formulations.

MAP (e.g. Mefp-1) and its derivatives and formulations containing it (e.g. medicine) (e.g. aqueous solutions, gels, creams, ointments, concentrates/lotions, pastes and And/or foam) can be further combined with a suitable matrix material to prepare a dressing or therapeutic patch for application on a biological surface, such as the skin or mucosal surface. Such formulations can therefore be used to impregnate matrix materials such as gauze, non-woven fabric or silk paper. The therapeutic patch may be, for example, a band-aid, facial mask, eye mask, hand mask, foot mask, or the like.

Vaseline can be used to apply such dressings to wounds, but we have also found that PEG-based ointments can be combined with matrix materials to prepare dressings without the use of vaseline.

MAP and its derivatives can also be combined with one or more growth factors selected from the group consisting of platelet-type growth factors (including platelet-derived growth factor, PDGF); osteosarcoma-derived Sexual growth factor (ODGF), epidermal growth factor (EGF), transforming growth factor (TGFα and TGFβ), fibroblast growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve Growth factor (NGF), interleukin-type growth factor (IL-1, IL-1, IL-3), erythropoietin (EPO) and colony stimulating factor (CSF).

The administration of the active ingredient can be continuous or intermittent. The mode of administration may also be determined by the timing and frequency of administration, but in the case of antiviral therapy also depends on the severity (eg, potential) of the condition.

Depending on the condition to be treated and the patient and the route of administration, the active ingredient can be administered to patients in need thereof in different therapeutically effective doses.

Similarly, the amount of active ingredient in the formulation will depend on the (eg, potential) severity of the condition, and on the patient to be treated, but can be determined by those of ordinary skill in the art.

In any case, depending on the severity (eg, potential) of the condition and the route of administration, medical practitioners or other technicians will be able to routinely determine the actual dose that is most appropriate for the individual patient. The dosages mentioned in this application are examples of general cases; of course there may be individual cases where higher or lower dosage ranges are advantageous, and these are also within the scope of the invention.

The agent can be administered once a day to four times a day.

The appropriate concentration of MAP and its derivatives in the aqueous solution product may be about 0.01 (eg, about 0.1) to about 15.0 (eg, about 1.5) mg/mL, and the appropriate pH range is about 1.0 to about 7.0 (eg, about 3.0 to about 6.5), regardless of whether the formulation used is a combination formulation described in this application or a kit of parts. Suitable commercial sources of such aqueous solutions include USUN Bio Co., Jiangyin, Jiangsu Province, China.

A suitable local dose of MAP and its derivatives is a treatment area of about 0.1 to about 50 μg/cm ² , such as a treatment area of about 0.5 to about 20 μg/cm ² , including a treatment area of about 1 to about 10 μg/cm ² , for example A treatment area of about 2 to about 8 μg/cm ² , such as a treatment area of about 5 μg/cm ² .

In any case, the dose administered to mammals (particularly humans) in the context of the present invention should be sufficient to produce an appropriate therapeutic response in the patient within a reasonable period of time (as described above). Those of ordinary skill in the art will recognize that the choice of the exact dosage and composition and the most appropriate delivery protocol will also be affected by (and others): the pharmacological properties of the formulation, the condition to be treated Nature and (eg, potential) severity, and the recipient's physical condition and mental sensitivity, as well as the age, condition, weight, sex, and response of the patient to be treated, and the stage and/or potential severity of any disease, and the patient Genetic differences.

In the uses and methods described in this application, MAP and its derivatives can also be combined with one or more active ingredients having antiviral properties. Such patients may therefore (and/or already) receive therapy based on the administration of one or more such other active ingredients, which means that before treatment with MAP/derivatives, in addition to treatment with MAP/derivatives, and/or Or receive a prescribed dose of one or more of the active ingredients mentioned in this application after treatment with MAP/derivatives.

Such antiviral agents that can be used in combination with MAP/derivatives in the treatment of viral infections include standard vaccines that can be used against the viral infection in question, as well as known and/or appropriate antiviral drugs, such as abacavir (abacavir), acyclovir, adefovir, amantadine, amprenavir, amprenavir, ampligen, arbidol , Atazanavir, atripla, Balavir, cidofovir, combivir, dolutegravir, darunavir, deira Delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabirie, enfuvirtide (enfuvirtide), entecavir, ecoliever, famciclovir, fomivirsen, fosamprenavir, foscamrena, fosfonet, fusion inhibitor, ganshine Ganciclovir, ibacitabine, isopropyl muscle Glucosides (imunovir), iodine (idoxuridine), imiquimod (imiquimod), indinavir (indinavir), inosine, integrase inhibitors, interferon, type I interferon, type II interferon, Type III interferon, lamivudine, lopinavir, loviride, maraviroc, moroxydine, indothiazine methisazone), nelfinavir, nevirapine, Nexavir, nitazoxanide, nucleoside analogues, Novir, oseltamivir, pegylated interferon alpha-2a , Penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, Pyramidine, raltegravir, reverse transcriptase inhibitors, Ribavirin, rimantadine, ritonavir, saquinavir, sofosbuvir, stavudine, synergistic enhancer ( (Retroviral), telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine ), trizivir, tromantadine, truvada, valaciclovir, valganciclovir, Vicriviroc, vidarabine , Viralidine (viramidine), zalcitabine (zalcitabine), zanamivir (zanamivir) and zidovudine (zidovudine). Antiviral drugs that may be specifically mentioned include acyclovir.

Other antiviral agents that can be used in combination include enzymes, such as trypsin, which can be combined with MAP or MAP derivatives, especially with Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys or salts thereof, Or Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys or a salt combination thereof.

When MAP/derivatives are "combined" with other antiviral agents in this way, the active ingredients can be co-administered in the same formulation, or administered separately (simultaneously or sequentially) in different formulations.

Such a combined product provides MAP and its derivatives to be co-administered with other antiviral agents, and thus can be in separate formulations, wherein at least one of these formulations includes MAP/derivatives, And at least one contains other antiviral agents, or may be (ie, formulated into) a combined preparation (ie, a single formulation containing MAP/derivative and other antiviral agents).

Therefore, there is further provided: (1) a pharmaceutical formulation comprising: at least one MAP or its derivative; another antiviral agent; and a pharmaceutically acceptable adjuvant, diluent, or carrier (the formulation is described below) (Referred to as "combination formulation"); and (2) a kit of parts, which contains the following components: (A) a pharmaceutical formulation, which contains: at least one MAP or its derivative and a pharmaceutically acceptable adjuvant, diluent Or a mixture of carriers; and (B) a pharmaceutical formulation comprising: a mixture of another antiviral agent and a pharmaceutically acceptable adjuvant, diluent or carrier, wherein components (A) and (B) are each suitable Provided in the form of a joint offer with another.

According to yet another aspect of the present invention, there is provided a method of preparing a component kit as defined above, the method comprising combining component (A) as defined above with component (B) as defined above, thereby making the two groups Points are suitable for joint giving to each other.

By "joining" the two components with each other, I request that the components (A) and (B) of the component set can: (i) be provided as separate formulations (ie independent of each other), then Place them together to be used in combination with each other in combination therapy; or (ii) packaged and presented together as individual components of a "combination pack" to be used in combination with each other in combination therapy.

Therefore, a kit of parts containing the following is further provided: (I) one of the components (A) and (B) as defined above; together with (II) instructions for use, to combine this component with the other of the two components Joint use.

When the word "about" is used in this application, for example, in the amount (such as the concentration of the active ingredient and /Or dosage, molecular weight or pH), it should be understood that such variables are approximate values, which in their own right can vary within the following range: the number specified in this application ± 10%, such as the number specified in this application ± 5%, preferably ±2% (eg ±1%). In this regard, the term “about 10%” means, for example, ±10% around the number 10, that is, between 9% and 11%.

The method described in this application may also have the advantage that, in the treatment of the conditions mentioned above, compared to similar methods (treatments) or others known in the prior art for the treatment of viral diseases or infections or others , The method described in this application may be more convenient for doctors and/or patients, more effective, less toxic, have a wider range of activities, stronger, produce fewer side effects, or may have other useful pharmacology nature.

The invention is illustrated by the following examples.

Example 1 Effect of MAP on the activity of human herpes simplex virus type II (HSV-2).

Serum-free 1640 medium (RPMI1640 medium; GIBCO/BRL; Thermo Fisher Scientific China, Nanjing, China) was prepared using standard methods. Newborn bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd. Luoshe, China) is formulated into a complete medium containing 10% serum before use, or it is formulated into a maintenance solution (maintenance solution) by adding 2% of the same serum ).

A MAP lyophilized powder was prepared by freeze-drying a MAP solution (USUN Bio Co., Ltd.), having a MAP purity of 87.04%, and the pH of the original solution was 4.5. Dissolve lyophilized powder (6.02mg) in 301 μL sodium chloride aqueous solution (in aqua pro injection, Jiangsu Hengrui Medicine Co., Ltd, Jiangsu Province, China) to prepare a 20 μg/μL stock solution.

0.05 mL of the stock solution was added to 1.95 mL of complete medium to prepare a 500 μg/mL drug solution (the maintenance solution was used instead of the complete medium in the antiviral tests No. 3 and 4 below). Then, the concentration of 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.625 μg/mL, 7.8125 μg/mL, 3.9063 μg/mL, 1.9531 μg/mL and 0.9766 μg/ were prepared by double dilution. mL of working solution.

20.34 mg of sodium lauryl sulfonate (SDS; manufactured by AMRESCO LLC, Solon, OH, USA, and packaged by Biosharp Company, Hefei, China; purity: 99%) was dissolved in 10.17 mL of medium (again, in the antiviral test Use a maintenance solution instead of the medium) to prepare a 2000 μg/mL stock solution. Then, a working solution having the above concentration is prepared by double dilution.

2.25 mg of acyclovir (ACV; Zhiyuan Pharmaceutical Co., Ltd, Wuxi City, China; purity: 99.3%) was dissolved in 2.25 mL medium (again, a maintenance solution was used instead of the medium in the antiviral test) to form 1000g/mL stock solution. Then, the 0.8 mL stock solution was doubled diluted to obtain concentrations of 500 μg/mL, 250 μg/mL, and 125 μg/mL. 0.2 mL of the stock solution was added to 1.95 mL of medium to obtain a concentration of 100 μg/mL, which was then diluted to 50 μg/mL, 25 μg/mL, and 12.5 μg/mL.

1. HSV-2 virus toxicity test

Inoculate 0.5 mL of a suspension of human herpes simplex virus type II (HSV-2; SAV strain; Shanghai Institute of Cell Biology) into a monolayer culture of Vero cells (Shanghai Institute of Cell Biology) and adsorb for 1 hour Then remove.

The maintenance solution was replaced and cultured at 37°C (under 5% CO ₂ ) until more than 95% of the cells showed obvious pathological changes under a microscope (Nikon ECLIPSE TS100 inverted phase control microscope (with imaging system)). The cells were harvested, repeatedly frozen and thawed, and centrifuged at 3000 rpm for 10 minutes in a 400C medical low-speed centrifuge (Beijing Baiyang Centrifuge Co., Ltd.). The supernatant was collected as a virus solution.

The Vero cell suspension with a density of 2×10 ⁵ was inoculated into a 96-well culture plate (Costar, Corning Inc., Oneonta, NY, USA) at 0.1 mL/well at 37° C. (under 5% CO ₂ , Thermo Scientific CO ₂ incubator) for 18 hours until a single layer is visible under the microscope. The virus collected above was diluted 10 times with the maintenance solution and seeded into monolayer Vero cells at 0.1 mL/well. Refill the maintenance solution and incubate it at 37°C (under 5% CO ₂ ). After culturing for 24 hours, the pathological changes of the cells were observed under a microscope. Each dilution factor was repeated in 3 wells. Normal cells were used as controls in the experiment. Repeat the virus virulence test 3 times.

For each well, three visual fields are observed. Determine the average percentage of pathological cells (P) in the field of view.

The median infectious dose (TCID ₅₀ ) of the virus is calculated according to the conventional method of Reed and Muench, that is, TCID ₅₀ , which is the logarithm of the next dilution rate of pathological changes higher than 50% + proportional distance (proportionate distance) , PD) × logarithm of the dilution factor. PD is (the percentage of pathological changes above 50% minus 50%) divided by (the percentage of pathological changes above 50% minus the percentage of pathological changes below 50%). The measured TCID ₅₀ values given by the three experiments were 4.89, 4.06 and 4.55, respectively.

2. Cytotoxicity of MAP and control drugs

Vero cells were seeded on 96-well culture plates and grown into monolayers. For each well, 0.2 mL of MAP or control drug was added at different concentrations (as described above). For each concentration, repeat this in 3 wells. The solvent and normal cell culture were used as negative controls. The cells were cultured at 37°C (5% CO ₂ ), and the growth and morphological changes of the cells were observed under the microscope for 2 days. Three fields of view were selected for each well, the percentage of pathological cells was counted, and the average value was calculated. Set the time point of the test to 24 hours, and calculate the half-toxic concentration (TC ₅₀ ) and the maximum non-toxic concentration (TC ₀ ). Repeat the experiment 3 times.

The cells were seeded as described above. The solvent and normal cell culture were used as negative controls. 24 hours after the addition of MAP and control drug, 5 mg/mL 3-(4,5-dimethyl-2-thiazolyl) in PBS (diluted from 10 x stock solution, Sigma-Aldrich (China)) was introduced -2,5-diphenyl-2-H-tetrazolium bromide (MTT; Sigma-Aldrich (China), Shanghai, China) (20 μL/well), and culture was continued for 4 hours. Then, the supernatant in each well was discarded, 150 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich (China)) was added, and then shaken in the dark at room temperature for 10 minutes.

The light absorption value (OD ₅₅₀ ) at 550 nm was measured by an enzyme-linked immunosorbent meter (MULTISKAN SPECTRUM; Thermo Scientific, Shanghai, China).

62.5μg/mL；SDS的TC₅₀>62.5μg/mL和TC₀

62.5μg/mL；ACV的TC₅₀>1000μg/mL和TC₀

250μg/mL。 The results of three parallel experiments performed by observing the cell morphology under the microscope showed that the TC _{50 of} MAP> 125 μg/mL and TC ₀

62.5μg/mL; TC _{50 of} SDS> 62.5μg/mL and TC ₀

62.5μg/mL; TC _{50 of} ACV>1000μg/mL and TC ₀

250μg/mL.

The results obtained from the MTT method indicate that MAP is not cytotoxic to Vero cells in the range from 500 to 15.625 g/mL. SDS at 62.5g/mL and below showed no cytotoxicity to Vero cells. The maximum concentration of ACV showing significant cytotoxicity to Vero cells is 100g/mL.

3. The effect of MAP and SDS on the cytopathic effect of virus after directly acting on HSV-2

The TCID ₅₀ stored at -80°C (in the Haier DW-86L486 ultra-low temperature freezer) with the determined HSV-2 virus was diluted to 200 TCID ₅₀ with a determined titer. The 200 TCID ₅₀ solution was mixed with an equal volume of MAP or SDS liquid, and the virus titer at this time was 100 TCID ₅₀ . The mixed solution was incubated in a water bath (DK-8B constant temperature electric hot water bath; Shanghai Jinghong Biotech Co., Ltd.) at 37°C for 1 hour, and then seeded into a 96-well culture plate containing monolayer Vero cells. Add 0.1 mL to each well.

After 1 hour of adsorption, the supernatant containing virus and drug was discarded. Then, the monolayer Vero cells were washed twice with the maintenance solution. Finally, a maintenance solution (0.2 mL/well) was added. The resulting mixture was continuously cultured at 37°C (5% CO ₂ ) until the cell-free rate of the drug-free medium reached 95% under the microscope. Set the evaluation time point of the test to 24 hours.

In addition to the experimental group, three control groups (solvent, no drug control (virus control) and normal cell control) were also tested in parallel. Each group consisted of 3 wells, and the experiment was repeated 4 times.

Dilute the virus to 0.1 TCID ₅₀ , 1 TCID ₅₀ , 10 TCID ₅₀ , 100 TCID ₅₀ and 1000 TCID ₅₀ and inoculate them into monolayer cell culture. Each dilution rate was repeated three times. Observe the cytopathic rate of each well (at 0.1 TCID _{50 there} should be no cytopathic effect, and the cytopathic effect should be visible at 100 TCID ₅₀ ; otherwise the neutralization test is not established).

The evaluation index is the same as that of the virus toxicity test. For each hole, observe three fields of view. Determine the average percentage of pathological cells (P) in the field of view. Median infective dose of virus (TCID ₅₀₎ calculated according to the Reed and Muencl conventional methods (as described above).

The toxicity of drugs to cells is determined by cell morphology, and antiviral tests are conducted at non-toxic concentrations. After culturing with different concentrations of MAP and SDS for 1 hour, 100 TCID ₅₀ HSV-2 (SAV strain) was inoculated into monolayer Vero cell culture. The cytopathic effect of cells caused by viral infection was suppressed to varying degrees, indicating that MAP has an inhibitory effect on HSV-2.

Using 250g/mL as a stock solution, the neutralization titers of MAP determined by these four experiments were 1:4.6 (54.88 μg/mL), 1:2.8 (88.39 μg/mL), 1:2.6 (96.39 μg/mL) ) And 1: 4.4 (56.61 μg/mL).

The control drug SDS also showed an inhibitory effect on HSV-2. Using 125g/mL as a stock solution, the neutralization titers of SDS are 1:5.7 (22.10μg/mL), 1:5.7 (21.93μg/mL), 1:5.8 (21.46μg/mL) and 1:6.0 (20.86) μg/mL).

4. The effect of MAP and ACV on HSV-2 (direct method)

The virus was diluted to 100 TCID ₅₀ and inoculated into monolayer Vero cell culture at 0.1 mL in each well. After 1 hour of adsorption, the supernatant was discarded and washed with the maintenance solution twice. Then, different concentrations of MAP or control drug ACV were added at 0.2 mL/well. The culture was continuously cultured at 37°C (5% CO ₂ ). Each concentration was repeated three times, and the solvent, virus without drug treatment (virus control) and normal cell culture (cell control) were set as controls.

During the cultivation period, the pathological changes were observed under a microscope, and the experiment was terminated when the cytopathic rate of the virus control reached >95%. The evaluation time point of the experiment was 24 hours, and the experiment was repeated 3 times.

The judgment index is the same as that of the virus toxicity test, that is, three fields of view are selected for microscopic examination in each well, the percentage of diseased cells (P) in the field of view is determined, and the average of the three fields of view is taken.

The linear regression equation was calculated according to the percentage of each reagent concentration group's effect on the cytopathy of drug concentration. Calculate the IC ₅₀ value and also the significance test of the correlation coefficient.

Antiviral tests are conducted at non-toxic concentrations. When treating HSV-2 infected Vero cells with different concentrations of MAP or ACV, MAP showed a certain degree of inhibitory effect on the cytopathic effect of Vero cells due to infection with 100 TCID ₅₀ HSV-2, where IC ₅₀ >125 μg/ mL. ACV control drugs showed IC ₅₀ value of 6.38μg / mL, 2.20μg / mL and 5.36μg / mL.

The results show that MAP powder has an effect on the activity of HSV-2, and can protect 50% of cells from pathological changes in the range of 54.88 μg/mL to 96.39 μg/mL.

Example 2 Effect of MAP on the activity of human herpes simplex virus type 1 (HSV-1)

Serum-free 1640 medium line used RPMI1640 powder (1000 mL dose; Thermo Fisher Scientific China), L-glutamine (0.29 g; Sinopharm Chemical Reagent Co. Ltd, Shanghai, China), sodium bicarbonate (2.2 g; Sinopharm Chemical Reagent Co.), HEPES (2.39 g; Thermo Fisher Scientific China) and deionized water (1000 mL) were prepared.

The reagents are mixed, dissolved and filtered to sterilize. This mixture is formulated into a complete medium containing 10% serum by adding newborn bovine serum before use, or it is formulated into a maintenance solution by adding 2% of the same serum.

The MAP stock solution was prepared as follows: A MAP solution with a concentration of 4.0 mg/mL was diluted with sodium chloride to prepare a 20 μg/μL stock solution.

After that, using the human herpes simplex virus type 1 (HSV-1; SAV strain; Shanghai Institute of Cell Biology), substantially the same test as described in Example 1 above was performed.

In the HSV-1 virus toxicity test, the measured TCID ₅₀ values given by the three tests were 4.09, 4.13 and 4.26, respectively.

The cytotoxicity of the MAP and control drug tests gave essentially the same results as reported in Example 1 above.

The results of the direct measurement show that the MAP solution has an effect on the activity of HSV-1, and can protect 50% of the cells from pathological changes within the range of 44.64 μg/mL to 58.14 μg/mL (MAP determined by these four experiments The neutralization titers are 1:5.6 (44.64 μg/mL), 1:4.9 (51.02 μg/mL), 1:4.3 (58.14 μg/mL) and 1:5.2 (48.08 μg/mL).

The results of indirect measurements indicate that the MAP solution has an inhibitory effect on the cytopathic effects of Vero cells caused by infection with HSV-1, and its value is 50.3 μg/mL.

Example 3 Effect of MAP on the activity of human influenza A virus H1N1

The attacking virus is human influenza A virus (H1N1) (Wuxi Center for Disease Control, Wuxi, China).

MDCK cells (ATCC CCL-34; Wuxi Center for Disease Control, Wuxi, China) in DEME medium (DEME high glucose medium containing L-glutamine; Thermo Fisher Scientific China) together with 10% FBS (fetal bovine serum, Thermo Fisher Scientific China). The culture medium system was prepared as follows: 50 mL of FBS and 5 mL of penicillin and streptomycin stock solutions (containing 10000 U/mL penicillin G and 10000 g/mL streptomycin sulfate) were added to 1.445 mL of DEME solution. The resulting material is sterilized by filtration.

The virus growth medium system was prepared as follows: trypsin (Thermo Fisher Scientific China) treated with 1 μg/mL TPCK [6-(1-toluenesulfonylamino-2-phenyl)ethyl chloromethyl ketone] was added Into DEME medium containing 1% HEPES (1M stock solution) and 1% glutamine.

6.4 mg of MAP lyophilized powder (prepared as described in Example 1 above) was dissolved in 1000 μL of aqueous sodium chloride solution to prepare 6.4 μg/μL of stock solution. 0.1 mL of stock solution was added to 0.9 mL of complete medium to prepare 640 μg/mL drug solution. Then, working solutions with concentrations of 320 μg/mL, 160 μg/mL, 80 μg/mL, 40 μg/mL, 20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, and 1.25 μg/mL were prepared by double dilution.

MDCK cells were seeded on 96-well culture plates and grown into monolayers. 0.2 mL of MAP at different concentrations (as described above) was added to each well. For each concentration, repeat this in 3 wells. The solvent and normal cell culture were used as negative controls. The cells were cultured at 35°C (5% CO ₂ ), and the growth and morphological changes of the cells were observed under the microscope for 2 days. Three fields of view were selected for each well, the percentage of pathological cells was counted, and the average value was calculated. Set the time point of the test to 24 hours, and calculate the half-toxic concentration (TC ₅₀ ) and the maximum non-toxic concentration (TC ₀ ). Repeat the experiment 3 times.

40μg/mL。直接測定的結果表明，MAP溶液對H1N1的活性具有影響，且在38.54μg/mL至31.14μg/mL的範圍內可以保 護50%的細胞免受病理變化。 The results obtained in three parallel tests conducted by morphological observation under a microscope showed that the cells, MAP of TC _50> 80μg / m and TC ₀

40μg/mL. The results of direct measurement show that MAP solution has an effect on the activity of H1N1, and can protect 50% of cells from pathological changes in the range of 38.54 μg/mL to 31.14 μg/mL.

Example 4 MAP hydrogel for the treatment of vaginal HPV infection

Prepare a 100g MAP hydrogel consisting of: MAP (0.1g; USUN Bio Co., Ltd.), methyl cellulose (2.5g), propylene glycol (11g), glycerin (11g), acetic acid (pH adjuster) 0 to 0.5 g (all from Sinopharm Chemical Reagent Co. Ltd.), supplemented with water for injection.

Recruit patients with HPV infection but no cytopathic changes. MAP hydrogel is used once a day for 30 days. Clean hands and perineum before use. The gel was placed in a storage tank of an Ollery medical compression nebulizer (Guangzhou Demi Medical Equipment Co. Ltd., China), and the nozzle was connected to the tube of the nebulizer.

Then, insert the catheter deep into the vagina and use an atomizer until all the gel has been atomized.

During this period, four visits were required at 0, 2, 3, and 4 weeks of treatment. At each visit, the vagina and cervix were cleaned with a saline cotton ball, and the cervical mucus and vaginal secretions were cleaned with a dry cotton ball. Dry cotton balls are also used to apply an appropriate amount of hydrogel on the surface of the cervix and cervical canal.

After a 30-day trial, colposcopy, leucorrhea, and cervical smears were performed to evaluate HPV inhibition.

Claims (17)

A mussel adhesion protein or derivative thereof, which is used to treat viral infections. A mussel adhesion protein or derivative thereof, which is used as an antiviral agent. A use of mussel adhesion protein or its derivatives, which is used to manufacture pharmaceuticals for the treatment of viral infections. A mussel adhesion protein or its derivative is used for manufacturing antiviral medicine. A method of treating a viral infection in a host, the method comprising administering at least one mussel adhesion protein or derivative thereof to the host. The compound for use according to claim 1, the use according to claim 3, or the method according to claim 5, wherein the treatment includes preventing the onset of a viral infection or disease, and protecting the cells of the host from viruses Infection, preventing the spread of viral infections (within or between hosts), and/or preventing reactivation of the virus after latent in the host. The compound for use according to any one of claims 1, 2, or 6, the use according to any one of claims 3, 4 or 6, or the one according to any one of claims 5 or 6. The method, wherein the at least one mussel adhesion protein is selected from the group consisting of mefp-1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6 and combinations thereof. The compound, use, or method for use according to claim 7, wherein the at least one mussel adhesion protein includes mefp-1. The compound for use according to any one of claims 1, 2, or 6, the use according to any one of claims 3, 4 or 6, or the one according to any one of claims 5 or 6. The method includes a derivative of mussel adhesion protein, the derivative being a peptide of the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys or a salt thereof. The compound for use as described in any one of claims 1, 2 or 6, as claimed 3. The use according to any one of 3, 4 or 6 or the method according to any one of claims 5 or 6, comprising a derivative of mussel adhesion protein, the derivative being the sequence Ala-Lys-Pro- Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys peptide or its salt. The compound, use or method for use according to any one of claims 1 to 10, wherein the virus is selected from herpes simplex virus type 1, herpes simplex virus type 2, human papilloma virus, influenza Viruses and parainfluenza viruses. The compound for use, use or method according to any one of claims 1 to 11, wherein the compound is topically administered to the skin. The compound for use, use or method according to any one of claims 1 to 11, wherein the compound is administered locally to the mucosal surface. The compound, use, or method for use according to claim 13, wherein the mucosal surface is in the lung, or is a nasal or vaginal mucosa. The compound, use or method for use according to claim 14, wherein the topical administration is by means of a powder aerosol spray containing the active ingredient. The compound, use or method for use according to claim 14, wherein the topical administration is by means of a water mist containing the active ingredient. The compound, use or method for use according to claim 16, wherein the water mist is formed by means of a sprayer or the like.