Pharmaceutical composition for treating or preventing inflammation caused by acanthamoeba protozoa

Abstract

The purpose of the present invention is to provide a pharmaceutical composition or the like for treating or preventing inflammation caused by acanthamoeba protozoa. Specifically, provided are: a pharmaceutical composition for treating or preventing inflammation caused by acanthamoeba protozoa, said pharmaceutical composition containing a complex in which lysozyme and chitosan are bound; an antiprotozoa composition containing a complex in which lysozyme and chitosan are bound; and an anti-adhesion agent for acanthamoeba protozoa, which contains a complex in which lysozyme and chitosan are bound to each other.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating or preventing inflammation caused by Acanthamoeba. Background Art

[0002] Acanthamoeba is a type of amoeba protozoan (protozoa), a microorganism that is often found in soil. Acanthamoeba infects the eye, especially the cornea, causing corneal inflammation. Inflammation caused by Acanthamoeba is particularly common in contact lens wearers, for example, they attach to contact lens cases through tap water, reproduce in the case, and cause corneal infection through the contact lenses.

[0003] Currently, various amebicides have been developed as countermeasures against Acanthamoeba, and biguanide-based fungicides are known as major examples (Patent Documents 1 to 3). However, compositions other than biguanide-based fungicides that exhibit strong anti-amoebic effects are not well known.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 02 / 4963 Pamphlet

[0007] Patent Document 2: International Publication No. WO2013 / 154016 Pamphlet

[0008] Patent Document 3: International Publication No. WO2014 / 192068 Pamphlet Summary of the invention

[0009] The object of the present invention is to provide a novel pharmaceutical composition for treating or preventing inflammation caused by Acanthamoeba. In addition, the present invention provides an antiprotozoal composition that exerts an effective therapeutic and preventive effect on protozoal infections such as Acanthamoeba, a method for treating and preventing protozoal infections such as Acanthamoeba, and a method for using the pharmaceutical composition in the manufacture of the antiprotozoal composition, etc.

[0010] The present inventors have verified the antiprotozoal and anti-adhesion properties of a complex of lysozyme and chitosan, which is highly safe and can be used as a food additive, against Acanthamoeba, and have found for the first time that the complex exhibits excellent antiprotozoal and anti-adhesion effects against Acanthamoeba, thereby completing the present invention. More specifically, the present invention is as follows.

[0011] [1] A pharmaceutical composition comprising a complex of lysozyme and chitosan for use in treating or preventing inflammation caused by Acanthamoeba.

[0012] [2] The pharmaceutical composition according to [1] above, wherein the chitosan is a water-soluble chitosan having a molecular weight of 1,000 to 30,000 Da.

[0013] [3] The pharmaceutical composition according to [1] or [2] above, wherein the pharmaceutical composition contains 0.01 μg / mL to 100,000 μg / mL of the complex.

[0014] [4] The pharmaceutical composition according to [1] or [2] above, wherein the pharmaceutical composition contains 0.0001% by mass to 5.0% by mass of the complex.

[0015] [5] The pharmaceutical composition according to any one of [1] to [4] above, further comprising an additional active ingredient selected from aqueous hydrogen peroxide solution, povidone iodine, polychloridium chloride and polyhexanide hydrochloride.

[0016] [6] The pharmaceutical composition according to any one of [1] to [5] above, wherein the inflammation caused by Acanthamoeba is dermatitis or keratitis.

[0017] [7] The pharmaceutical composition according to any one of [1] to [6] above, wherein the treatment or prevention of inflammation caused by the Acanthamoeba is achieved by inhibiting the adhesion of the Acanthamoeba.

[0018] [8] Use of a complex of lysozyme and chitosan for the manufacture of a pharmaceutical composition for treating or preventing inflammation caused by Acanthamoeba.

[0019] [9] An antiprotozoal composition comprising a complex of lysozyme and chitosan.

[0020]

[10] The antiprotozoan composition according to [9] above, wherein the protozoan is Acanthamoeba.

[0021]

[11] An anti-attachment agent for Acanthamoeba comprising a complex of lysozyme and chitosan.

[0022]

[12] A method for preventing Acanthamoeba from attaching to the surface of a material, comprising applying a complex composed of lysozyme and chitosan to the surface of the material.

[0023]

[13] The method for preventing the attachment of Acanthamoeba according to

[12] above, wherein the substance is selected from polyethylene, polypropylene, polyurethane and polycarbonate.

[0024] According to the present invention, performance removes the effect of acanthamoeba, antiprotozoa or killing protozoa, and suppresses the effect of its breeding. Particularly the complex that lysozyme of the present invention is combined with chitosan can not only suppress the breeding of acanthamoeba, but also has the effect of significantly killing acanthamoeba. Therefore, according to the present invention, can cure and / or prevent acanthamoeba infection. In addition, lysozyme also can be used as the food additive from natural products with high safety and utilize, and the antiprotozoa composition using the complex of this lysozyme and chitosan can make the patient of utilization feel at ease, and alleviate its burden. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the reaction when synthesizing a complex of lysozyme and chitosan.

[0026] Figure 2 This is a biological microscope photograph of Acanthamoeba observed when measuring the "number of all Acanthamoeba adhesion" in Table 1.

[0027] Figure 3 This is a biological microscope photograph showing a state in which dead Acanthamoeba observed when measuring the "number of dead Acanthamoeba" in Table 1 are stained with trypan blue and appear darker.

[0028] Figure 4 This is a graph showing the total number of Acanthamoeba attached when using the mixture of lysozyme and chitosan in Tables 2 to 4, and each solution of lysozyme monomer and chitosan monomer in comparison with the case of using the complex of lysozyme and chitosan in Table 1.

[0029] Figure 5 This is a graph showing the mortality rate of Acanthamoeba when using the mixture of lysozyme and chitosan in Tables 2 to 4, and each solution of lysozyme monomer and chitosan monomer in comparison with the case of using the complex of lysozyme and chitosan in Table 1.

[0030] Figure 6 This is a graph showing the total number of Acanthamoeba attached when the lysozyme-chitosan complex (LYZOX (registered trademark)) in Table 5 and the additional active ingredient were used in combination.

[0031] Figure 7 This is a graph showing the mortality rate of Acanthamoeba when the lysozyme-chitosan complex (LYZOX (registered trademark)) in Table 5 and the additional active ingredient were used in combination. DETAILED DESCRIPTION

[0032] An embodiment of the present invention is described in detail below. It should be noted that the preferred embodiment or more preferred embodiment illustrated below has nothing to do with expressions such as "preferably" or "more preferably", and can be appropriately used in combination with each other. In addition, the description of the numerical range is only an example, and the range formed by appropriately combining the upper and lower limits of each range and the numerical values ​​of the embodiments can also be preferably used. In addition, the terms such as "containing" or "comprising" can be interpreted as "essentially" or "consisting only of...".

[0033] <Pharmaceutical Composition>

[0034] The present invention relates to a pharmaceutical composition for treating or preventing inflammation caused by Acanthamoeba, comprising a complex of lysozyme and chitosan. The pharmaceutical composition will be described in detail below.

[0035] The "complex formed by combining lysozyme and chitosan" refers to a complex formed by combining lysozyme and chitosan by, for example, the Maillard reaction (see Figure 1 ). It has the following characteristics: lysozyme and water-soluble chitosan are combined through the Maillard reaction, so that most or all of the antigenic structure in the lysozyme is masked, so that even if people ingest the lysozyme-chitosan complex, it is not easy to cause allergies. In addition, the above-mentioned complex can also be obtained by covalently bonding lysozyme and chitosan using a cross-linking agent.

[0036] Here, "lysozyme" is an enzyme that hydrolyzes mucopolysaccharides, and chicken-derived lysozyme or human-derived lysozyme can be preferably used.

[0037] The upper limit of the molecular weight of lysozyme can be, for example, less than 30000Da, more preferably less than 25000Da, less than 20000Da, less than 18000Da, less than 15000Da. In addition, the lower limit of the molecular weight of lysozyme is not particularly limited, for example, it can be more than 1000Da, preferably more than 5000Da, more than 10000Da, more than 12000Da. The range of the molecular weight of the lysozyme can be the range between any of the upper and lower limits mentioned above, for example, it can be 1000Da to 30000Da, preferably 5000Da to 20000Da, more preferably 10000Da to 15000Da.

[0038] "Chitosan" is a poly-β1→4-glucosamine ((C 6 H 11 NO 4 ) n , CAS registration number 9012-76-4).

[0039]

[0040] The chitosan is water-soluble. The upper limit of the molecular weight of chitosan can be, for example, less than 30000Da, more preferably less than 20000Da, less than 15000Da, less than 10000Da, less than 7000Da. In addition, the lower limit of the molecular weight of chitosan is not particularly limited, for example, it can be more than 300Da, preferably more than 500Da, more than 1000Da, more than 3000Da. The range of the molecular weight of the chitosan can be the range between any of the above-mentioned upper and lower limits, for example, 300Da to 30000Da, preferably 500Da to 15000Da, more preferably 1000Da to 10000Da, and particularly preferably 3000Da to 7000Da. If the antiprotozoal property is focused on, chitosan with a large molecular weight is advantageous, and if the ease of manufacture is focused on, the solubility and stability of chitosan with a small molecular weight are good, and chitosan with a low molecular weight is advantageous.

[0041] It should be noted that when chitosan is mentioned here, it means that in addition to the above chitosan, it also includes chitosan oligosaccharides and glucosamine. Chitosan oligosaccharides refer to the substance represented by the above formula (I) formed by connecting several or so D-glucosamines, which is a low-molecular chitosan, or a substance formed by hydrolyzing chitosan in a narrow sense with hydrochloric acid or enzymes.

[0042] Examples of the cross-linking agent include amine-reactive cross-linking agents (eg, alkoxyamines), carbonyl-reactive cross-linking agents (eg, hydrazine compounds), and mercapto-reactive cross-linking agents.

[0043] The mass ratio of lysozyme / chitosan is, for example, 99 / 1 to 1 / 99, preferably 90 / 10 to 10 / 90, more preferably 80 / 20 to 20 / 80, further preferably 60 / 40 to 40 / 60, and particularly preferably 50 / 50.

[0044] As a specific method for producing a complex formed by combining lysozyme and chitosan, for example, the following method can be cited. First, lysozyme and chitosan in the above-mentioned mass ratio are mixed and dissolved in water, and the total mass of lysozyme and chitosan in the obtained aqueous solution is prepared in a manner of 5 to 30 mass%. The obtained aqueous solution is freeze-dried to make it powder. The obtained powder is subjected to a Maillard reaction for example for 2 to 20 days, more preferably 7 to 14 days, at a temperature of 50 to 80° C., preferably 55 to 65° C., and a relative humidity of 50 to 80%, preferably 60 to 70%, thereby producing a complex formed by combining lysozyme and chitosan of the present invention.

[0045] Whether the lysozyme-chitosan complex of the present embodiment is generated can be confirmed by various known methods, for example, by staining a plate obtained by SDS (Sodium dodecyl sulfate) or SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide) polyacrylamide electrophoresis to confirm the generation of a high molecular weight substance that is a protein-chitosan complex.

[0046] "Acanthamoeba" (Acanthamoeba polyphaga) is a protozoan, a kind of ameba classified as protozoa, especially rhizopods, for example, existing in soil or fresh water. As mentioned above, Acanthamoeba infects the skin, eyes, especially the cornea of ​​the eyes, causing inflammation of the skin and cornea. Inflammation caused by Acanthamoeba is particularly common in contact lens wearers. For example, Acanthamoeba attaches to the contact lens case through tap water, Acanthamoeba reproduces in the case, and Acanthamoeba infects the cornea through contact lens storage solution or contact lenses, causing inflammation. As inflammation, dermatitis and keratitis of the eyes can be cited. In particular, the present invention can be expected to have the effect on eye diseases such as corneal congestion, corneal opacity, radial keratoneuritis, discoid corneal ulcer, scleritis, and endophthalmitis.

[0047] The present invention has antiprotozoal properties to "Acanthamoeba". "Protozoa" includes amoebae such as Acanthamoeba. Here, antiprotozoal properties broadly include protozoan prevention, removal, protozoicide, antiprotozoan, etc., and when the number of protozoa is the same or less, it refers to the whole state in which the protozoa are at least not reproduced. It should be noted that the removal of protozoa refers to all the removal of protozoa, including protozoicide. Protozoicide means killing at least a part of the protozoa. Therefore, the antiprotozoal composition in the present invention means including a protozoan removal composition, a protozoicide composition, a protozoan removal agent, a protozoicide, and an antiprotozoan agent.

[0048] In particular, the present invention can also be used as an anti-adhesion agent to prevent acanthamoeba from adhering to materials such as contact lens cases. Acanthamoeba has the property of being easily attached to the plastic surface of a contact lens case, and by making the present invention present in a cleaning solution for contact lenses, etc., the attachment of acanthamoeba can be suppressed. The above-mentioned treatment or prevention of inflammation caused by the above-mentioned acanthamoeba can also be achieved by suppressing the attachment of acanthamoeba. The present invention has an effect on the surface of various materials such as polypropylene used for contact lens cases, and polyethylene, polyurethane, polycarbonate, polyvinyl polymer, melamine resin, urea resin, epoxy resin, acrylic resin, silicone, elastomer, rubber, etc., contact lens cases, cleaning liquid bottles, blister packaging for sealing unworn contact lenses, toys, etc., polymer resin compositions, hydroxyethyl methacrylate, silicone hydrogel, polymethyl methacrylate, contact lens materials such as glass, paper, metal, cloth, wood, ceramics, leather, etc. It should be noted that the substances mentioned here do not include human bodies or animals such as skin and their parts.

[0049] The present invention is further effective for the treatment and prevention of inflammation caused by "Acanthamoeba". Here, "treatment" includes completely curing the inflammation that is the object of the present invention, and also includes suppressing inflammation to reduce the severity of injury. "Prevention" includes the case where there is no onset of inflammation that is the object of the present invention, and also includes preventing the recurrence of the inflammation that is the object of the present invention after curing the inflammation.

[0050] In addition to the above-mentioned complex, the pharmaceutical composition of the present invention may also contain one or more additional active ingredients. As additional active ingredients, in addition to the active ingredients themselves acting as active ingredients, substances (adjuvants) that do not act as active ingredients themselves but exert their effects by being used in combination with the above-mentioned complex as the active ingredient of the present invention are also included. As additional active ingredients, for example, biguanide-based bactericides, aqueous hydrogen peroxide solution, povidone iodine (a complex of a polymer of 1-vinyl-2-pyrrolidone and iodine), polychloride (POLYQUAD (registered trademark)), polyhexanide hydrochloride (PHMB), terpene alcohols, fatty acids and / or salts of the fatty acids can be cited. By adding one or more of these additional active ingredients, it is possible to cooperate with the complex of the present invention to obtain a strengthening effect and / or a synergistic effect. Aqueous hydrogen peroxide solution, povidone iodine (a complex of a polymer of 1-vinyl-2-pyrrolidone and iodine), polychloride (POLYQUAD (registered trademark)), and polyhexanide hydrochloride (PHMB) are particularly preferred.

[0051] As terpene alcohol, for example, terpene-4-ol, hinokitiol, geraniol, and menthol etc. can be mentioned. As fatty acid, for example, fatty acid with carbon number 8 to 12 can be mentioned, preferably capric acid (decanoic acid) or lauric acid. As salt of fatty acid, for example, sodium salt, potassium salt, magnesium salt and calcium salt etc. can be mentioned. In addition, polyhexanide hydrochloride, lactoferrin, boric acid and / or its salt, carbofungin and / or its salt, voriconazole, miconazole, econazole, sulconazole, fluconazole, quaternary ammonium disinfectant, chitinase etc. known to be effective for inflammation caused by Acanthamoeba can also be added as the additional active ingredient.

[0052] The pharmaceutical composition of the present invention may contain one or more other components in addition to the above-mentioned complex. Examples of other components include additives such as excipients, binders, emulsifiers, solvents, adhesives, disintegrants, thickeners, lubricants, colorants, flow agents, and humectants.

[0053] Examples of the moisturizer include glycerin, butylene glycol, collagen, hyaluronic acid, and ceramide.

[0054] As the binder, for example, cellulose, methyl cellulose, hydroxyethyl cellulose and sodium carboxymethyl cellulose can be cited.

[0055] Examples of the emulsifier include lecithin, polyethylene glycol (PG), PG-hydrogenated castor oil, fatty acid glycerides, sorbitan fatty acid esters, and ceteareth-23.

[0056] Examples of the solvent include water, ethanol, 1-butanol, 2-butanol, 1-propanol, 2-propanol, and 1-pentanol.

[0057] In 1 mL of the pharmaceutical composition of the present invention, the lysozyme-chitosan complex of the present invention is preferably contained at least 0.01 μg / mL, at least 0.05 μg / mL, at least 0.1 μg / mL, at least 0.5 μg / mL, at least 1 μg / mL, at least 10 μg / mL, at least 50 μg / mL, or at least 100 μg / mL. In addition, in 1 mL of the pharmaceutical composition of the present invention, the lysozyme-chitosan complex of the present invention is preferably contained at least 100,000 μg / mL, at least 50,000 μg / mL, at least 10,000 μg / mL, at least 5,000 μg / mL, or at least 1,000 μg / mL. The preferred content range of the lysozyme-chitosan complex of the present invention can be a range obtained by appropriately combining these lower limits and upper limits, for example, 0.1 μg / mL to 10000 μg / mL, preferably 0.5 μg / mL to 5000 μg / mL, 50 μg / mL to 5000 μg / mL, more preferably 1 μg / mL to 1000 μg / mL, 100 μg / mL to 1000 μg / mL is appropriate.

[0058] In addition, relative to the mass of the entire pharmaceutical composition of the present invention, the lysozyme-chitosan complex of the present invention, for example, contains 0.0001 mass% or more, 0.001 mass% or more, 0.005 mass% or more, 0.01 mass% or more, 0.05 mass% or more, 0.1 mass% or more, 0.5 mass% or more, or 1 mass% or more is appropriate. In addition, relative to the mass of the entire pharmaceutical composition of the present invention, the lysozyme-chitosan complex of the present invention, for example, contains 5.0 mass% or less, 1 mass% or less, 0.5 mass% or less, 0.1 mass% or less, 0.05 mass% or less, 0.01 mass% or less, 0.005 mass% or less is appropriate. The preferred content range of the lysozyme-chitosan complex of the present invention can be a range obtained by appropriately combining these lower limits and upper limits, for example, 0.001 mass% to 5.0 mass%, preferably 0.005 mass% to 1.0 mass%, more preferably 0.01 mass% to 0.5 mass% is appropriate.

[0059] In addition, in the pharmaceutical composition of the present invention, the additional active ingredient is, for example, 0.00001% to 20% by mass, preferably 0.00005% to 10% by mass, more preferably 0.0001% to 5% by mass, further preferably 0.0005% to 3% by mass, and particularly preferably 0.001% to 1% by mass. The mass ratio of the lysozyme-chitosan complex of the present invention to the additional active ingredient is, for example, 99 / 1 to 1 / 99, preferably 90 / 10 to 10 / 90, more preferably 80 / 20 to 20 / 80, further preferably 60 / 40 to 40 / 60, and particularly preferably 50 / 50.

[0060] In addition, the appropriate content of other components needs to be appropriately adjusted according to each component, for example, it is appropriate to contain 0.1% to 90% by mass, preferably 1% to 70% by mass, and more preferably 10% to 50% by mass. It should be noted that most of the pharmaceutical composition of the present invention can be a solvent such as water. For example, in the pharmaceutical composition of the present invention, it is appropriate that the solvent contains 95% by mass or more, preferably 98% by mass or more, more preferably 99% by mass or more, and further preferably 99.5% by mass or more.

[0061] <Antiprotozoal composition>

[0062] The present invention further relates to an antiprotozoal composition containing a complex formed by combining lysozyme and chitosan. The details of the content of the complex formed by combining lysozyme and chitosan that can be contained in the antiprotozoal composition, the details of the additional active ingredients, other ingredients, etc. are all as described in the above-mentioned description of the pharmaceutical composition.

[0063] <Anti-adhesion agent>

[0064] The present invention further relates to an anti-adhesion agent containing a complex formed by combining lysozyme and chitosan. Detailed information such as the content of the complex formed by combining lysozyme and chitosan that may be contained in the anti-adhesion agent, additional active ingredients, details of other ingredients, etc. are all as described in the description of the above-mentioned pharmaceutical composition. It is believed that the anti-adhesion property is due to the fact that the complex formed by combining lysozyme and chitosan combines with Acanthamoeba through an electrostatic mechanism, thereby inhibiting the attachment of Acanthamoeba to the surface of plastics, etc., but it is not limited to this theory. It is believed that the complex formed by combining lysozyme and chitosan is positively charged, while the Acanthamoeba is negatively charged, so the attachment of Acanthamoeba to the surface of the material competes with the attachment of the complex, thereby obtaining an anti-adhesion effect.

[0065] It should be noted that the anti-adhesion agent is usually contained in the cleaning solution or storage solution of the contact lens. The content of the anti-adhesion agent in the cleaning solution or storage solution can be determined according to the ratio of the complex formed by the lysozyme and chitosan contained in the anti-adhesion agent. In 1 mL of the cleaning solution or storage solution, the complex formed by the lysozyme and chitosan is, for example, 0.001 μg / mL or more, 0.01 μg / mL or more, 0.1 μg / mL or more, 0.5 μg / mL or more, 1 μg / mL or more, 10 μg / mL or more, 50 μg / mL or more, 100 μg / mL or more. In addition, in 1 mL of the cleaning solution or storage solution of the present invention, the lysozyme-chitosan complex of the present invention is, for example, 100000 μg / mL or less, 50000 μg / mL or less, 10000 μg / mL or less, 5000 μg / mL or less, 1000 μg / mL or less. The preferred content range of the lysozyme-chitosan complex of the present invention may be a range formed by appropriately combining these lower limits and upper limits, and is, for example, 0.01 μg / mL to 100,000 μg / mL, preferably 0.5 μg / mL to 10,000 μg / mL, and more preferably 0.1 μg / mL to 1,000 μg / mL.

[0066] Here, the contact lenses may be generally used soft contact lenses or hard contact lenses. The cleaning solution or storage solution may be a solution commonly used for cleaning or storing contact lenses, such as purified water represented by physiological saline, containing components such as surfactants, thickeners, and wetting agents, or a storage solution in a blister package used for new contact lenses that have not been worn.

[0067] <Treatment methods and prevention methods>

[0068] The present invention may include a method for treating or preventing inflammation caused by Acanthamoeba by administering a pharmaceutical composition containing a complex formed by combining lysozyme and chitosan to a subject. The treatment or prevention is, for example, directed to infection, corneal inflammation and / or disease of the skin or eyes, especially the cornea of ​​the eye. It can be said that the treatment or prevention effect is brought about by the above-mentioned antiprotozoal and / or anti-adhesion properties of the complex formed by combining lysozyme and chitosan, but it is not limited to this theory.

[0069] Here, the pharmaceutical composition used in the treatment or prevention method and the details of various definitions are as described above, and aspects such as the additional active ingredients that may be included, as well as the definitions of lysozyme or chitosan, the content and content ratio of the additional active ingredients or other ingredients are all as described in the above description of the pharmaceutical composition.

[0070] Here, the “subject” includes not only humans but also mammals such as cats, dogs, monkeys, cows, and horses.

[0071] <Dosage, dosage form, and administration method>

[0072] The pharmaceutical composition of the present invention varies according to its dosage form, administration target, administration route, target disease, symptoms, etc. For example, in the case of a spray sprayed on the skin of a human body, hand, finger, etc., the daily dosage is, for example, 0.1 to 20 mg / kg body weight, preferably 0.2 to 10 mg / kg body weight, and more preferably 0.5 to 10 mg / kg body weight, preferably once a day to several times (for example, 2 times, 3 times, 4 times or 8 times). In addition to the spray, the dosage can also be preferably used in other preparations such as the cream described below.

[0073] The pharmaceutical composition of the present invention can be administered orally or parenterally, and their preparation does not require special technology, and can be prepared using general technology. As dosage forms, creams, ointments, cataplasms, liquids, sprays, gels, injections, tablets, suppositories, capsules, granules, powders, eye drops, eye ointments, etc. are mentioned, and creams, ointments, cataplasms, liquids, and sprays are particularly preferred.

[0074] The pharmaceutical composition of the present invention can be administered by a method appropriately selected according to the above-mentioned administration form. The administration method may be a known administration method, for example, in the case of a cream, the pharmaceutical composition of the present invention can be applied to the inflammation site in the above-mentioned dosage.

[0075] <Anti-protozoal method, dosage, form of use, and method of use>

[0076] The present invention can use the antiprotozoal composition containing the complex formed by the combination of lysozyme and chitosan for the object to prevent, remove, kill protozoa, and antiprotozoa such as Acanthamoeba. The meaning of antiprotozoal property is as described above. When the object is a mammal such as a human, the instructions for the treatment method, prevention method, dosage, dosage form, and administration method involved in the above-mentioned pharmaceutical composition can be used for the antiprotozoal method. When the object is non-human, the amount, form, and method of use can be appropriately selected according to the treatment method, prevention method, dosage, dosage form, and administration method involved in the above-mentioned pharmaceutical composition. For example, when the object is a contact lens case, the antiprotozoal composition can be spread on the contact lens case in the form of a liquid or a spray.

[0077] <Anti-adhesion method, dosage, form of use, and method of use>

[0078] The present invention uses an anti-adhesion agent containing a complex formed by combining lysozyme and chitosan on an object to prevent and remove the attachment of protozoa such as Acanthamoeba. The meaning of anti-adhesion and the object of attachment are as described above. The amount, form of use, and method of use of the anti-adhesion agent can adopt the instructions for the treatment method, prevention method, dosage, dosage form, and method of administration involved in the above-mentioned pharmaceutical composition. For example, when the object of anti-adhesion is a contact lens case, the anti-adhesion agent can be mixed in the cleaning solution or storage solution of the contact lens in the form of a liquid.

[0079] <Application>

[0080] In addition, the present invention may include the use of the complex formed by combining lysozyme and chitosan in the manufacture of a pharmaceutical composition, an antiprotozoan composition and an anti-adhesion agent containing the complex formed by combining lysozyme and chitosan for treating or preventing inflammation caused by Acanthamoeba.

[0081] In the manufacture of the pharmaceutical composition, antiprotozoal composition and anti-adhesion agent, the complex formed by the combination of lysozyme and chitosan can be mixed arbitrarily with additional active ingredients or other ingredients other than the complex to prepare the pharmaceutical composition, etc. As the mixing method, a known method can be used. For example, in the case of a liquid agent, the complex formed by the combination of lysozyme and chitosan and any other ingredients mentioned above can be added to a solvent such as water and mixed, and an emulsifier can be added as needed to mix to prepare a dispersion or emulsion, thereby preparing a liquid agent.

[0082] The pharmaceutical composition, antiprotozoan composition and anti-adhesion agent can be made into cleaning water used in an atomizer. Thus, by spraying the pharmaceutical composition, antiprotozoan composition and anti-adhesion agent at a required location, the reproduction and adhesion of protozoa such as Acanthamoeba can be inhibited, and the protozoa can be killed.

[0083] In addition, the pharmaceutical composition, antiprotozoal composition, and anti-adhesion agent of the present embodiment have, for example, antiprotozoal effects and anti-adhesion effects on Acanthamoeba, and have therapeutic, inhibitory, or preventive effects on inflammation caused by them.

[0084] The present invention is described in more detail with reference to the following examples, but the present invention is not limited to these examples. It should be noted that in this specification and the accompanying drawings, the complex formed by combining lysozyme and chitosan is also referred to as lysozyme-chitosan complex or LYZOX (registered trademark of WAKO FILTER TECHNOLOGY Co., Ltd.).

[0085] Example

[0086] [Preparation of sample]

[0087] ·Lysozyme-chitosan complex (LYZOX (registered trademark)) solution

[0088] As a solution of a complex (lysozyme-chitosan complex) formed by combining lysozyme and chitosan, commercially available LYZOX (registered trademark, manufactured by WAKO FILTER TECHNOLOGY Co., Ltd.) is used. Commercially available LYZOX (registered trademark) is a powder containing lysozyme (about 14000 Da) from chicken and water-soluble chitosan (chitosan oligosaccharide) of about 5000 Da in a mass ratio of 1:1. Specifically, the above-mentioned lysozyme and water-soluble chitosan are mixed and dissolved in water, and then freeze-dried to prepare a powder, and the Maillard reaction is further carried out under conditions of temperature, humidity and days sufficient for the Maillard reaction to be completely completed, thereby obtaining the lysozyme-chitosan complex (LYZOX (registered trademark)) used in the present invention. The lysozyme-chitosan complex (LYZOX (registered trademark)) was mixed with sterile water so that the lysozyme-chitosan complex had a desired concentration in each experimental example to prepare a target lysozyme-chitosan complex (LYZOX (registered trademark)) solution.

[0089] Solution of lysozyme monomer

[0090] The lysozyme monomer solution was prepared by mixing egg white lysozyme having a molecular weight of about 14,300 (manufactured by BIOCON (JAPAN) Co., Ltd., Aichi Prefecture) with sterile water so that the lysozyme concentration was desired in each experimental example to prepare a target lysozyme monomer solution.

[0091] Chitosan monomer solution

[0092] The chitosan monomer solution was prepared by using chitosan oligosaccharide (KIMICA Chitosan Oligosaccharide COS-A manufactured by KIMICA Corporation) as chitosan and mixing the chitosan oligosaccharide with sterile water so that the chitosan oligosaccharide had a desired concentration in each experimental example to prepare a target chitosan monomer solution.

[0093] · Solution of a mixture of lysozyme and chitosan

[0094] The mixture of lysozyme and chitosan was prepared by mixing the two so that the mass ratio of the lysozyme monomer to the chitosan monomer was 1:1, and further adding sterile water so that the mixture, lysozyme and chitosan had desired concentrations.

[0095] Acanthamoeba solution (suspension)

[0096] Acanthamoeba polyphaga (source: American Type Culture Collection (ACC), 30872 TM ) were dispersed in a phosphate-buffered saline solution to prepare a solution containing 1 × 10 5 Acanthamoeba solution containing 100% Acanthamoeba.

[0097] Test piece

[0098] The polypropylene test piece used in the adhesion test described below was purchased from JOHOKU Co., Ltd., product number: PP_A3. A test piece of 15 mm in length, 15 mm in width, and 0.25 mm in thickness cut from a test piece of 320 mm in length × 440 mm in width × 0.25 mm in thickness was used for each experiment.

[0099] The detailed steps of each experimental example are as follows.

[0100] Experimental Example 1 (Adhesion Test and Cell Death Test)

[0101] In order to verify the anti-adhesion and antiprotozoal properties of the lysozyme-chitosan complex (LYZOX (registered trademark)), an adhesion test and a cell death test were performed.

[0102] Specifically, the above-mentioned Acanthamoeba solution was added to the above-mentioned lysozyme-chitosan complex (LYZOX (registered trademark)) solution, and the mixed solution was prepared in such a way that the lysozyme-chitosan complex had a concentration of 0.1 μg, 1 μg, 10 μg, 100 μg and 1000 μg in 1 mL of the mixed solution. In addition, a control solution with a concentration of 0 μg / mL without lysozyme-chitosan complex was prepared. The concentration was adjusted using the same sterile water as that used when preparing the lysozyme-chitosan complex as the diluent. All of these 6 solutions contained 1×10 5 Acanthamoeba.

[0103] The above-mentioned polypropylene test piece was immersed in the prepared mixed solution and allowed to stand. After 60 minutes of immersion, the test piece was taken out from the mixed solution, and the test piece was washed with phosphate buffered saline (PBS, purchase source: FUJIFILM WakoChemicals, product number: 166-23555) and dried. The surface of the obtained test piece was observed with a biological microscope (purchase source: Nikon, product number: ECLIPSEE-200), and the Acanthamoeba adhered within the observation range of 5×5 mm on the surface of the test piece was counted. Thereafter, the dead Acanthamoeba were stained with trypan blue in the observation range, and the number of dead Acanthamoeba was counted. The results are shown in the following Tables 1 and Figure 2 and Figure 3 .

[0104] Table 1

[0105]

[0106] As shown in Table 1, the lysozyme-chitosan complex causes cell death of Acanthamoeba in a concentration-dependent manner. The effect of the lysozyme-chitosan complex in inhibiting the adhesion of Acanthamoeba was observed starting from 0.1 μg / mL, and a significant adhesion inhibition effect was observed at concentrations of 1 μg / mL and above. At 100 μg / mL and 1000 μg / mL, the adhesion of most Acanthamoeba was inhibited, and a very excellent adhesion inhibition effect was obtained. Regarding the inhibitory effect of the lysozyme-chitosan complex on Acanthamoeba (antiprotozoal effect), from the number of dead Acanthamoeba and the mortality rate of Acanthamoeba in Table 1, it can be seen that almost 100% mortality was achieved at 100 μg / mL and 1000 μg / mL, and it has a significant antiprotozoal effect.

[0107] Experimental Example 2 (Comparative test of adhesion test and cell death test)

[0108] The above-mentioned solution of the mixture of lysozyme and chitosan (Table 2), lysozyme monomer (Table 3), and chitosan monomer (Table 4) were used instead of the lysozyme-chitosan complex (LYZOX (registered trademark)) solution of Experimental Example 1. The Acanthamoeba were counted in the same manner as in Experimental Example 1, and the number of all Acanthamoeba attached, the number of dead Acanthamoeba, and the mortality rate of Acanthamoeba were measured. The results are shown in the following Tables 2 to 4. Figure 4 and Figure 5 .

[0109] Table 2

[0110]

[0111] Table 3

[0112]

[0113] Table 4

[0114]

[0115] From Tables 1 to 4 and Figure 4It can be seen that the lysozyme-chitosan complex of the present invention shows an excellent adhesion inhibition effect on Acanthamoeba compared to a mixture of lysozyme and chitosan of the same mass, a lysozyme monomer of the same mass as the lysozyme in the lysozyme-chitosan complex of the present invention, and a chitosan monomer of the same mass as the chitosan in the lysozyme-chitosan complex of the present invention. In particular, at a concentration of 1 μg / mL or more, a significant adhesion inhibition effect was observed compared to the mixture of lysozyme and chitosan, the lysozyme monomer, and the chitosan monomer. In addition, as shown in Tables 2 to 4 and Figure 5 It can be seen that the lysozyme-chitosan complex shows an excellent mortality rate of Acanthamoeba, i.e., an inhibitory effect (antiprotozoal effect) on Acanthamoeba, compared to a mixture of lysozyme and chitosan of the same mass, a lysozyme monomer of the same mass as the lysozyme in the lysozyme-chitosan complex of the present invention, and a chitosan monomer of the same mass as the chitosan in the lysozyme-chitosan complex of the present invention. In particular, the lysozyme-chitosan complex of the present invention achieves almost 100% mortality rate at 100 μg / mL and 1000 μg / mL, while the inhibitory effect of the mixture of lysozyme and chitosan, the lysozyme monomer and the chitosan monomer on Acanthamoeba is incomplete.

[0116] Experimental Example 3 (Comparative test of adhesion test and cell death test)

[0117] In order to verify the effects of using a lysozyme-chitosan complex (LYZOX (registered trademark)) with an additional active ingredient, in particular the synergistic anti-adhesion and antiprotozoal properties, an adhesion test and a cell death test were performed.

[0118] First, a contact lens storage solution containing Acanthamoeba was prepared. Specifically, Acanthamoeba polyphaga (purchased from American Type Culture Collection (ACC), 30872 TM ) were dissolved in each of the commercially available contact lens storage solutions listed in Table 5, and 1 mL of the storage solution was prepared to contain 1×10 6 Acanthamoeba-containing contact lens storage solution.

[0119] The above-mentioned lysozyme-chitosan complex (LYZOX (registered trademark)) solution was added to the prepared contact lens storage solution containing Acanthamoeba, and a mixed solution was prepared in which the lysozyme-chitosan complex was 100 μg (mass 0.01%) in 1 mL of the mixed solution. Distilled water was also used as a control. The concentration was adjusted using the same sterile water as that used when preparing the lysozyme-chitosan complex as a diluent.

[0120] A polypropylene test piece was immersed in the prepared mixed solution in the same manner as in Experimental Example 1, and the number of Acanthamoeba adhering to the test piece was counted, and the number of dead Acanthamoeba was counted. The results are shown in the following Table 5. Figure 6 and Figure 7 .

[0121] Table 5

[0122]

[0123] *: In Table 5, the upper part of each storage solution is only the storage solution, and the lower part is the storage solution + lysozyme-chitosan complex

[0124] *1: ClearCare is a registered trademark, source: Japan Alcon Co., Ltd., product name: AOSEPT ClearCare (registered trademark)

[0125] Cleadew is a registered trademark. Purchase source: OPHTECS Co., Ltd. Product name: Cleadew First Care (registered trademark)

[0126] OptiFree is a registered trademark. Purchase source: Japan Alcon Co., Ltd. Product name: OptiFree Plus (registered trademark)

[0127] renu is a registered trademark. Purchase source: Bausch & Lomb Japan Co., Ltd. Product name: renu MultiPlus (registered trademark)

[0128] As shown in Table 5 and Figure 6 As shown in Table 5, the adhesion inhibition effect on Acanthamoeba was limited in the case of each contact lens storage solution alone, but a significant adhesion inhibition effect was observed when the contact lens storage solution was combined with the lysozyme-chitosan complex. In addition, regarding the inhibitory effect (antiprotozoal effect) of the lysozyme-chitosan complex on Acanthamoeba, the number of dead Acanthamoeba and the mortality rate of Acanthamoeba in Table 5 and the Figure 7 In particular, when povidone iodine and polyhexanide hydrochloride were used as additional active ingredients in combination with the lysozyme-chitosan complex, a better inhibitory effect (antiprotozoal effect) against Acanthamoeba was obtained compared to the case of using only the contact lens storage solution.

[0129] Industrial Applicability

[0130] According to the present invention, a novel pharmaceutical composition, an antiprotozoan composition, an anti-adhesion agent, and the like against Acanthamoeba can be provided.

Claims

1. A pharmaceutical composition comprising a complex of lysozyme and chitosan for treating or preventing inflammation caused by Acanthamoeba.

2. The pharmaceutical composition according to claim 1, wherein The chitosan is water-soluble chitosan with a molecular weight of 1000Da to 30000Da.

3. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition contains 0.01 μg / mL to 100,000 μg / mL of the complex.

4. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition contains 0.0001% by mass to 5.0% by mass of the complex.

5. The pharmaceutical composition according to claim 1, wherein The composition further contains an additional active ingredient selected from the group consisting of aqueous hydrogen peroxide solution, povidone iodine, polychloridium chloride and polyhexanide hydrochloride.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein Inflammation caused by Acanthamoeba is dermatitis or keratitis.

7. The pharmaceutical composition according to claim 6, wherein The treatment or prevention of inflammation caused by the Acanthamoeba is achieved by inhibiting the attachment of the Acanthamoeba.

8. Use of a complex of lysozyme and chitosan in the manufacture of a pharmaceutical composition for treating or preventing inflammation caused by Acanthamoeba.

9. An antiprotozoal composition comprising a complex of lysozyme and chitosan.

10. The antiprotozoal composition according to claim 9, wherein The protozoa is Acanthamoeba.

11. An anti-attachment agent for Acanthamoeba, comprising a complex of lysozyme and chitosan.

12. A method for preventing Acanthamoeba from attaching to the surface of a substance, comprising applying a complex formed by combining lysozyme and chitosan to the surface of the substance.

13. The method for preventing the attachment of Acanthamoeba according to claim 12, wherein: The material is selected from the group consisting of polyethylene, polypropylene, polyurethane and polycarbonate.

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