WO2012161112A1 - 新規金属タンパク質及びその製造方法、並びに前記金属タンパク質を含有する角結膜疾患の予防又は治療剤 - Google Patents
新規金属タンパク質及びその製造方法、並びに前記金属タンパク質を含有する角結膜疾患の予防又は治療剤 Download PDFInfo
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- selenium
- lactoferrin
- apolactoferrin
- corneal
- eye
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Definitions
- the present invention relates to a novel metal protein, a method for producing the same, and a preventive or therapeutic agent for keratoconjunctival diseases containing the metal protein. More specifically, the present invention relates to a protein comprising selenium and apolactoferrin, in which selenium is bound to apolactoferrin, that is, selenium-lactoferrin, a method for producing the same, and a keratoconjunctival disease containing the metalloprotein. The present invention relates to a preventive or therapeutic agent.
- the selenium-lactoferrin of the present invention is useful for the prevention or treatment of keratoconjunctival diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or corneal ulcer.
- the cornea is a thin tissue with a thickness of approximately 1 mm and no blood vessels, but has a very regular and fine structure consisting of an epithelial layer, Bowman's membrane, parenchyma, Descemet's membrane, and endothelial cell layer.
- This cornea is located at the forefront of the eyeball, and is a highly differentiated tissue that has permeability and refractive power to guide light from the outside world to the photoreceptors in the retina. Since it is in direct contact, it also functions as a barrier against chemical invasion and biological invasion of microorganisms.
- corneal epithelial layer On the corneal epithelial layer, there is a tear film that keeps the eye wet, prevents adhesion between the eyelid and the conjunctiva, and maintains the physiological state of the cornea and conjunctiva.
- the corneal epithelial cells have a columnar shape at the base, but become flat as they approach the surface, and the epithelial cells that have divided at the base gradually move upward, eventually drop off, and are carried away by tears. Since corneal endothelial cells do not divide, they are not regenerated even if they fall off. If the keratoconjunctival disorder caused by various corneal diseases such as dry eye, corneal ulcer, corneal epithelial detachment, keratitis, etc.
- Examples of the therapeutic agent for corneal epithelial wound include components such as fibronectin, EGF (Epidermal Growth Factor), and hyaluronic acid.
- fibronectin is a blood product that must be purified from the patient's own plasma using a special purification kit, which is laborious and burdensome for the patient, and is not used clinically.
- EGF has a mitotic and proliferative action on corneal epithelial cells, but in the case of inflammation or in diabetic keratopathy, side effects that cause angiogenesis are problematic and are rarely used clinically.
- Hyaluronic acid is a glucosaminoglycan with a molecular weight of several millions, which is composed of N-acetyl-D-glucosamine and D-glucuronic acid, and is used in dry eye patients because of its water retention effect.
- hyaluronic acid also acts on the adhesion and migration of corneal epithelial cells, the proliferation effect of epithelial cells is weak. There is also a problem that the viscosity becomes high at a high concentration.
- lactoferrin is an iron-binding glycoprotein that exists mainly in milk of mammals and is also found in neutrophils, tears, saliva, nasal discharge, bile, semen and the like. Lactoferrin is an important protein for maintaining the health and development of infants and has recently been found to have antibacterial and antibacterial effects, and is used in various fields in addition to the food industry.
- lactoferrin as a tear fluid component as an active ingredient of eye drops for treating dry eye, and apolactoferrin from which iron is removed from lactoferrin are effective ingredients of ophthalmic agents and contact lens compositions It is proposed to use as (refer patent document 2, 3).
- Lactoferrin is an iron-binding glycoprotein having a molecular weight of about 80,000, and in its three-dimensional structure, there are two iron-binding pockets that can bind to one iron.
- bacteriostatic (antibacterial) action of lactoferrin it is considered as follows. That is, empty iron-binding pockets present in lactoferrin have a chelating action on iron, but the action deprives iron necessary for the growth of microorganisms and restricts their growth. For this reason, microorganisms that strongly require iron during growth receive the bacteriostatic (antibacterial) action of lactoferrin. Such a bacteriostatic (antibacterial) action of lactoferrin has been considered particularly in the intestinal environment.
- keratoconjunctival disease therapeutic agents As described above, conventionally, various keratoconjunctival disease therapeutic agents have been known, but those having high therapeutic effects and suitable for industrial large-scale production are not known. In view of the above, a further excellent therapeutic agent has been desired. In addition, a better therapeutic agent has been desired for ophthalmic disorders such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, and corneal ulcer.
- the present inventors have found that the metal protein in which selenium is bound to apolactoferrin has an excellent curative effect on keratoconjunctival disorders, and have led to the present invention.
- the present invention provides the following (1) to (11).
- a preventive or therapeutic agent for keratoconjunctival disease comprising the metal protein of (1), (2) or (6) as an active ingredient.
- a preventive or therapeutic agent for dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or corneal ulcer comprising the metal protein of (1), (2) or (6) as an active ingredient.
- An ophthalmic composition comprising the metal protein of the above (1), (2) or (6) and an ophthalmic carrier acceptable for an ophthalmic preparation.
- the present invention provides the following (12) to (15).
- (12) Use of the metal protein of (1), (2) or (6) above as an active ingredient of a preventive or therapeutic agent for keratoconjunctival disease.
- (12) Use of the metal protein of (1), (2) or (6) for the manufacture of a preventive or therapeutic agent for keratoconjunctival disease.
- (14) The metal protein of the above (1), (2) or (6) for use in the prevention or treatment of keratoconjunctival disease.
- a method for preventing or treating keratoconjunctival diseases such as erosion or corneal ulcer, preferably dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, corneal ulcer and the like accompanied by keratoconjunctival epithelial disorder.
- Lactoferrin is readily available in large quantities because it is present in large quantities in mammalian body fluids, such as milk, and can also be produced by genetic engineering techniques. Therefore, the selenium-lactoferrin of the present invention using this as a raw material can also be produced on a large scale industrially at a low cost.
- the selenium-lactoferrin of the present invention has a high therapeutic effect on keratoconjunctival diseases. Furthermore, since selenium-lactoferrin has an antibacterial action, it can be expected to reduce the amount of preservatives during formulation or to avoid the use of preservatives itself, thereby reducing side effects caused by the preservatives. be able to. That is, according to the present invention, it is possible to provide a keratoconjunctival disease therapeutic agent that has both high therapeutic effect and safety and is suitable for industrial large-scale production.
- FIG. 1 shows the result of comparison of the amount of selenium contained in the selenium-lactoferrin of the present invention when manufactured using apolactoferrin as a raw material and when manufactured using lactoferrin.
- FIG. 1 (a) shows the results when excess selenium that was not incorporated into the protein was removed using a dialysis membrane, and (b) shows the results when a UF membrane was used.
- FIG. 2 shows the results of comparing the degree of prevention of corneal epithelial damage by selenium-lactoferrin of the present invention with phosphate buffered saline (PBS) using an extraorbital lacrimal gland excision dry eye model. Is.
- PBS phosphate buffered saline
- FIG. 2A shows a comparison with 1% selenium-lactoferrin
- FIG. 2B shows a comparison with 5% selenium-lactoferrin
- FIG. 2C shows a result in a normal group.
- the vertical axis indicates the fluorescence score value
- the left column indicates the result of the left eye administered with PBS (control)
- the right column ((a) and (b) of FIG. 2) indicates selenium-lactoferrin.
- the result of the administered right eye is shown.
- FIG. 3 shows the results of comparing the degree of prevention of corneal epithelial damage by selenium-lactoferrin of the present invention with the same concentration of apolactoferrin using an extraorbital lacrimal gland excision dry eye model.
- FIG. 4 shows the results of comparing the degree of prevention of corneal epithelial damage by selenium-lactoferrin of the present invention with PBS using a smoking treated dry eye model.
- the vertical axis shows the average fluorescence score value
- the left column shows the PBS eyedrop side of the control
- the right column shows the 0.1% selenium-lactoferrin eyedrop side.
- FIG. 5 shows the results of comparing the degree of prevention of corneal epithelial damage by selenium-lactoferrin of the present invention with PBS using a dry room-treated dry eye model.
- the vertical axis represents the average fluorescence score value.
- FIG. 5A shows the score of the normal environment, and the right column shows the score of the dry room treatment (PBS eye drop side).
- the left column in FIG. 5 (b) shows the PBS ophthalmic side of the control, and the right column shows the 0.1% selenium-lactoferrin ophthalmic side.
- the “selenium-lactoferrin” referred to in the present invention means a metal protein composed of selenium and apolactoferrin, wherein selenium is bound to apolactoferrin.
- selenium-lactoferrin of the present invention selenium is not removed by dialysis or ultrafiltration, but is removed only by making the pH of the solution strong acid. As such, selenium is not present in mere mixture form in apolactoferrin but is bound to apolactoferrin with some strength. Therefore, the selenium-lactoferrin of the present invention is distinguished from a simple mixture of selenium and apolactoferrin.
- selenium and apolactoferrin are considered to be bound to selenium by the chelating action of the iron binding pocket in the apolactoferrin, but the present invention is bound by such a theory. It is not a thing.
- “Apolactoferrin” referred to in the present invention means a product obtained by removing iron from lactoferrin.
- the apolactoferrin referred to in the present invention may be derived from natural lactoferrin obtained from animal body fluids such as milk, or may be synthesized by genetic engineering, and some of the amino acids may be converted to other amino acids. It may be substituted, a part of the amino acid may be deleted, or a few amino acids may be added.
- the selenium-lactoferrin of the present invention uses a solution containing lactoferrin and / or apolactoferrin as a raw material, and by adding selenium salt thereto, selenium is bound to apolactoferrin, and then the solution is dialyzed or limited. It can be produced by subjecting it to external filtration to remove excess selenium.
- the selenium-lactoferrin of the present invention is not limited by theory, it is considered that selenium binds to an empty iron-binding pocket present in lactoferrin or apolactoferrin.
- any solution containing either one or both of lactoferrin and apolactoferrin can be used as a raw material.
- production efficiency since the binding of selenium to empty iron-binding pockets present in lactoferrin or apolactoferrin is considered to occur more easily than substitution with iron in lactoferrin, it is called production efficiency. From the viewpoint, it is desirable to use a solution containing apolactoferrin as a raw material.
- the content of apolactoferrin is preferably as high as possible.
- the content of apolactoferrin is 90 mol relative to the total amount of lactoferrin and apolactoferrin. % Or more, particularly 95 mol% or more, and most preferably 97 mol% or more.
- the solution containing the said lactoferrin and / or apolactoferrin can be prepared using what is marketed as a lactoferrin or apolactoferrin as a pharmaceutical, a reagent, etc.
- the solution containing lactoferrin and / or apolactoferrin may be prepared from an animal-derived one obtained from milk (eg, milk) or whey (whey), or produced by genetic engineering techniques.
- a solution containing lactoferrin and apolactoferrin an acid such as hydrochloric acid or citric acid is added to a solution in which the concentration of apolactoferrin is improved by various known methods, for example, a lactoferrin-containing solution extracted from whey, etc. Then, it is also possible to use those prepared by adjusting the pH to about 2 and dissociating iron. Furthermore, as an example in which the concentration of apolactoferrin is improved, for example, the method described in Japanese Patent No. 4634809, that is, a solution containing lactoferrin and an acid are mixed, and the mixture is used as an ultrafiltration membrane.
- the non-permeate For the non-permeate, add acid again, perform ultrafiltration, and repeat the procedure until the target protein concentration and separation rate are achieved. It can be suitably manufactured also by performing.
- the acid inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and carbonic acid, and organic acids such as acetic acid, benzoic acid and citric acid are used.
- ACP-0013 hollow fiber module: inner diameter of membrane
- An ultrafiltration device incorporating 0.8 mm, effective membrane area 170 cm 2 , membrane material: polyacrylonitrile, nominal molecular weight cut-off: 13,000 may be used.
- the selenium salt used in the production method of the present invention is not particularly limited as long as it is water-soluble, and any selenium salt may be used, but selenium chloride, selenium bromide, and the like can be used.
- the amount of selenium salt added is not particularly limited, but selenium is considered to bind to the iron binding pocket of lactoferrin or apolactoferrin, and there are two iron binding pockets per molecule of the protein. It is preferable to add an amount of selenium salt in the reaction system such that about 2 mol or more of selenium is present per mol of the protein.
- the pH of the reaction solution is preferably 3 to 9.
- the pH of the reaction solution can also be adjusted by using various known buffering agents.
- the reaction temperature and time between the protein and the selenium salt should be adjusted as appropriate according to the materials and amounts used, but in general, mixing at 10 ° C. to 35 ° C. for about 5 minutes to 2 hours. Is desirable.
- lactoferrin and apolactoferrin have a molecular weight of about 80,000, so that the fractional molecular weight is about 5,000 to about 80,000.
- the material for the dialysis membrane include regenerated cellulose, cellulose acetate, and polyacrylonitrile.
- the material for the ultrafiltration membrane include cellulose acetate, polysulfone, polyethersulfone, polyacrylamide, polyimide, aromatic polyamide, Examples thereof include organic films of natural or synthetic polymers such as polyacrylonitrile and hydrophilic polyolefin, and ceramic inorganic films such as alumina, zirconia, and titanium.
- ultrafiltration membrane examples include hollow fiber module type, flat plate module type, and flat membrane type
- the hollow fiber module type is preferably selected from the viewpoint of filtration speed.
- the selenium-lactoferrin of the present invention has an excellent therapeutic effect on a rat corneal epithelial disorder model, as shown in the experimental examples described later. Therefore, a medicament containing these is a disease associated with inflammation or defect in the cornea or conjunctiva, for example, keratoconjunctival disease, more specifically, dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or cornea It is useful as a prophylactic or therapeutic agent for ulcers and the like, particularly as a prophylactic or therapeutic agent for these diseases associated with keratoconjunctival epithelial disorder.
- keratoconjunctival disease more specifically, dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or cornea
- cornea conjunctiva means the cornea and / or conjunctiva
- keratoconjunctival disease is a disease in the cornea and / or conjunctiva.
- Diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or corneal ulcer in the present invention are generally diseases included in keratoconjunctival diseases, but are not necessarily caused by keratoconjunctival diseases. It doesn't have to be a thing.
- the dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, corneal ulcer and the like in the present invention include those that do not depend on keratoconjunctival disease.
- the “pharmaceutically acceptable carrier” in the present invention refers to a carrier for a formulation that is acceptable for producing a preparation for internal use such as a tablet, a capsule, a liquid, etc.
- An “acceptable ophthalmic carrier” refers to a carrier for a formulation that is acceptable for producing an ophthalmic formulation such as eye drops or eye ointments.
- the pharmaceutical composition containing selenium-lactoferrin of the present invention as an active ingredient may further contain a pharmaceutically acceptable carrier and may contain an active ingredient other than selenium-lactoferrin.
- the selenium-lactoferrin of the present invention can be directly administered to the eye as an ophthalmic preparation, or can be used as a contact lens preservation solution. Furthermore, the selenium-lactoferrin of the present invention may be used as an internal preparation.
- Examples of internal preparations include oral solid preparations such as tablets, capsules, granules, film coating agents and powders, and oral liquid preparations such as liquids and syrups.
- oral solid preparations such as tablets, capsules, granules, film coating agents and powders
- oral liquid preparations such as liquids and syrups.
- a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, a flavor, etc. tablets, granules, Powders, capsules and the like can be produced.
- Such additives may be those commonly used in the art, such as lactose, sodium chloride, glucose, starch, microcrystalline cellulose, silicic acid, etc.
- excipients Water, ethanol, propanol, simple syrup, gelatin solution, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinyl pyrrolidone and the like;
- lubricants include purified talc, stearate, borax, and polyethylene glycol;
- colorants include ⁇ -carotene, yellow ferric oxide, and carmela;
- flavoring agents include sucrose and orange peel.
- oral liquids, syrups, elixirs and the like can be produced by conventional methods with the addition of flavoring agents, buffers, stabilizers, preservatives and the like.
- Such additives may be those commonly used in the art, such as sucrose as a flavoring agent; sodium citrate as a buffer; tragacanth as a stabilizer;
- preservatives include paraoxybenzoic acid esters.
- the pharmaceutical composition containing the selenium-lactoferrin of the present invention as an active ingredient is preferably an ophthalmic preparation, particularly an eye drop preparation.
- eye drops include aqueous eye drops, non-aqueous eye drops, and suspension eye drops. Any of emulsion eye drops, eye ointments and the like may be used.
- Such a preparation is prepared as a composition suitable for the dosage form, if necessary, as a pharmaceutically acceptable carrier, for example, an isotonic agent, a chelating agent, a stabilizer, a pH adjusting agent, a preservative, an antioxidant. , A solubilizing agent, a thickening agent, and the like, and can be produced by a method known to those skilled in the art.
- Isotonic agents include glucose, trehalose, lactose, fructose, mannitol, xylitol, sorbitol and other sugars, glycerin, polyethylene glycol, propylene glycol and other polyhydric alcohols, sodium chloride, potassium chloride, calcium chloride and other inorganic salts
- the blending amount is preferably 0 to 5% by weight based on the total amount of the composition.
- chelating agents include edetates such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate Citric acid and the like, and the blending amount is preferably 0 to 0.2% by weight based on the total amount of the composition.
- edetates such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate Citric acid and the like, and the blending amount is preferably 0 to 0.2% by weight based on the total amount of the composition.
- the stabilizer examples include sodium bisulfite and the like, and the blending amount is preferably 0 to 1% by weight with respect to the total amount of the composition.
- Examples of the pH adjuster include acids such as hydrochloric acid, carbonic acid, acetic acid and citric acid, and further alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, Examples thereof include alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like.
- the blending amount is preferably 0 to 20% by weight based on the total amount of the composition.
- the selenium-lactoferrin of the present invention itself can be expected to have antibacterial action.
- Paraoxybenzoic acid esters such as propyl acid and butyl paraoxybenzoate
- quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene Biguanides, chlorhexidine and the like
- the blending amount thereof is preferably 0 to 0.2% by weight based on the total amount of the composition.
- antioxidant examples include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like, and the blending amount is preferably 0 to 0.4% by weight with respect to the total amount of the composition.
- solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc., and the blending amount is based on the total amount of the composition. 0 to 3% by weight is preferred.
- the thickening agent examples include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, polyvinyl alcohol, and the like.
- the content is preferably 0 to 70% by weight based on the total amount of the composition.
- the desired components described above are dissolved or suspended in an aqueous solvent such as sterilized purified water or physiological saline, or a non-aqueous solvent such as vegetable oil such as cottonseed oil, soybean oil, sesame oil, or peanut oil.
- an ointment base can be included in addition to the various components described above.
- the ointment base is not particularly limited, but is an oily base such as petrolatum, liquid paraffin, or polyethylene; an emulsion base obtained by emulsifying an oil phase and an aqueous phase with a surfactant; hydroxypropyl methylcellulose, carboxymethylcellulose A water-soluble base composed of polyethylene glycol or the like is preferred.
- the dose varies depending on the body weight, age, sex, symptom, dosage form and number of administrations of the patient, but usually For adults, 0.2 to 1000 ⁇ g, preferably 2 to 200 ⁇ g per day as selenium-lactoferrin may be administered once or divided into several times.
- 0.01 to 50 mg. / ML, preferably 0.1 to 10 mg / mL may be instilled 1 to several drops at a time several times a day.
- Example 1 Preparation of selenium-lactoferrin 1 g of apolactoferrin (manufactured by Upwell Co., Ltd.) was added to an aqueous solution containing 5.4 mg of selenium chloride (I) (manufactured by Wako Pure Chemical Industries, Ltd.), and the volume was adjusted to 10 mL. The pH of the aqueous solution was confirmed to be 3 or higher, and selenium was bound to apolactoferrin by stirring at 25 ° C. for 30 minutes using a stirrer at 240 times / minute.
- I selenium chloride
- the above mixed solution of selenium chloride (I) and apolactoferrin is transferred to a dialysis cellulose tube (fractional molecular weight: 12,000 to 14,000; manufactured by ASONE Co., Ltd.), and 6 L of pure water (17 M ⁇ or more) Then, dialysis was performed at 4 ° C. for 48 hours while exchanging pure water every 12 hours to remove excess selenium that did not bind to apolactoferrin. After dialysis, freeze-drying was performed using a freeze dryer FDU-12AS (manufactured by ASONE Co., Ltd.).
- Example 2 The process of Example 1 was repeated except that lactoferrin (manufactured by Tatsua Japan Co., Ltd.) was used as a raw material.
- Example 3 Although the process of Example 1 was repeated, in this example, instead of dialysis, the removal of excess selenium was performed by the following procedure. That is, in this example, a UF membrane (Microza AP-0013 (UF) molecular weight cut off 6,000; manufactured by Asahi Kasei Co., Ltd.) is a pencil-type module tabletop filtration device (Microza UF • M FPS-24001; Asahi Kasei). (Made by Co., Ltd.). Then, using the apparatus (operation pressure: 40 kPa at the module outlet pressure), 10 mL of a mixed solution of selenium chloride (I) and apolactoferrin was ultrafiltered three times at room temperature (20 ° C. or higher).
- a UF membrane Molecular weight cut off 6,000; manufactured by Asahi Kasei Co., Ltd.
- Example 4 The process of Example 3 was repeated except that lactoferrin (manufactured by Tatsua Japan Co., Ltd.) was used as a raw material.
- Example 5 Measurement of selenium bond (experimental method) 1 g of selenium-lactoferrin lyophilized product prepared in Examples 1 to 4 was placed in a Kjeldahl flask, 10 mL of nitric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was allowed to stand at room temperature for 4 hours. Next, it was heated gently for 15 minutes with direct fire, and further heated for 10 minutes with slightly strong fire. After natural cooling, 5 mL of 70% perchloric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added and concentrated by heating. Heating was continued for another 15 minutes after white perchloric acid smoke was generated. The degradation product turned slightly yellow.
- PH was adjusted to 1.0 to 1.5 using 10% hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 10% aqueous ammonia (manufactured by Wako Pure Chemical Industries, Ltd.). To this, 5 mL of a 0.1% 2,3-diaminonaphthalene (manufactured by Wako Pure Chemical Industries, Ltd.) solution was added and mixed. The lid was capped and warmed in a 50 ° C. water bath for 30 minutes in the dark.
- FIG. 1 shows the results of removing excess selenium using a dialysis membrane, and (b) shows the result of removing excess selenium by ultrafiltration using a UF membrane.
- the vertical axis shows the amount of selenium (mg) bound in 100 g of apolactoferrin
- the left column shows the selenium binding amount when apolactoferrin is used as the raw material
- the right column uses lactoferrin as the raw material. Of selenium bonds.
- the selenium bond amounts are 142 mg / 100 g and 114 mg / 100 g, respectively, while using lactoferrin as a raw material (a), ( In the right column of b), the amount of selenium bonds was 64 mg / 100 g and 28 mg / 100 g. From this result, it was confirmed that the production efficiency of the final product, selenium-lactoferrin, was higher when apolactoferrin was used than when lactoferrin was used as a raw material.
- the damaged part of the corneal epithelium was stained with the fluorescent dye fluorescein.
- the degree of corneal epithelial damage was determined by dividing the entire cornea into nine parts, upper, middle, lower, and left middle right, and scoring the damage for each part according to the following criteria, and obtaining the total value. Thereafter, in each group, the total score value of the left eye and the total score value of the right eye were compared by Student's t-test. In order to make a fair evaluation, from the start of instillation to scoring of corneal epithelial disorder, the contents of the drug solution administered to each group were blinded and collated after scoring.
- the vertical axis indicates the fluorescence score value
- the left column is the left eye, ie, PBS eye drop side of the control
- the right column is the right eye, ie, selenium-lactoferrin (in the figure, expressed as Se-ApoL; The side which instilled a), (b)) or PBS (c) is shown.
- Each fluorescence score value is shown as an average value.
- the average value of the fluorescence score is 6.1 to 6.3, and corneal epithelial damage has progressed by PBS instillation for 2 weeks after lacrimation. It became clear.
- the average value of the fluorescence score was 3.3 (left eye) and 3.3 (right eye) by PBS instillation for 2 weeks.
- the average value of the fluorescence score is 4.7 for the S1 group of (a) and the same for the S5 group of (b). 3.7, confirming that the progression of corneal epithelial injury was suppressed as compared to the left eye.
- Example 2 The pharmacological test of Experimental Example 1 was repeated. In addition to the normal group, 4 groups were provided as drug administration groups, and concentrations of 0.01%, 0.1%, 1%, and 5% were set in the right eye, respectively. Of selenium-lactoferrin and the same concentration of apolactoferrin was administered to the left eye.
- (result) The results are shown in FIG.
- (a) is a 0.01% selenium-lactoferrin (PBS solution) administration group (hereinafter referred to as S0.01 group), and (b) is a 0.1% selenium-lactoferrin (PBS solution) administration group (hereinafter referred to as S0.00).
- (Group 1) (c) is a group administered with 1% selenium-lactoferrin (PBS solution) (hereinafter referred to as S1 group), (d) is a group administered with 5% selenium-lactoferrin (PBS solution) (hereinafter referred to as S5 group), and (e) is It is a normal group (hereinafter referred to as N group).
- the vertical axis indicates the fluorescence score value
- the left column is the left eye, that is, the side instilled with apolactoferrin (indicated as ApoL in the figure; (a) to (d)) or PBS (e)
- the right column shows the right eye, that is, the side instilled with selenium-lactoferrin (denoted as Se-ApoL in the figure; (a) to (d)) or PBS (e).
- Each fluorescence score value is shown as an average value. In the left column of (a), the average value of the fluorescence score is 4.6, indicating that the corneal epithelial disorder has progressed.
- FIG. 4 The results are shown in FIG. In the figure, the vertical axis represents the average fluorescence score value.
- the left column shows the PBS ophthalmic side of the control, and the right column shows the 0.1% selenium-lactoferrin ophthalmic side.
- keratoconjunctival epithelial disorder induced by smoking treatment for 5 days was caused by instillation of 0.1% selenium-lactoferrin (fluorescence score average value: 2.6) and control (PBS ophthalmic side, fluorescence score). It was revealed that the average value was significantly (p ⁇ 0.001) suppressed as compared with 5.6).
- Example 4 Curing efficacy test against Dry Room Mouse A keratoconjunctival epithelial disorder model by dry eye was prepared by the following method, and the healing effect on keratoconjunctival epithelial disorder was evaluated using selenium-lactoferrin produced in Example 3. In this experiment, both eyes of each individual were used, and one eye was used as a control, and a drug solution was administered to the other eye, so that comparison could be made in the same individual.
- a dry eye model was prepared by treating a male ICR mouse (6 weeks old) in a dry room. That is, keratoconjunctival epithelial injury was induced by treating mice in a dry room (humidity 20 ⁇ 5%, temperature 23 ⁇ 4 ° C.) for 2 weeks. As instillation conditions, PBS was applied to one eye, and 0.1% selenium-lactoferrin (PBS solution) was applied to the other eye at 5 ⁇ L once a day, 4 times a day for 2 weeks. For comparison, an animal in a normal environment (humidity 50 ⁇ 5%, temperature 23 ⁇ 4 ° C.) was also prepared (no instillation). Eight animals were used in the dry room and normal environment.
- FIG. 5A shows the score of the normal environment, and the right column shows the score of the dry room treatment (PBS eye drop side).
- the left column in FIG. 5 (b) shows the PBS ophthalmic side of the control, and the right column shows the 0.1% selenium-lactoferrin ophthalmic side.
- FIG. 5 (a) shows the keratoconjunctival epithelial disorder was significantly (p ⁇ 0.05) induced by the dry room treatment.
- FIG. 5 (a) shows the keratoconjunctival epithelial disorder was significantly (p ⁇ 0.05) induced by the dry room treatment.
- this dry room-induced keratoconjunctival epithelial disorder was caused by instillation of 0.1% selenium-lactoferrin (fluorescence score average value: 2.1) and control (PBS ophthalmic side, fluorescence score average). It was revealed that the value was significantly (p ⁇ 0.05) suppressed compared with the value: 3.4).
- selenium-lactoferrin is a prophylactic or therapeutic agent for keratoconjunctival diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratosis, corneal erosion, or corneal ulcer, especially for the prevention or It is useful as a therapeutic agent.
- Table 1 below shows typical formulation examples of eye drops containing selenium-lactoferrin of the present invention.
- the concentration of each component means the amount of each component contained per 100 mL of sterilized purified water.
- the selenium-lactoferrin of the present invention has an effect of suppressing the progression of corneal epithelial disorder, it prevents or treats keratoconjunctival diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, corneal ulcer and the like.
- keratoconjunctival diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, corneal ulcer and the like.
- the selenium-lactoferrin of the present invention can be produced industrially on a large scale at low cost because lactoferrin is used as a raw material and the lactoferrin can be easily obtained in large quantities. That is, the present invention provides a therapeutic agent for keratoconjunctival disease that has both high therapeutic effect and safety and is suitable for industrial large-scale production, and is industrially useful.
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Abstract
Description
ヒアルロン酸はN-アセチル-D-グルコサミンとD-グルクロン酸を構成糖とする分子量数百万のグルコサミノグリカンで、保水効果を持つことからドライアイ患者に用いられている。ヒアルロン酸は角膜上皮細胞の接着、移動にも働くものの、上皮細胞の増殖効果は弱い。また、高濃度では粘度が高くなってしまう問題もある。
ラクトフェリンは、分子量約80,000の鉄結合性の糖タンパク質であり、その三次元構造中には、1個の鉄と結合することができる、鉄結合ポケットが2つ存在する。ラクトフェリンの静菌(制菌)作用に関して、以下のように考えられている。すなわち、ラクトフェリンに存在する空の鉄結合性ポケットは、鉄に対するキレート作用を有するものであるが、当該作用によって、微生物の生育に必要とする鉄分が奪われ、その増殖が制限される。このため、生育の際に鉄分を強く要求する微生物が、ラクトフェリンの静菌(制菌)作用を受ける。このようなラクトフェリンの静菌(制菌)作用は、特に腸内環境において考察されている。このように、ラクトフェリンの静菌(制菌)作用に関して、同タンパク質中に存在する鉄結合性ポケットのキレート作用が注目されている。
しかしながら、一般的に、ラクトフェリンに存在する鉄結合性ポケットの鉄に対する結合性は、他のカチオンに対して2000倍程度高く、鉄に対して特異的であると考えられていたことから(非特許文献4)、従来、ラクトフェリン中の鉄を他の金属に置換した金属タンパク質は知られていなかった。さらに、本発明で述べるようなセレン-ラクトフェリンの眼部疾患に関わるような作用を類推することは難しく、実際に、そのような作用を見出した報告もない。
(1)セレン-ラクトフェリンである金属タンパク質。
(2)セレンが、アポラクトフェリン中に存在する鉄結合性ポケットに結合している(1)の金属タンパク質。
(3)上記(1)又は(2)に記載の金属タンパク質の製造方法であって、ラクトフェリン及び/又はアポラクトフェリンを含有する溶液に、セレン塩を添加する工程、及び得られた混合溶液を透析又は限外濾過に付する工程を含む前記製造方法。
(4)アポラクトフェリンを含有する溶液を用いる、(3)の方法。
(5)上記(4)の金属タンパク質の製造方法であって、アポラクトフェリンを、ラクトフェリンと酸とを含有する溶液を限外濾過膜に供給し、限外濾過を行う工程、及び前記限外濾過膜を透過しなかった溶液を回収する工程を含む方法により製造する前記製造方法。
(6)上記(3)~(5)のいずれかの方法により製造される、金属タンパク質。
(7)上記(1)、(2)又は(6)の金属タンパク質を有効成分とする角結膜疾患の予防又は治療剤。
(8)角結膜疾患が、ドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍である、(7)の予防又は治療剤。
(9)上記(1)、(2)又は(6)の金属タンパク質を有効成分とするドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍の予防又は治療剤。
(10)点眼剤又は眼軟膏剤である、上記(7)~(9)のいずれかの予防又は治療剤。
(11)上記(1)、(2)又は(6)の金属タンパク質、及び眼科用製剤に許容される眼科用担体を含有してなる眼科用組成物。
(12)角結膜疾患の予防又は治療剤の有効成分としての上記(1)、(2)又は(6)の金属タンパク質の使用。
(13)上記(1)、(2)又は(6)の金属タンパク質の角結膜疾患の予防又は治療剤の製造のための使用。
(14)角結膜疾患の予防又は治療に使用するための上記(1)、(2)又は(6)の金属タンパク質。
(15)ドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍等の角結膜疾患、好ましくは角結膜上皮障害を伴う、ドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍等を患っている患者又はこれらの疾患を患うおそれのある患者に、有効量のセレン-ラクトフェリンを投与することからなるドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍等の角結膜疾患、好ましくは角結膜上皮障害を伴う、ドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍等を予防・治療する方法。
本明細書における「角結膜」とは、角膜及び/又は結膜の意味であり、「角結膜疾患」とは角膜及び/又は結膜における疾患である。本発明におけるドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍等の疾患は、一般的には角結膜疾患に包含される疾患であるが、必ずしも角結膜疾患に起因するものである必要はない。従って、本発明におけるドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍等は、角結膜疾患によらないものをも包含するものである。
また、本発明における「製剤学的に許容される担体」とは、錠剤、カプセル剤、液剤などの内服用製剤を製造するために許容されている製剤用の担体をいい、「眼科用製剤に許容される眼科用担体」とは、点眼剤や眼軟膏などの眼科用製剤を製造するために許容されている製剤用の担体をいう。
経口用固形製剤を調製する場合は、賦形剤、必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味剤、矯臭剤等を加えた後、常法により錠剤、顆粒剤、散剤、カプセル剤等を製造することができる。そのような添加剤としては、当該分野で一般的に使用されているものでよく、例えば、賦形剤としては乳糖、塩化ナトリウム、ブドウ糖、デンプン、微結晶セルロース、珪酸等を;結合剤としては水、エタノール、プロパノール、単シロップ、ゼラチン液、ヒドロキシプロピルセルロース、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドン等を;崩壊剤としてはカンテン末、炭酸水素ナトリウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド等を;滑沢剤としては精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコール等を;着色剤としてはβ-カロチン、黄色三二酸化鉄、カルメラ等を;矯味剤としては白糖、橙皮等を例示できる。
尚、眼軟膏剤を調製する場合は、前記各種の成分の他に、軟膏基剤を含むことができる。前記軟膏基剤としては、特に限定されないが、ワセリン、流動パラフィン、ポリエチレン等の油性基剤;油相と水相とを界面活性剤等により乳化させた乳剤性基剤;ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、ポリエチレングリコール等からなる水溶性基剤等が好ましく挙げられる。
セレン-ラクトフェリンの調製
5.4mgの塩化セレン(I)(和光純薬(株)製)を含有する水溶液にアポラクトフェリン((株)アップウェル製)1gを添加し、液量を10mLに調整し、水溶液のpHが3以上であることを確認し、25℃で30分間、撹拌子を用いて240回/分の条件で撹拌することにより、アポラクトフェリンにセレンを結合させた。次に、前記の塩化セレン(I)とアポラクトフェリンの混合溶液を透析用セルローズチューブ(分画分子量12,000~14,000;アズワン(株)製)に移し、6Lの純水(17MΩ以上)を用い、12時間ごとに純水を交換しながら、4℃、48時間透析を行い、アポラクトフェリンに結合しなかった過剰のセレンを除去した。透析後、凍結乾燥をフリーズドライヤーFDU-12AS(アズワン(株)製)を用いて行った。
原料として、ラクトフェリン(タツア・ジャパン(株)製)を使用したこと以外は、実施例1の工程を繰り返した。
実施例1の工程を繰り返したが、本実施例においては、透析に代えて、過剰セレンの除去を以下の手順で行った。すなわち、本実施例においては、UF膜(マイクローザ AP-0013(UF)分画分子量6,000;旭化成(株)製)をペンシル型モジュール卓上濾過装置(マイクローザUF・M FPS-24001;旭化成(株)製)にセットした。そして、前記装置(操作圧力:モジュール出口圧で40kPa)を用い、塩化セレン(I)とアポラクトフェリンの混合溶液10mLを、室温(20℃以上)で3回限外濾過した。
原料として、ラクトフェリン(タツア・ジャパン(株)製)を使用したこと以外は、実施例3の工程を繰り返した。
セレン結合量の測定
(実験方法)
実施例1~4で製造したセレン-ラクトフェリンの凍結乾燥品1gをケルダールフラスコに入れ、硝酸(和光純薬(株)製)10mLを加え、室温に4時間放置した。次に、直火で15分間ゆるやかに加熱し、更に、やや強火で10分間加熱した。自然冷却後、70%過塩素酸(和光純薬(株)製)5mLを加え、加熱濃縮した。過塩素酸の白煙が発生してから更に15分間加熱を継続した。分解物はわずかに黄色になった。自然冷却後、水1mLを加え、加熱濃縮して白煙が認められてから更に2分間加熱した。自然冷却後、更に同じ操作を繰り返した。次に、10%塩酸(和光純薬(株)製)1mLを加え、沸騰水浴中で30分間加熱した。これを全体で5mLとなるように純水で希釈した。本希釈液を試験溶液とした。
検量線用溶液と試験溶液をそれぞれ2mL採って、これに0.1mol/LのEDTA(和光純薬(株)製)溶液4mL、20%塩酸ヒドロキシルアミン(和光純薬(株)製)溶液2mLを加え、10%塩酸(和光純薬(株)製)及び10%アンモニア水(和光純薬(株)製)を用いてpH1.0~1.5に調整した。これに0.1%2,3-ジアミノナフタレン(和光純薬(株)製)溶液5mLを加え混和した。蓋をして、暗所で50℃水浴中30分間加温した。自然冷却後、液全体を分液漏斗に移し、シクロヘキサン(和光純薬(株)製)10mLを加え、5分間振盪の後、シクロヘキサン層を分取した。このシクロヘキサン溶液を0.1mol/Lの塩酸(和光純薬(株)製)25mLで2回洗浄した。シクロヘキサンのみを取りだし、分光蛍光光度計(FP6000;日本分光(株)製)を用いて、励起波長378nm、蛍光波長520nmで蛍光強度を測定した。検量線の値から、試験溶液のセレン濃度を測定した。
結果を図1に示す。図1において、(a)は、透析膜を用いて過剰セレンを除去した結果を示し、(b)は、UF膜を用いた限外濾過により過剰セレンを除去した結果を示す。図中、縦軸はアポラクトフェリン100g中に結合したセレン量(mg)を示し、左側のカラムが原料としてアポラクトフェリンを使用した場合のセレン結合量を、右側のカラムが原料としてラクトフェリンを使用した場合のセレン結合量を示す。
原料としてアポラクトフェリンを使用した(a)、(b)の左側カラムにおいて、セレン結合量は、それぞれ、142mg/100g、114mg/100gであるのに対し、原料としてラクトフェリンを使用した(a)、(b)の右側カラムにおいて、セレン結合量は、64mg/100g、28mg/100gであった。この結果から、最終物であるセレン-ラクトフェリンの生産効率は、原料としてラクトフェリンを使用するよりも、アポラクトフェリンを使用した方が高いことが確認された。
角結膜上皮障害の治癒効力試験
藤原らの方法(Invest.
Ophthalmol. Vis. Sci. 42, 96-100, 2001)
を参考に、以下に示す方法でドライアイによる角結膜上皮障害モデルを作製し、実施例3で製造したセレン-ラクトフェリンを用いて、角結膜上皮障害に対する治癒効果を評価した。この実験は、各個体の両眼を用いて、片眼をコントロールとし、他方の眼に薬液を投与することにより、同一個体での比較ができるようにした。
雄性SDラット(7週齢)にペントバルビタールを腹腔内に投与(35mg/kg)して、全身麻酔後、両側の眼窩外涙腺を摘出しドライアイモデルを作製した。
涙腺摘出翌日より、いずれの群にも左眼にはPBSを、右眼にはPBS(Control群)、1%セレン-ラクトフェリン(PBS溶液)、又は5%セレン-ラクトフェリン(PBS溶液)を、それぞれ1回5μLで1日4回、2週間連日点眼した。また、正常群として、涙腺摘出を施さない動物を用意し、同一の条件にて両眼に共にPBSを点眼した(正常群)。なお、動物は各群とも9~10匹使用した。
3週間の点眼終了後、角膜上皮の障害部を蛍光色素フルオレセインにて染色した。
角膜上皮の障害の程度は、角膜全体を上中下及び左中右の合わせて9つの部分に分割して、各部分ごとに下記の基準で障害をスコア化し、その合計値を求めた。その後、各群において、左眼の合計スコア値と右眼の合計スコア値との比較をステューデントのt検定にて行った。なお、公平な評価を行うため、点眼開始から角膜上皮障害のスコア化まで、各群に投与した薬液の内容にブラインドをかけ、スコア化後に照合した。
0:染色されない(点状蛍光なし)
1:わずかに点状蛍光がみられる
2:比較的多く点状蛍光がみられる
3:密に点状蛍光がみられる
結果を図2に示す。図2において、(a)は1%セレン-ラクトフェリン(PBS溶液)投与群(以下S1群)、(b)は5%セレン-ラクトフェリン(PBS溶液)投与群(以下S5群)、(c)は正常群(以下N群)である。図中、縦軸は蛍光スコア値を示し、左側のカラムが左眼、即ちコントロールのPBS点眼側を、右側のカラムが右眼、すなわち、セレン-ラクトフェリン(図中、Se-ApoLと表記;(a)、(b))、又はPBS(c)を点眼した側を示す。また、各蛍光スコア値は、平均値で示してある。
(a)、(b)の左側カラムにおいて、蛍光スコアの平均値は6.1~6.3を示しており、涙腺摘出後、2週間のPBS点眼により、角膜上皮の障害が進行していることが明らかとなった。なお、涙腺摘出を施さない(c)のN群において、2週間のPBS点眼により、蛍光スコアの平均値は3.3(左眼)、及び3.3(右眼)となった。
一方、(a)、(b)の右側カラムに見られるように、蛍光スコアの平均値は、(a)のS1群では蛍光スコアの平均値は4.7、(b)のS5群では同じく3.7となり、左眼と比較して角膜上皮障害の進行を抑制することが確認された。
実験例1の薬理試験を繰り返したが、正常群の他に、薬剤投与群として、4群を設け、右眼に、それぞれ、0.01%、0.1%、1%、5%の濃度のセレン-ラクトフェリンを投与し、左眼にこれと同濃度のアポラクトフェリンを投与した。
結果を図3に示す。図3において、(a)は0.01%セレン-ラクトフェリン(PBS溶液)投与群(以下S0.01群)、(b)は0.1%セレン-ラクトフェリン(PBS溶液)投与群(以下S0.1群)、(c)は1%セレン-ラクトフェリン(PBS溶液)投与群(以下S1群)、(d)は5%セレン-ラクトフェリン(PBS溶液)投与群(以下S5群)、(e)は正常群(以下N群)である。図中、縦軸は蛍光スコア値を示し、左側のカラムが左眼、すなわち、アポラクトフェリン(図中、ApoLと表記;(a)~(d))又はPBS(e)を点眼した側を、右側のカラムが右眼、すなわち、セレン-ラクトフェリン(図中、Se-ApoLと表記;(a)~(d))、又はPBS(e)を点眼した側を示す。また、各蛍光スコア値は、平均値で示してある。
(a)の左側カラムにおいて、蛍光スコアの平均値は4.6を示しており、角膜上皮の障害が進行していることが明らかとなった。一方、(a)の右側カラムに見られるように、蛍光スコアの平均値は、3.2となり、左眼と比較して角膜上皮障害の進行を抑制することが確認された。また、同様の傾向が(b)~(d)にも認められた。
Smoker Ratに対する治癒効力試験
樋口らの方法(Higuchi
A, Ito K, Dogru M, Kitamura M, Mitani F,
Kawakita T, Ogawa Y, Tsubota K., Corneal damage and
lacrimal glands dysfunction in a smoking rat model.
Free Radic Biol Med., 2011;51:2210-16.)を参考に、以下に示す方法でドライアイによる角結膜上皮障害モデルを作製し、実施例3で製造したセレン-ラクトフェリンを用いて、角結膜上皮障害に対する治癒効果を評価した。この実験は、各個体の両眼を用いて、片眼をコントロールとし、他方の眼に薬液を投与することにより、同一個体での比較ができるようにした。
雄性SDラット(7週齢)に喫煙処理を施し、ドライアイモデルを作製した。
即ち、ラットを入れたチャンバー中に主流煙(300mL)を30分毎に6回添加し、5日間処理することにより角結膜上皮障害を惹起した。点眼条件として、片眼にPBSを、他眼に0.1%セレン-ラクトフェリン(PBS溶液)を、それぞれ1回5μLで喫煙処理前に1回、処理後に3回の都合1日4回、5日間連日点眼した。なお、動物は9匹使用した。
5日間の喫煙処理並びに点眼終了後、実施例1と同様に角膜上皮の障害部を蛍光色素フルオレセインにて染色し、スコア化して評価した。PBS点眼側の合計スコア値と0.1%セレン-ラクトフェリンの合計スコア値との比較をステューデントのt検定にて行った。
結果を図4に示す。図中、縦軸は蛍光スコア値の平均値を示す。左側のカラムがコントロールのPBS点眼側を、右側のカラムが0.1%セレン-ラクトフェリン点眼側を示す。
図4に示すとおり、5日間の喫煙処理により惹起された角結膜上皮障害は、0.1%セレン-ラクトフェリンの点眼により(蛍光スコア平均値:2.6)、コントロール(PBS点眼側、蛍光スコア平均値:5.6)と比較して有意に(p<0.001)抑制されることが明らかとなった。
Dry Room Mouseに対する治癒効力試験
以下に示す方法でドライアイによる角結膜上皮障害モデルを作製し、実施例3で製造したセレン-ラクトフェリンを用いて、角結膜上皮障害に対する治癒効果を評価した。この実験は、各個体の両眼を用いて、片眼をコントロールとし、他方の眼に薬液を投与することにより、同一個体での比較ができるようにした。
雄性ICRマウス(6週齢)をドライルーム処置することにより、ドライアイモデルを作製した。
即ち、マウスをドライルーム(湿度20±5%、温度23±4℃)にて2週間処置することにより、角結膜上皮障害を惹起した。点眼条件として、片眼にPBSを、他眼に0.1%セレン-ラクトフェリン(PBS溶液)を、それぞれ1回5μLで1日4回、2週間連日点眼した。また、比較のため、正常環境(湿度50±5%、温度23±4℃)の動物も用意した(点眼は行わない)。なお、動物はドライルーム、正常環境共に8匹使用した。
2週間のドライルーム処置並びに点眼終了後、実施例1と同様に角膜上皮の障害部を蛍光色素フルオレセインにて染色し、スコア化して評価した。PBS点眼側の合計スコア値と0.1%セレン-ラクトフェリンの合計スコア値との比較をステューデントのt検定にて行った。
結果を図5に示す。図中、縦軸は蛍光スコア値の平均値を示す。図5(a)の左側カラムは正常環境のスコアを示し、右側カラムはドライルーム処置のスコア(PBS点眼側)を示す。図5(b)の左側カラムはコントロールのPBS点眼側を、右側カラムは0.1%セレン-ラクトフェリン点眼側を示す。
図5(a)に示すとおり、ドライルーム処置により角結膜上皮障害が有意に(p<0.05)惹起された。このドライルーム惹起角結膜上皮障害は、図5(b)に示すように、0.1%セレン-ラクトフェリンの点眼により(蛍光スコア平均値:2.1)、コントロール(PBS点眼側、蛍光スコア平均値:3.4)と比較して有意に(p<0.05)抑制されることが明らかとなった。
すなわち、本発明は、高い治療効果と安全性とを兼ね備え、かつ、工業的大規模生産に適した角結膜疾患治療剤を提供するものであり、産業上有用なものである。
Claims (11)
- セレン-ラクトフェリンである金属タンパク質。
- セレンが、アポラクトフェリン中に存在する鉄結合性ポケットに結合している、請求項1記載の金属タンパク質。
- 請求項1に記載の金属タンパク質の製造方法であって、ラクトフェリン及び/又はアポラクトフェリンを含有する溶液に、セレン塩を添加する工程、及び得られた混合溶液を透析又は限外濾過に付する工程、を含む、前記製造方法。
- アポラクトフェリンを含有する溶液を用いる、請求項3記載の方法。
- 請求項4記載の金属タンパク質の製造方法であって、アポラクトフェリンをラクトフェリンと酸とを含有する溶液を限外濾過膜に供給し、限外濾過を行う工程、及び前記限外濾過膜を透過しなかった溶液を回収する工程を含む方法により製造する、前記製造方法。
- 請求項3に記載の方法により製造される、金属タンパク質。
- 請求項1に記載の金属タンパク質を有効成分とする角結膜疾患の予防又は治療剤。
- 角結膜疾患が、ドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍である、請求項7記載の予防又は治療剤。
- 点眼剤又は眼軟膏剤である、請求項7に記載の予防又は治療剤。
- 請求項1に記載の金属タンパク質を有効成分とするドライアイ、乾性角結膜炎、点状表層角膜症、角膜びらん、又は角膜潰瘍の予防又は治療剤。
- 請求項1に記載の金属タンパク質、及び眼科用製剤に許容される眼科用担体を含有してなる眼科用組成物。
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CN201280024177.4A CN103998464A (zh) | 2011-05-20 | 2012-05-18 | 新型金属蛋白质及其制造方法以及含有所述金属蛋白质的角结膜疾病的预防或治疗剂 |
EP12789433.5A EP2711374A4 (en) | 2011-05-20 | 2012-05-18 | NOVEL METALLOPROTEIN AND PROCESS FOR PRODUCTION THEREOF, AND PROPHYLACTIC OR THERAPEUTIC AGENT FOR CORNEAL AND CONJUNCTIVE DISEASES COMPRISING SAID METALLOPROTEIN |
MX2013013480A MX2013013480A (es) | 2011-05-20 | 2012-05-18 | Metaloproteina innovadora y proceso para producir la misma y agente profilactico o terapeutico para enfermedades corneales y conjuntivales que comprende dicha metaloproteina. |
CA2835816A CA2835816A1 (en) | 2011-05-20 | 2012-05-18 | Novel metalloprotein and process for producing same, and prophylactic or therapeutic agent for corneal and conjunctival diseases comprising said metalloprotein |
AU2012259925A AU2012259925A1 (en) | 2011-05-20 | 2012-05-18 | Novel metalloprotein and process for producing same, and prophylactic or therapeutic agent for corneal and conjunctival diseases comprising said metalloprotein |
BR112013029592A BR112013029592A2 (pt) | 2011-05-20 | 2012-05-18 | "metaloproteína e processo para produção desta, e a gente profilático ou terapêutico para doenças corneanas e conjuntivais compreendendo a dita metaloproteína" |
US14/118,879 US20140080765A1 (en) | 2011-05-20 | 2012-05-18 | Novel metalloprotein and process for producing same, and prophylactic or therapeutic agent for corneal and conjunctival diseases comprising said metalloprotein |
EA201391595A EA201391595A1 (ru) | 2011-05-20 | 2012-05-18 | Новый металлопротеин, способ его получения и профилактический или терапевтический агент при заболеваниях роговицы или конъюнктивы, содержащий указанный металлопротеин |
KR1020137032428A KR20140039217A (ko) | 2011-05-20 | 2012-05-18 | 신규 금속 단백질 및 그 제조 방법, 그리고 상기 금속 단백질을 함유하는 각결막 질환의 예방 또는 치료제 |
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US9463201B2 (en) | 2014-10-19 | 2016-10-11 | M.G. Therapeutics Ltd | Compositions and methods for the treatment of meibomian gland dysfunction |
US10688122B2 (en) | 2015-09-28 | 2020-06-23 | Azura Ophthalmics Ltd. | Thiol and disulfide-containing agents for increasing meibomian gland lipid secretion |
US11040062B2 (en) | 2016-04-14 | 2021-06-22 | Azura Ophthalmics Ltd. | Selenium disulfide compositions for use in treating meibomian gland dysfunction |
US11517586B2 (en) | 2020-01-10 | 2022-12-06 | Azura Ophthalmics Ltd. | Instructions for composition and sensitivity |
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US9463201B2 (en) | 2014-10-19 | 2016-10-11 | M.G. Therapeutics Ltd | Compositions and methods for the treatment of meibomian gland dysfunction |
US10034887B2 (en) | 2014-10-19 | 2018-07-31 | M.G. Therapeutics, Ltd. | Compositions and methods for the treatment of meibomian gland dysfunction |
US10588915B2 (en) | 2014-10-19 | 2020-03-17 | Azura Ophthalmics Ltd. | Compositions and methods for the treatment of meibomian gland dysfunction |
US10772899B2 (en) | 2014-10-19 | 2020-09-15 | Azura Ophthalmics Ltd. | Compositions and methods for the treatment of meibomian gland dysfunction |
US11013749B2 (en) | 2014-10-19 | 2021-05-25 | Azura Ophthalmics Ltd. | Compositions and methods for the treatment of meibomian gland dysfunction |
US11633410B2 (en) | 2014-10-19 | 2023-04-25 | Azura Ophthalmics Ltd | Compositions and methods for the treatment of meibomian gland dysfunction |
US12048707B2 (en) | 2014-10-19 | 2024-07-30 | Azura Ophthalmics Ltd. | Compositions and methods for the treatment of meibomian gland dysfunction |
US10688122B2 (en) | 2015-09-28 | 2020-06-23 | Azura Ophthalmics Ltd. | Thiol and disulfide-containing agents for increasing meibomian gland lipid secretion |
US11040062B2 (en) | 2016-04-14 | 2021-06-22 | Azura Ophthalmics Ltd. | Selenium disulfide compositions for use in treating meibomian gland dysfunction |
US12011457B2 (en) | 2016-04-14 | 2024-06-18 | Azura Ophthalmics Ltd | Selenium disulfide compositions for use in treating meibomian gland dysfunction |
US11517586B2 (en) | 2020-01-10 | 2022-12-06 | Azura Ophthalmics Ltd. | Instructions for composition and sensitivity |
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EA201391595A1 (ru) | 2014-06-30 |
KR20140039217A (ko) | 2014-04-01 |
EP2711374A1 (en) | 2014-03-26 |
MX2013013480A (es) | 2014-02-06 |
US20140080765A1 (en) | 2014-03-20 |
CA2835816A1 (en) | 2012-11-29 |
BR112013029592A2 (pt) | 2019-09-24 |
AU2012259925A1 (en) | 2013-11-28 |
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EP2711374A4 (en) | 2014-05-21 |
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