Pharmaceutical composition for inactivating norovirus and method for producing same

Abstract

To provide effective antiviral agents against norovirus.SOLUTION: Disclosed is a pharmaceutical composition for inactivating norovirus, which comprises a polypeptide having an anti-norovirus activity as an effective ingredient at a pH of 6.2-9.0, where the polypeptide is: (1) a polypeptide comprising an amino acid sequence corresponding to an amino acid sequence from position 5 to position 35 of a specific amino acid sequence; or (2) a polypeptide comprising an amino acid sequence having one or more amino acid substitution, deletion or addition in the amino acid sequence from position 5 to position 35 of the specific amino acid sequence, or an amino acid sequence having 80% or more identity to the amino acid sequence from position 5 to position 35 of the specific amino acid sequence, where the polypeptide has a norovirus-inactivating activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for inactivating norovirus and a method for producing the same. [Background technology]

[0002] Norovirus is known to be highly infectious to humans and to cause food poisoning or acute viral gastroenteritis. Currently, no effective antiviral agent has been developed against norovirus, and therefore no effective prevention or treatment method has been established. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2015 / 125961 [Patent Document 2] International Publication No. 2016 / 017784 [Patent Document 3] JP 2016-141659 A Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have found that norovirus can be inactivated by heat-denaturing lysozyme (Patent Documents 1 and 2). Furthermore, they have found that a polypeptide containing a partial amino acid sequence of lysozyme can inactivate norovirus (Patent Document 3). However, there has been a demand for the development of more effective antiviral agents against norovirus. Therefore, an object of the present invention is to provide an effective antiviral agent against norovirus. [Means for solving the problem]

[0005] Means for Solving the Problems The present inventors have conducted extensive research into antiviral agents effective against norovirus and have surprisingly found that the antiviral effect can be improved by adjusting the pH of a pharmaceutical composition to a higher value than in the past. The present invention is based on these findings. Thus, the present invention provides [1] A pharmaceutical composition for inactivating norovirus, comprising as an active ingredient: (1) a polypeptide comprising the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1; or (2) an amino acid sequence in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence which has an identity of 80% or more with the amino acid sequence of amino acids 5 to 35 and has norovirus inactivation activity; and having a pH of 6.2 to 9.0. [2] The pharmaceutical composition for inactivating norovirus according to [1], wherein the polypeptide is a polypeptide consisting of the amino acid sequence of 1 to 39 in the amino acid sequence represented by SEQ ID NO: 1, a polypeptide consisting of the amino acids represented by SEQ ID NO: 2, or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. [3] The pharmaceutical composition for inactivating norovirus according to [1] or [2], wherein the pH is from pH 7.0 to pH 9.0. [4] The pharmaceutical composition for inactivating norovirus according to any one of [1] to [3], wherein the polypeptide is a heat-denatured polypeptide in a solution having a pH of 6.2 to 9.0. [5] A method for producing a norovirus-inactivating medicinal composition, comprising a step of heat-denaturing, in a solution of pH 6.2 to pH 9.0, (1) a polypeptide comprising the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence which has an identity of 80% or more to the amino acid sequence of amino acids 5 to 35 and has norovirus-inactivating activity; [6] The method for producing a norovirus-inactivating medicinal composition according to [4], wherein the polypeptide is a polypeptide consisting of the amino acid sequence of 1 to 39 in the amino acid sequence represented by SEQ ID NO: 1, a polypeptide consisting of the amino acids represented by SEQ ID NO: 2, or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1; and [7] The method for producing a norovirus-inactivated pharmaceutical composition according to [5] or [6], wherein the pH is from pH 7.0 to pH 9.0. Regarding. Effect of the Invention

[0006] According to the pharmaceutical composition for inactivating norovirus of the present invention, norovirus can be inactivated efficiently in a short time with a small amount of the composition. Moreover, according to the method for producing the pharmaceutical composition for inactivating norovirus of the present invention, a pharmaceutical composition for inactivating norovirus with excellent effects can be obtained. [Brief description of the drawings]

[0007] [Figure 1] 1 is a graph showing the reduction in the infectivity titer of norovirus when the peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were used at a concentration of 1% by weight. [Diagram 2] 1 is a graph showing the reduction in the infectivity titer of norovirus when the peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were used at a concentration of 0.5% by weight or 0.1% by weight. [Diagram 3] 1 is a graph showing the inactivation of norovirus by polypeptide 1-129, polypeptide 5-39, polypeptide 1-35, polypeptide 5-39R3, and polypeptide 88-125. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] [1] Pharmaceutical composition for inactivating norovirus The pharmaceutical composition for inactivating norovirus of the present invention comprises (1) a polypeptide comprising an amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence that has an identity of 80% or more to the amino acid sequence of amino acids 5 to 35 and has norovirus inactivation activity, and has a pH of 6.2 to 9.0.

[0009] Active ingredients: The active ingredient of the pharmaceutical composition for inactivating norovirus of the present invention is not particularly limited as long as it is a specific polypeptide containing the amino acid sequence of the N-terminus of lysozyme, but is preferably a specific polypeptide containing the amino acid sequence of the N-terminus of chicken lysozyme, such as egg white lysozyme or human lysozyme. Specifically, examples of the active ingredient include a polypeptide consisting of the amino acid sequence of amino acids 5 to 35 of egg white lysozyme, or a polypeptide consisting of the amino acid sequence of amino acids 5 to 35 of human lysozyme. In the case of egg white lysozyme, examples of the active ingredient include a polypeptide consisting of the amino acid sequence of amino acids 1 to 35 (KVFGRCELAA AMKRHGLDNY RGYSLGNWVC AAKFE: SEQ ID NO: 4) and a polypeptide consisting of the amino acid sequence of amino acids 5 to 39 (RCELAAAMKR HGLDNYRGYS LGNWVCAAKFE SNFN: SEQ ID NO: 5) in addition to the polypeptide consisting of the amino acid sequence of amino acids 5 to 35 (RCELAAAMKR HGLDNYRGYS LGNWVCAAKFE SNFN: SEQ ID NO: 5).

[0010] The active ingredient polypeptide (hereinafter, sometimes referred to as "active ingredient polypeptide") may be a modified polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the active ingredient polypeptide, or an amino acid sequence having 80% or more identity with the amino acid sequence of the active ingredient polypeptide, so long as it has norovirus inactivation activity. For example, modified polypeptides include polypeptides at the N-terminus of lysozymes other than egg white lysozyme or human lysozyme, polypeptides in which substitutions have been introduced by methods such as site-directed mutagenesis, or polypeptides in which several amino acid substitutions have been introduced to the extent that they may occur naturally. The number of amino acid modifications is preferably 1 to 5, more preferably 1 to 4, even more preferably 1 to 3, even more preferably 1 to 2, and most preferably 1. The identity with the amino acid sequence of the active ingredient polypeptide is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more. A specific example of the mutant polypeptide is the polypeptide 5-39R3 (RCNLAAAMKR HGLNNYRGYS LGNWVCAAKFN SNFN: SEQ ID NO: 2) in which the three amino acid substitutions described in Example 7 have been introduced. In the technical field of polypeptides (proteins), it is known that a polypeptide loses its function due to the substitution of one amino acid. That is, it is common technical knowledge that there are important amino acids in the amino acid sequence constituting the polypeptide. However, it is also known that a polypeptide in which one or more amino acids are substituted, deleted, or added maintains its function. That is, it is common technical knowledge that there are amino acids in the amino acids constituting the polypeptide that have little effect on the expression of the function of the polypeptide. For example, the lysozymes of many organisms are lysozymes consisting of different amino acid sequences. However, these lysozymes have the same function of hydrolyzing polysaccharides, and the polypeptide has amino acids that are important for the expression of the function and amino acids that have little effect on the expression of the function.

[0011] An example of the modified amino acid sequence of the modified polypeptide of the present invention may be an amino acid sequence having one, two, three, four, or five conservative substitutions. In the present specification, the term "conservative substitution" means that some amino acids are substituted while the modified polypeptide of the present invention has norovirus inactivation activity. For example, it means replacing one or several amino acid residues with other chemically similar amino acid residues. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Even if they are not chemically similar, amino acids may be replaced with amino acid residues that have a similar structure. Examples include substitution of aspartic acid (D) with alanine (A), asparagine (E) with alanine (A), and tyrosine (Y) with phenylalanine (F).

[0012] As used herein, the term "norovirus inactivation activity" is not particularly limited as long as it can inhibit norovirus, but may include, for example, the activity of reducing the number of plaques formed by norovirus after contacting a polypeptide with norovirus, as shown in the Examples below. The reduction rate of the number of plaques formed is not limited, but is, for example, 80% or less, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less.

[0013] Polypeptides The polypeptide contained in the pharmaceutical composition for inactivating norovirus of the present invention is not particularly limited as long as it contains the above-mentioned active ingredient polypeptide. It may be a polypeptide consisting of the above-mentioned active ingredient polypeptide, or it may be a polypeptide containing the active ingredient polypeptide. That is, a polypeptide containing an active ingredient polypeptide includes a polypeptide in which an arbitrary peptide (amino acid sequence) is added to the N-terminus and / or C-terminus of the above-mentioned active ingredient polypeptide (hereinafter, sometimes referred to as an added polypeptide). Specifically, an example of the added polypeptide is full-length egg white lysozyme consisting of 129 amino acids represented by SEQ ID NO: 1. However, the arbitrary polypeptide bound to the N-terminus and / or C-terminus of the active ingredient polypeptide is not limited to peptides other than the active ingredient polypeptide of egg white lysozyme, and any polypeptide can be used. The pharmaceutical composition for inactivating norovirus of the present invention has a pH of 6.2 to 9.0. The full-length egg white lysozyme is a polypeptide (protein) with a strong structure having many S-S bonds and many hydrophobic amino acids. However, at a pH of 6.2 to 9.0, the S-S bonds are cleaved, the strong structure is loosened, and the active ingredient polypeptide region is exposed on the surface, thereby exhibiting excellent norovirus inactivation activity. Therefore, even if an additional polypeptide in which any peptide is bound to the N-terminus and / or C-terminus of an active ingredient polypeptide is used, the active ingredient polypeptide is exposed on the surface at a pH of 6.2 to 9.0, and excellent norovirus inactivation activity can be exhibited. For example, when a polypeptide is used in which nucleotides encoding an active ingredient polypeptide are inserted into a vector and expressed in E. coli, yeast, etc., it may be expressed as a fusion protein by binding with a protein such as GST or TrpE in order to easily express it in large quantities. These fusion proteins also have an active ingredient polypeptide exposed on the surface by being kept at pH 6.2 to pH 9.0, and can exhibit excellent norovirus inactivation activity.

[0014] The additional polypeptide may have amino acid substitution, deletion, or addition in any polypeptide region other than the active ingredient polypeptide. For example, when the polypeptide added to the C-terminus of the active ingredient polypeptide is a polypeptide consisting of the amino acid sequence of 36 to 129 of SEQ ID NO: 1, or a polypeptide consisting of the amino acid sequence of 40 to 129 of SEQ ID NO: 1, the additional polypeptide may have an amino acid sequence in which one or more amino acids are substituted, deleted, or added, or may have an identity of 80% or more with the amino acid sequence. The number of amino acid modifications is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, even more preferably 1 to 4, and most preferably 1 to 2. The identity with the amino acid sequence of the active ingredient polypeptide is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.

[0015] The polypeptide contained in the pharmaceutical composition for inactivating norovirus of the present invention may be in the form of a salt, which may be, for example, a salt of an inorganic acid such as hydrochloric acid, carbonic acid, phosphoric acid, boric acid, hexametaphosphoric acid, nitric acid, or sulfuric acid, or a salt of an organic acid such as citric acid, tartaric acid, succinic acid, malic acid, acetic acid, glutamic acid, glycerophosphoric acid, or gluconic acid.

[0016] 《pH》 The pH of the pharmaceutical composition for inactivating norovirus of the present invention is pH 6.2 to pH 9.0, more preferably pH 7.0 to pH 9.0, even more preferably pH 7.5 to pH 9.0, and most preferably pH 8.0 to pH 9.0. When the pH is within the above range, norovirus can be efficiently inactivated. The buffer solution used in the pharmaceutical composition for inactivating norovirus of the present invention is not particularly limited as long as it can be adjusted to a pH of 6.2 to 9.0. Examples of the buffer solution include phosphate buffer, bicarbonate buffer, citrate buffer, lactate buffer, acetate buffer, adipic acid buffer, tartrate buffer, fumarate buffer, malate buffer, Tris buffer, and HEPES buffer.

[0017] Pharmaceutical Composition The pharmaceutical composition of the present invention can inactivate norovirus in vivo or ex vivo. When used ex vivo, for example, the composition can be made into a liquid with a sprayable viscosity and filled into a spray container such as a trigger sprayer, squeeze container, or aerosol container, and droplets containing the norovirus inactivating agent can be sprayed onto fingers, food, cooking utensils, living environment, vomit, excrement, etc., to easily inactivate norovirus. The norovirus can also be easily inactivated by immersing an object to be inactivated (e.g., fingers, food, medical equipment, medical equipment, cooking utensils, living environment) in the liquid containing the norovirus inactivating agent. In addition, the liquid containing the norovirus inactivating agent can be used as a sheet moistened with the liquid. Furthermore, the composition can be used as a skin external preparation by making it into a lotion, cream, etc., and can be used as an infection preventive drug that inactivates norovirus on the spot even if it adheres to fingers, and prevents oral infection with norovirus.

[0018] When used in vivo, the dosage form of the pharmaceutical composition is not particularly limited, and examples thereof include oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, and pills, and parenteral preparations such as injections, external liquid preparations, ointments, suppositories, creams for topical administration, and eye drops, with oral preparations being preferred. Oral preparations can be manufactured in accordance with standard methods using excipients such as gelatin, sodium alginate, starch, corn starch, white sugar, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, flow enhancers, diluents, preservatives, colorants, fragrances, flavorings, stabilizers, moisturizers, preservatives, or antioxidants.

[0019] (Other Ingredients) The pharmaceutical composition of the present invention may contain other ingredients. Examples of the other components include food ingredients and food additives such as edible oils and fats, water, emulsifiers such as edible oils and fats, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerin organic acid fatty acid esters, polyglycerin fatty acid esters, calcium stearoyl lactylate, sodium stearoyl lactylate, and polyoxyethylene sorbitan fatty acid esters, thickening stabilizers such as locust bean gum, carrageenan, alginic acids, pectin, xanthan gum, crystalline cellulose, carboxymethylcellulose, methylcellulose, agar, glucomannan, gelatin, starch, and modified starch, salting agents such as salt and potassium chloride, acidulants such as acetic acid, lactic acid, and gluconic acid, sweeteners such as sugars or sugar alcohols, stevia, and aspartame, colorants such as beta-carotene, caramel, and red koji pigment, antioxidants such as tocopherol and tea extract, flavorings, pH adjusters, food preservatives, and shelf life enhancers. It is also possible to add functional ingredients such as various vitamins, coenzyme Q, plant sterols, or milk fat cell membranes.

[0020] 《Content》 The content of the polypeptide in the pharmaceutical composition is not particularly limited as long as the effects of the present invention can be obtained. For example, when the composition is used in vitro, a pharmaceutical composition containing about 1 to 100% by weight of the polypeptide may be appropriately diluted before use. When administered in vivo, the pharmaceutical composition should contain an amount of the active ingredient polypeptide that allows an adult to ingest 10 mg to 4 g, more preferably 20 mg to 2 g, of the active ingredient polypeptide per day. Specifically, the active ingredient polypeptide is preferably present in an amount of 0.1 to 100% by mass, more preferably 0.5 to 99% by mass, and most preferably 1 to 80% by mass in the pharmaceutical composition.

[0021] The subjects to which the pharmaceutical composition of the present invention is to be administered are not particularly limited, and examples thereof include pets such as humans, dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; livestock such as cows and pigs; laboratory animals such as mice and rats; and fish.

[0022] <Heat denaturation> The polypeptide contained in the pharmaceutical composition of the present invention is preferably, but not limited to, a polypeptide heat-denatured at pH 6.2 to pH 9.0. This is because it is believed that the active ingredient polypeptide assumes a structure that exhibits excellent norovirus inactivation activity by being heat-denatured at pH 6.2 to pH 9.0. The conditions for heat denaturation can be the same as those described in the "heat denaturation step" of the production method for the pharmaceutical composition for inactivating norovirus described below.

[0023] [2] Method for producing pharmaceutical composition for inactivating norovirus The method for producing a pharmaceutical composition for inactivating norovirus of the present invention includes the step of heat-denaturing, in a solution of pH 6.2 to pH 9.0, (1) a polypeptide comprising the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence in which 1 to 5 amino acids have been substituted, deleted, or inserted in the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence which has an identity of 80% or more to the amino acid sequence of amino acids 5 to 35 and which has norovirus inactivation activity.

[0024] In the method for producing a pharmaceutical composition for inactivating norovirus of the present invention, the polypeptide used can be the polypeptide described in the above section "[1] Pharmaceutical composition for inactivating norovirus." Therefore, the terms "active ingredient," "polypeptide," "active ingredient polypeptide," and "additional polypeptide" are the same as those described in the above section "[1] Pharmaceutical composition for inactivating norovirus."

[0025] <Heat denaturation process> The production method of the present invention includes a step of heat-denaturing a polypeptide containing an active ingredient polypeptide in a solution of pH 6.2 to pH 9.0. The pH for heat denaturation is pH 6.2 to pH 9.0, preferably pH 7.0 to pH 9.0, more preferably pH 7.5 to pH 9.0, and most preferably pH 8.0 to pH 9.0. By setting the pH within the above range, a pharmaceutical composition capable of efficiently inactivating norovirus can be produced. In heat denaturation, the solution in which the polypeptide is dissolved is not particularly limited as long as it can be adjusted to a pH of 6.2 to 9.0. For example, the polypeptide can be dissolved in a phosphate buffer, bicarbonate buffer, citrate buffer, lactate buffer, acetate buffer, adipic acid buffer, tartrate buffer, fumarate buffer, malate buffer, Tris buffer, or HEPES buffer.

[0026] The temperature for heat denaturation is not particularly limited as long as the effects of the present invention can be obtained, but for example, the lower limit is 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, and most preferably 95° C. or higher. The upper limit of the heat denaturation temperature is, for example, 140° C. or lower, more preferably 130° C. or lower, even more preferably 120° C. or lower, and most preferably 110° C. or lower. The heat denaturation time is not particularly limited as long as the effects of the present invention can be obtained, but is, for example, 1 to 120 minutes, preferably 10 to 90 minutes, more preferably 20 to 60 minutes, and even more preferably 30 to 50 minutes.

[0027] Furthermore, the combination of the heat denaturation temperature and the heat denaturation time can be appropriately changed. For example, when the heat denaturation temperature is low, the effect of the present invention can be obtained by extending the heat denaturation time. When the heat denaturation time is short, the effect of the present invention can be obtained by increasing the heat denaturation temperature. Furthermore, the effect of the present invention can be obtained by increasing the pH, lowering the heat denaturation temperature, and shortening the heat denaturation time.

[0028] In the production method of the present invention, heat denaturation is performed at pH 6.2 to pH 9.0. The full-length egg white lysozyme is a polypeptide (protein) with a strong structure having many S-S bonds and many hydrophobic amino acids. However, by heat denaturation at pH 6.2 to pH 9.0, the S-S bonds are broken and the strong structure is loosened, and the active ingredient polypeptide region is exposed on the surface, thereby exhibiting excellent norovirus inactivation activity. Therefore, even in the case of a polypeptide in which any peptide other than egg white lysozyme is bound to the N-terminus and / or C-terminus of the active ingredient polypeptide, the active ingredient polypeptide is exposed on the surface at pH 6.2 to pH 9.0, and the polypeptide can exhibit excellent norovirus inactivation activity. For example, when a polypeptide is used that has been expressed in Escherichia coli, yeast, etc. by incorporating nucleotides encoding the active ingredient polypeptide into a vector, the polypeptide may be expressed as a fusion protein by binding with a protein such as GST or TrpE in order to easily express it in large quantities. These fusion proteins can also be denatured by heating at pH 6.2 to pH 9.0 to expose the active ingredient polypeptide on the surface, thereby exhibiting excellent norovirus inactivation activity.

[0029] 《Effect》 The mechanism by which the pharmaceutical composition for inactivating norovirus of the present invention exhibits excellent norovirus inactivation activity has not been fully elucidated, but can be presumed as follows, although the present invention is not limited to the following presumption. It is believed that the active ingredient polypeptide contained in the pharmaceutical composition for inactivating norovirus of the present invention does not have a fully exposed region effective for inactivating norovirus at a pH of less than 6.2, but it is presumed that by increasing the pH of the pharmaceutical composition to pH 6.2 or higher, the region effective for inactivating norovirus becomes fully exposed. Furthermore, in the case of an additional polypeptide, the region effective in inactivating norovirus may be covered by peptides other than the active ingredient polypeptide, but it is presumed that by heat denaturation at pH 6.2 or higher, the region effective in inactivating norovirus becomes sufficiently exposed. EXAMPLES

[0030] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0031] Example 1 In this example, a pharmaceutical composition having a pH of 6.5 was prepared using full-length egg white lysozyme (129 amino acids). Egg white lysozyme was dissolved in distilled water to a concentration of 2% by weight. An appropriate amount of NaOH was added to adjust the pH to 6.5. The resulting solution was heated at 100°C for 40 minutes to obtain a pharmaceutical composition with a pH of 6.5.

[0032] Example 2 In this example, a pharmaceutical composition with a pH of 7.5 was prepared using full-length egg white lysozyme (129 amino acids). The procedure of Example 1 was repeated to obtain a pharmaceutical composition with a pH of 7.5, except that the pH was adjusted to 7.5 instead of 6.5.

[0033] Example 3 In this example, a pharmaceutical composition with a pH of 8.5 was prepared using full-length egg white lysozyme (129 amino acids). The procedure of Example 1 was repeated to obtain a pharmaceutical composition with a pH of 8.5, except that the pH was adjusted to 7.5 instead of 6.5.

[0034] Comparative Example 1 In this comparative example, a pharmaceutical composition with a pH of 3.7 was prepared using full-length egg white lysozyme (129 amino acids). The procedure of Example 1 was repeated without adjusting the pH to 3.7 to obtain a pharmaceutical composition with a pH of 3.7.

[0035] Comparative Example 2 In this comparative example, a pharmaceutical composition with a pH of 4.5 was prepared using full-length egg white lysozyme (129 amino acids). The procedure of Example 1 was repeated, except that the pH was adjusted to 4.5 instead of 6.5, to obtain a pharmaceutical composition with a pH of 4.5.

[0036] Comparative Example 3 In this comparative example, a pharmaceutical composition with a pH of 5.5 was prepared using full-length egg white lysozyme (129 amino acids). The procedure of Example 1 was repeated, except that the pH was adjusted to 5.5 instead of 6.5, to obtain a pharmaceutical composition with a pH of 5.5.

[0037] 《Analysis example 1》 The inactivation activity against norovirus was measured for the pharmaceutical compositions obtained in Examples 1 to 3 and Comparative Examples 1 to 3. MNV-1 (provided by Dr. Herbert W. Virgin of Washington University) was used as the norovirus, and a norovirus solution (MNV-1 solution) was prepared from which medium components had been removed. Each pharmaceutical composition was mixed with an MNV-1 solution in equal amounts and exposed for 1 minute. The infectivity titer of MNV-1 (PFU / mL) was measured by plaque assay. Mouse macrophage cell line (RAW264.7 cells) was cultured in a 6-well plate until it reached 60-80% confluence. After exposure, each sample was diluted, the reaction was stopped, and then inoculated into the cultured cells at 500 μL / well. The cells inoculated with each sample were incubated at room temperature for 1 hour to infect with norovirus. After that, the entire amount of the inoculum on the plate was removed, and 1.5% Sea Plaque Agarose-DMEM (37°C) was overlaid at 2 mL / well and cultured at 37°C and 5% CO2 for 2 days. After culture, the cells were stained by adding 0.03% neutral red solution to the plate, and the number of plaques was counted. The infectious titer (PFU / mL) was calculated from the number of plaques obtained and the dilution ratio. Sterile distilled water and the infectious titer (10 6 ~10 7The reduction in the infectivity titer (PFU / mL) was calculated by subtracting the infectivity titer of each sample to which the MNV-1 solution was added from the infectivity titer (approximately 1.0 PFU / mL). The results are shown in Figure 1.

[0038] 1, the pharmaceutical compositions of Examples 1 to 3 (pH 6.5 to 8.5) showed a reduction in norovirus of 4.5 log PFU / mL or more. On the other hand, the pharmaceutical compositions of Comparative Examples 1 to 3 (pH 3.7 to 5.5) showed a reduction in norovirus of about 0.5 to 1.5 log PFU / mL, a difference of more than 1000 times.

[0039] 《Analysis example 2》 In this analytical example, the pharmaceutical composition was diluted to adjust the egg white lysozyme to 0.1% by weight or 0.5% by weight, and the inactivation activity was examined. The procedures of Analysis Example 1 were repeated, except that the pharmaceutical compositions obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were diluted to 0.1% by weight or 0.5% by weight of egg white lysozyme, and the exposure time to the MNV-1 solution was changed from 1 minute to 60 minutes. The results are shown in Figure 2.

[0040] As shown in FIG. 2, even when the egg white lysozyme content was 0.1% by weight or 0.5% by weight, the pharmaceutical compositions of Examples 1 to 3 showed superior reductions in norovirus compared to the pharmaceutical compositions of Comparative Examples 1 to 3.

[0041] Example 4 In this example, a pharmaceutical composition was prepared at pH 6.5 using full-length egg white lysozyme (129 amino acids). The procedure of Example 1 was repeated to obtain a pharmaceutical composition.

[0042] Example 5 In this example, a pharmaceutical composition having a pH of 6.5 was prepared using polypeptide 5-39 (RCELAAAMKR HGLDNYRGYS LGNWVCAAKFE SNFN: SEQ ID NO: 5) consisting of the amino acid sequence of 5 to 39 as the polypeptide. A pharmaceutical composition was obtained by repeating the procedure of Example 1, except that polypeptide 5-39 was used instead of full-length egg white lysozyme (129 amino acids).

[0043] Example 6 In this example, a pharmaceutical composition having a pH of 6.5 was prepared using polypeptide 1-35 (KVFGRCELAA AMKRHGLDNY RGYSLGNWVC AAKFE: SEQ ID NO: 4) consisting of the amino acid sequence of 1 to 35 as the polypeptide. A pharmaceutical composition was obtained by repeating the procedure of Example 1, except that polypeptide 1-35 was used instead of full-length egg white lysozyme (129 amino acids).

[0044] Example 7 In this example, a pharmaceutical composition of pH 6.5 was prepared using polypeptide 5-39R3 (RCNLAAAMKR HGLNNYRGYS LGNWVCAAKFN SNFN: sequence number 2) in which three amino acid substitutions were introduced into the amino acid sequence of 1 to 35. A pharmaceutical composition was obtained by repeating the procedure of Example 1, except that the polypeptide 5-39R3 was used instead of the full-length egg white lysozyme (129 amino acids).

[0045] Comparative Example 4 In this comparative example, a pharmaceutical composition of pH 6.5 was prepared using polypeptide 88-125 (ITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIR: SEQ ID NO: 6) consisting of the amino acid sequence of 88-125 as the polypeptide. A pharmaceutical composition was obtained by repeating the procedure of Example 1, except that polypeptide 88-125 was used instead of full-length egg white lysozyme (129 amino acids).

[0046] 《Analysis example 3》 In this analytical example, the inactivation activity of the pharmaceutical compositions obtained in Examples 5 to 7 and Comparative Example 4 against norovirus was measured. The procedure of Analysis Example 1 was repeated, except that the pharmaceutical compositions obtained in Examples 5 to 7 and Comparative Example 4 were used. The results are shown in FIG.

[0047] As shown in FIG. 3, the pharmaceutical compositions containing the polypeptide 5-39, the polypeptide 1-35, and the polypeptide 5-39R3 having an amino acid substitution exhibited excellent reduction in the amount of norovirus. [Industrial Applicability]

[0048] The pharmaceutical composition of the present invention can be used to suppress norovirus infection.

Claims

1. (1) A polypeptide comprising the amino acid sequence of 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or (2) A polypeptide comprising an amino acid sequence having an identity of 90% or more with the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO:1 and having norovirus inactivation activity. as an active ingredient, and having a pH of 7.5 to 9.0, A pharmaceutical composition for inactivating norovirus, wherein the polypeptide is heat-denatured at 70 to 140°C.

2. The pharmaceutical composition for inactivating norovirus according to claim 1, wherein the polypeptide is a polypeptide consisting of the amino acid sequence of 1 to 39 in the amino acid sequence represented by SEQ ID NO: 1, a polypeptide consisting of the amino acids represented by SEQ ID NO: 2, or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:

1.

3. The pharmaceutical composition for inactivating norovirus according to claim 1 or 2, wherein the polypeptide is a heat-denatured polypeptide in a solution having a pH of 7.5 to 9.

0.

4. A pharmaceutical composition for inactivating norovirus, comprising as an active ingredient a polypeptide having an amino acid sequence represented by SEQ ID NO:2, wherein the polypeptide has been heat-denatured at 70 to 140°C.

5. (1) A polypeptide comprising the amino acid sequence of 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1, or (2) A polypeptide comprising an amino acid sequence having an identity of 90% or more with the amino acid sequence of amino acids 5 to 35 in the amino acid sequence represented by SEQ ID NO: 1 and having norovirus inactivation activity, A method for producing a norovirus-inactivated pharmaceutical composition, comprising a step of heat-denaturing the composition at 70 to 140°C in a solution having a pH of 7.5 to 9.

0.

6. The method for producing a norovirus-inactivating pharmaceutical composition according to claim 5, wherein the polypeptide is a polypeptide consisting of the amino acid sequence of 1 to 39 in the amino acid sequence represented by SEQ ID NO: 1, a polypeptide consisting of the amino acids represented by SEQ ID NO: 2, or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

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