JP2015140327A - Phacosclerosis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop an effective therapeutic and preventive agent for presbyopia and the like caused by phacosclerosis.SOLUTION: The above subject is solved by providing a therapeutic and/or preventive agent for diseases involved in phacosclerosis which contains selenium-lactoferrin or a salt thereof as an active ingredient, as well as a nonhuman model animal preparation method useful for developing the therapeutic and preventive agent for the diseases, and a method using the animal for screening the therapeutic and preventive agent.

Description

  The present invention includes a lens hardening inhibitor, a therapeutic and / or preventive agent for a disease involving lens hardening, a method for producing a model animal of the disease, and a selenium compound such as selenium lactoferrin or a salt thereof as an active ingredient, The present invention relates to a screening method for a lens sclerosis inhibitor using an animal.

  The lens is one of the tissues with the highest protein ratio in humans, comprising 33% protein, 66% water, and 1% mineral. As main proteins, α-, β-, and γ-crystallins account for 1/3 to 1/2 of the total protein in the lens by weight. Although the lens adjusts its focus by adjusting its thickness, it is believed that the lens hardens with age and fails to adjust, resulting in presbyopia. To date, effective therapeutic and preventive drugs for presbyopia have not been marketed.

  Lactoferrin is an iron ion-binding glycoprotein having a molecular weight of about 80,000, which is contained in breast milk, blood, mucosal secretion (tears, saliva, semen), mucosal surfaces, special granules of neutrophils, etc. Since it exhibits the action of endotoxin and the like, it is used as an antiseptic in eye drops and contact lens preservation solutions (Patent Document 1). In addition, it has been reported that lactoferrin is effective for the treatment of dry eye (Patent Document 2). Furthermore, it has been reported that apolactoferrin obtained by removing iron from lactoferrin also has an antibacterial action and improves the wettability of the eyeball and contact lens surface (Patent Document 3).

Selenium lactoferrin is a combination of selenium and apolactoferrin, and the present inventors disclosed a method for producing selenium lactoferrin and that selenium lactoferrin has a therapeutic effect on keratoconjunctival diseases such as dry eye. (Patent Documents 4 and 5).
However, the effect of selenium lactoferrin on the lens is not known.

By the way, smoking and passive smoking cause various diseases such as cancer and respiratory diseases, and are known to be risk factors such as cataract and hyperopia in the ophthalmic field. The present inventors have found that corneal damage accompanied by oxidative DNA damage and lacrimal gland dysfunction occur in rats exposed to mainstream smoke (Non-patent Document 1), and using this model animal, selenium lactoferrin is dried. It was proved that it is effective in the treatment of keratoconjunctival epithelial diseases such as eye (Patent Documents 4 and 5).
However, nothing is known about whether smoking treatment can cause lens hardening in experimental animals.

JP-A-9-30966 Japanese Patent No. 4634809 JP 2007-137817 A International Publication No. 2012/161112 JP 2013-006829 A

Free Radic. Biol. Med., 2011, Vol. 51, pages 2210-2216

  As the population ages, the demand for therapeutic and preventive drugs for various disorders associated with aging is expected to increase in the future, but so far it has been effective for presbyopia caused by lens hardening. There is no effective therapeutic or preventive drug, and its development is required.

  The present inventors have intensively studied to solve the above-mentioned problems, and succeeded in causing hardening of the lens by giving a non-human animal a smoking treatment under certain conditions. Furthermore, using the model animal, it was found that selenium lactoferrin suppresses lens hardening, and the present invention was completed.

That is, the present invention relates to the following.
[1] A lens hardening inhibitor comprising selenium lactoferrin or a salt thereof as an active ingredient.
[2] The agent according to [1], which is a therapeutic and / or prophylactic agent for a disease involving lens hardening.
[3] The agent according to [2], wherein the disease involving lens hardening is presbyopia.
[4] The agent according to any one of [1] to [3], which is an eye drop or an eye ointment.
[5] A non-human animal model of a disease involving lens hardening, wherein the non-human animal is exposed to an atmosphere containing 50 to 250 ppm of tobacco smoke at a carbon monoxide concentration of 30 hours or more in total. Manufacturing method.
[6] The method according to [5], wherein the disease involving lens hardening is presbyopia.
[7] (a) A step of administering a test substance to the non-human model animal produced by the method according to [5], and (b) evaluating a change in a disease state of a disease associated with lens hardening in the animal. Process,
A method for screening candidate substances for therapeutic and / or prophylactic agents for the disease.

  According to the present invention, it is possible to treat and / or prevent a disease associated with lens hardening such as presbyopia, for which no effective therapeutic and / or preventive drug has existed. In addition, since a model animal that causes lens hardening by a simple smoking treatment is provided, it is useful for the development of a therapeutic and / or prophylactic agent for a disease involving lens hardening.

FIG. 1 is a diagram showing changes in lens hardness due to mainstream smoke exposure. FIG. 2 is a diagram showing that lens hardening due to mainstream smoke exposure is suppressed by selenium lactoferrin.

  The present invention will be described below. The terms used in this specification have meanings commonly used in the art unless otherwise specified.

  The present invention provides a lens hardening inhibitor containing selenium lactoferrin or a salt thereof as an active ingredient.

The selenium lactoferrin used in the present invention (hereinafter also simply referred to as “the selenium lactoferrin of the present invention”) is a metal protein composed of selenium and apolactoferrin, wherein selenium is bound to apolactoferrin. Apolactoferrin means a product obtained by removing iron from lactoferrin.
In the selenium lactoferrin of the present invention, selenium is considered to be bound by chelating action in the iron binding pocket of apolactoferrin, but is not limited thereto. In the present specification, the iron-binding pocket refers to a portion that binds to an iron ion existing in the structure of apolactoferrin, and apolactoferrin has two iron-binding pockets per molecule in the structure. ing. The portion forms a globular domain, the N-terminal domain is called the N lobe and the C-terminal domain is called the C lobe. The selenium lactoferrin of the present invention may be one in which selenium is bound in two iron-binding pockets, one in which selenium is bound in one iron-binding pocket, or a mixture thereof.

  The lactoferrin and apolactoferrin used in the present invention (hereinafter sometimes abbreviated as “the lactoferrin of the present invention”) are animal milk, whey, blood, mucosal secretion (tears, saliva, semen), mucosal surface. Even those derived from natural lactoferrin obtained from special granules of neutrophils, etc. were synthesized and purified by genetic engineering using techniques known to those skilled in the art using E. coli, yeast, insect cells, animal cells, etc. It may be a thing. In the case of preparing lactoferrin, which is a naturally occurring glycoprotein, as a glycoprotein, it is preferable to use yeast, insect cells, or animal cells that undergo post-translational modification as a host in the genetic engineering method. It is more preferable to use animal cells, particularly human-derived cells, that can be expected to undergo sugar chain modification similar to the modification mode. In addition, in order to perform mass production at a low cost, it is also preferable to carry out genetic engineering production using E. coli. The selection of the host cell is not particularly limited as long as the desired effect of the present invention can be obtained. Or since lactoferrin and apolactoferrin are marketed as a pharmaceutical, a supplement, a reagent, etc., these commercial items can also be used conveniently for this invention.

  For example, the lactoferrin of the present invention is prepared from natural materials such as milk of the animal by a method of adsorbing to a cation exchange resin and desorbing with a high concentration salt solution, a separation method by electrophoresis, a separation method by affinity chromatography, etc. can do. In the case of synthesis by genetic engineering, a tag sequence may be added to the lactoferrin polypeptide in order to facilitate purification after synthesis. As tags, Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (for example, GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. The position where the tag sequence is added is preferably the N-terminus or C-terminus of the lactoferrin polypeptide.

  Alternatively, the lactoferrin of the present invention has a binding property to selenium and has a natural amino acid sequence of 1 or 2 as long as it has a therapeutic and / or preventive effect on a disease involving lens hardening. The above amino acid substitutions, deletions, insertions, additions, or combinations thereof (for example, about 1 to 50, preferably about 1 to 10, more preferably about 1 to 5) may be included.

  The lactoferrin of the present invention is preferably derived from a mammal (eg, human, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.).

  The method for obtaining the apolactoferrin used in the present invention is not particularly limited as long as a desired effect is obtained, but a method known to those skilled in the art (for example, a lactoferrin-containing solution obtained by the above method) A method in which an acid is added to make the solution acidic (for example, adjusted to pH 1 to 3), a method in which a lactoferrin-containing solution is dialyzed against an acid solution (for example, an acid solution having a pH of 0.5 to 2), and It can be obtained by dissociating iron from lactoferrin by a method of contacting lactoferrin with a chelating agent such as EDTA). Examples of the acid include 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.

Alternatively, apolactoferrin used in the present invention is
(A) a step of subjecting a solution containing lactoferrin and an acid to ultrafiltration, and (b) a step of recovering the solution that did not permeate the ultrafiltration membrane in (a),
Can be produced by a method comprising: The method can be, for example, the method described in Japanese Patent No. 4634809. Specifically, a solution containing lactoferrin and an acid are mixed, the mixture is applied to an ultrafiltration membrane, the non-permeate and the permeate are removed from the system, and the acid is again added to the non-permeate. It is possible to manufacture by repeating the operation until the desired amount of apolactoferrin is obtained by adding ultrafiltration. As 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 can be preferably used. In ultrafiltration, any membrane (for example, cellulose acetate, polysulfone, polyether, etc.) may be used as long as it uses a membrane that does not permeate lactoferrin and apolactoferrin and permeates iron dissociated from lactoferrin. Organic films of natural or synthetic polymers such as sulfone, polyacrylamide, polyimide, aromatic polyamide, polyacrylonitrile, hydrophilic polyolefin, ceramics such as alumina, zirconia and titanium, inorganic films such as carbon and glass) can also be used. The type of membrane (for example, hollow fiber membrane, tubular membrane, spiral membrane, flat membrane, etc.) is not particularly limited. In addition, for example, an ultrafiltration method using a centrifugal operation or a cross flow can be used, but the method is not limited thereto as long as a desired effect can be obtained. After performing the ultrafiltration, apolactoferrin can be obtained by collecting the solution that did not permeate the membrane.

  Dialysis can be used in place of the ultrafiltration in the method for producing apolactoferrin described above. When dialysis is performed, it is desirable to use a dialysis membrane having a pore size that does not permeate lactoferrin and apolactoferrin having a molecular weight of about 80,000 and permeates iron. Various materials (for example, regenerated cellulose, cellulose acetate, polyacrylonitrile, etc.) and pore size dialysis membranes are commercially available, and any dialysis membrane can be used in the present invention as long as the above effects are exhibited. Dialysis is preferably performed for 2 to 24 hours with respect to a solution that does not contain iron at least 100 times as much as the solution containing lactoferrin, and the dialysis operation is performed once or more (for example, 1 to 5 times). preferable.

The selenium lactoferrin of the present invention or a salt thereof is, for example,
(A) a step of adding selenium salt to a solution containing lactoferrin and / or apolactoferrin, and (b) a step of subjecting the solution obtained in the step (a) to dialysis or ultrafiltration,
Can be produced by a method comprising: In this method, selenium is bound to apolactoferrin by adding a selenium salt to a solution containing lactoferrin and / or apolactoferrin, and then the solution is subjected to dialysis or ultrafiltration to remove excess selenium. To produce selenium lactoferrin.
In the production method, lactoferrin and / or apolactoferrin can be used. When lactoferrin is used, selenium lactoferrin is produced by replacing the iron bound to the iron binding pocket with selenium in the solution, and when apolactoferrin is used, selenium binds to the empty iron binding pocket. Selenium lactoferrin is thought to be produced. Therefore, from the viewpoint of production efficiency of selenium lactoferrin, it is preferable to use apolactoferrin, but a lactoferrin solution or a mixed solution of lactoferrin and apolactoferrin can also be used.
The selenium salt used in the method is not particularly limited as long as it is water-soluble and can provide a desired effect. For example, selenium fluoride, selenium chloride, selenium bromide, and the like can be used. As described above, since apolactoferrin has two iron-binding pockets in one molecule, the number of moles of selenium salt added by the method is at least twice the number of moles of lactoferrin and / or apolactoferrin. Preferably there is. In addition, since lactoferrin and apolactoferrin are denatured in a strongly basic solution having a high pH and do not bind to selenium in a strongly acidic solution having a low pH, the pH of the solution used in the method is, for example, pH 3 to 3. 8 is preferable. The pH of the solution can be adjusted by methods known to those skilled in the art. The reaction temperature and reaction time of lactoferrin and / or apolactoferrin and selenium salt in the method need to be appropriately set according to the amount of lactoferrin and / or apolactoferrin and selenium salt used, but usually 10 It is carried out at ˜35 ° C. for 10 minutes to 2 hours.

  Since excessive intake of selenium is toxic to the living body, in the above method, it is necessary to remove selenium that has not bound to lactoferrin or apolactoferrin. As a method for removing excess selenium, dialysis or ultrafiltration can be used. When dialysis is performed, it is desirable to use a dialysis membrane having a pore size that does not permeate lactoferrin and apolactoferrin having a molecular weight of about 80,000 and permeates selenium. Various materials (for example, regenerated cellulose, cellulose acetate, polyacrylonitrile, etc.) and pore size dialysis membranes are commercially available, and any dialysis membrane can be used in the present invention as long as the above effects are exhibited. Dialysis is preferably performed for 2 to 24 hours against a solution that does not contain selenium at least 100 times the amount of lactoferrin and / or apolactoferrin and a selenium salt, and the dialysis operation is performed once or more (for example, 1 to 5 times). As for ultrafiltration, any material (for example, cellulose acetate, polysulfone, polyethersulfone, polysulfone, etc.) may be used as long as it uses a membrane that does not permeate lactoferrin and apolactoferrin and has a property of permeating selenium. Organic membranes of natural or synthetic polymers such as acrylamide, polyimide, aromatic polyamide, polyacrylonitrile, hydrophilic polyolefin, ceramics such as alumina, zirconia and titanium, inorganic membranes such as carbon and glass) can also be used. (For example, hollow fiber membranes, tubular membranes, spiral membranes, flat membranes, etc.) are not particularly limited. For example, an ultrafiltration method using a centrifugal operation or a cross flow may be used, but the ultrafiltration may be performed by any method as long as a desired effect can be obtained.

  The selenium lactoferrin of the present invention may be produced by any method other than the above method as long as the effects of the present invention are not impaired.

  The selenium lactoferrin of the present invention may form a salt with an inorganic base, organic base, inorganic acid, organic acid or the like. Examples of the salt with the inorganic base include, for example, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; and aluminum salt and ammonium salt. Examples of the salt with the organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N, N′-dibenzylethylenediamine. Examples of the salt with the inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of the salt with the organic acid include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p- And a salt with toluenesulfonic acid. Of these salts, pharmacologically acceptable salts are preferred.

  The selenium lactoferrin of the present invention has a therapeutic and / or preventive effect on diseases involving lens hardening by suppressing lens hardening. In the present specification, the suppression of the hardening of the crystalline lens includes not only suppressing the hardening of the crystalline lens and maintaining the hardness of the crystalline lens but also recovering the hardening of the crystalline lens and imparting elasticity. In the present specification, a disease involving lens hardening means any disease in which hardening of the lens contributes to its onset / progression or causes hardening of the lens accompanying the onset / progress. Examples of the disease include presbyopia, hyperopia and cataract, and a preferred example is presbyopia. The lens adjusts the focus of the eye by changing its thickness. Therefore, a disease state in which lens hardening is involved is that the lens is hardened and loses elasticity, making it difficult to adjust the focus of the eye.

Since selenium is one of trace essential elements and lactoferrin is a body component and is also known as a supplement, the selenium lactoferrin of the present invention has low toxicity and is pharmaceutically acceptable as it is or according to a method known per se. As a pharmaceutical composition mixed with additives, it can be safely administered orally or parenterally to humans and other mammals (eg, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) Can be administered.
The amount of selenium lactoferrin or a salt thereof contained in the lens hardening inhibitor or the therapeutic and / or preventive agent for diseases associated with lens hardening of the present invention (hereinafter collectively referred to as “the agent of the present invention”) There is no particular limitation as long as it is sufficient to suppress curing and does not exhibit cytotoxicity, but it is usually 0.001 to 100 w / v%, preferably 0.01 to 1 w / v%. The content of selenium lactoferrin can be appropriately increased or decreased depending on the purpose of use, dosage form, degree of disease state, and the like.

  The dosage form of the agent of the present invention is not particularly limited, and various dosage forms suitable for oral administration or parenteral administration can be appropriately selected. As preparations for parenteral administration, for example, eye drops, ointments, lotions, creams, injections, suppositories, etc. are used, preferably dosage forms suitable for topical administration, such as eye drops Agents (aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, etc.), ointments, lotions, creams and the like. When the agent of the present invention is an eye drop, a substrate can be appropriately used. Examples of the base material used for the eye drops include phosphate buffer, Hanks buffer, physiological saline, perfusate, artificial tears and the like.

In the agent of the present invention, in addition to the active ingredient selenium lactoferrin, in the case of a pharmaceutically acceptable additive, for example, a preparation for topical ocular administration, for example, a buffer, an isotonic agent, a solubilizing agent, a preservative, Viscosity bases, chelating agents, cooling agents, pH adjusters, antioxidants and the like can be appropriately selected and added.
Examples of the buffer include a phosphate buffer, a borate buffer, a citrate buffer, a tartrate buffer, an acetate buffer, and an amino acid.
Examples of the isotonic agent include saccharides such as sorbitol, glucose and mannitol, polyhydric alcohols such as glycerin and propylene glycol, salts such as sodium chloride, boric acid and the like.
Examples of solubilizers include polyoxyethylene sorbitan monooleate (for example, polysorbate 80), polyoxyethylene hydrogenated castor oil, nonionic surfactants such as tyloxapol and pluronic, and polyhydric alcohols such as glycerin and macrogol. It is done.
Examples of the preservative include quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, paraoxybenzoic acid such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate. Examples include esters, benzyl alcohol, sorbic acid and salts thereof (sodium salt, potassium salt, etc.), thimerosal (trade name), chlorobutanol, sodium dehydroacetate and the like.
Examples of the viscous base include water-soluble polymers such as polyvinyl pyrrolidone, polyethylene glycol, and polyvinyl alcohol, and celluloses such as hydroxyethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl cellulose.
Examples of chelating agents include sodium edetate and citric acid.
Examples of the refreshing agent include l-menthol, borneol, camphor, and eucalyptus oil.
Examples of the pH adjuster include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, boric acid or a salt thereof (borax), hydrochloric acid, citric acid or a salt thereof (sodium citrate, sodium dihydrogen citrate). Etc.), phosphoric acid or a salt thereof (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or a salt thereof (sodium acetate, ammonium acetate, etc.), tartaric acid or a salt thereof (sodium tartrate, etc.), and the like.
Examples of the antioxidant include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherol.

  The pH of the agent of the present invention is usually adjusted to about 5.0 to about 8.5, preferably about 6.0 to about 8.0, preferably sterilization treatment such as filtration sterilization using a membrane filter or the like. I do.

  When the agent of the present invention is prepared as an eye ointment, it may further contain an ointment base. Although it does not specifically limit as this ointment base, Generally fats and oils, wax, a hydrocarbon compound, etc. as a hydrophobic base can be used. Specific examples include mineral bases such as yellow petrolatum, white petrolatum, paraffin, liquid paraffin, plastibase, and silicone, and animal and plant bases such as beeswax and animal and vegetable fats and oils.

  On the other hand, when the agent of the present invention is a preparation for oral administration, for example, tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, It can be formulated into an emulsion, suspension or the like. These preparations can be manufactured by a method known per se, for example, the method described in the 14th revised Japanese Pharmacopoeia, General Rules for Preparations, and the above-mentioned pharmaceutically acceptable additives that can be used for preparations for topical ophthalmic administration. In addition to products, excipients, lubricants, binders, disintegrants, water-soluble polymers, basic inorganic salts and the like that are usually used in the pharmaceutical field may be contained.

Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, titanium oxide and the like.
Examples of the lubricant include magnesium stearate, sucrose fatty acid ester, polyethylene glycol, talc, stearic acid and the like.
Examples of the binder include hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, starch, polyvinylpyrrolidone, gum arabic powder, gelatin, pullulan, and low-substituted hydroxypropylcellulose.
Disintegrants include (1) crospovidone, (2) disintegrants called super disintegrants such as croscarmellose sodium (FMC-Asahi Kasei), carmellose calcium (Gotoku Pharmaceutical), (3) sodium carboxymethyl starch ( Examples include Matsutani Chemical Co., Ltd.), (4) Low-substituted hydroxypropyl cellulose (eg, Shin-Etsu Chemical Co., Ltd.), and (5) Corn starch. The “crospovidone” is crosslinked with a chemical name of 1-ethenyl-2-pyrrolidinone homopolymer, including polyvinyl polypyrrolidone (PVPP) and 1-vinyl-2-pyrrolidinone homopolymer. Specific examples include Kollidon CL (manufactured by BASF), Polyplaston XL (manufactured by ISP), Polyplaston XL-10 (manufactured by ISP), and Polyplastidone. INF-10 (manufactured by ISP) or the like.
Examples of the water-soluble polymer include ethanol-soluble water-soluble polymers [eg, cellulose derivatives such as hydroxypropyl cellulose (hereinafter sometimes referred to as HPC), polyvinylpyrrolidone, etc.], ethanol-insoluble water-soluble polymers [for example, , Hydroxypropylmethylcellulose (hereinafter sometimes referred to as HPMC), cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum and the like].
Examples of the basic inorganic salt include basic inorganic salts of sodium, potassium, magnesium and / or calcium. Preferred is a basic inorganic salt of magnesium and / or calcium. More preferred is a basic inorganic salt of magnesium. Examples of the basic inorganic salt of sodium include sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate and the like. Examples of the basic inorganic salt of potassium include potassium carbonate and potassium hydrogen carbonate. Examples of the basic inorganic salt of magnesium include heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg ₆ Al ₂ ( OH) ₁₆ · CO ₃ · 4H ₂ O] and alumina / magnesium hydroxide, preferably heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide and the like. Examples of the basic inorganic salt of calcium include precipitated calcium carbonate and calcium hydroxide.

  If necessary, in order to facilitate transport of the pharmaceutical composition into cells, it can also be encapsulated in liposomes. Preferred liposomes are positively charged liposomes, positively charged cholesterol, membrane-permeable peptide-bound liposomes and the like (Nakanishi Mamoru et al., Protein Nucleic Acid Enzyme, 44: 1590-1596 (1999), Shiro Futaki, Chemistry and Biology, 43: 649- 653 (2005), Clinical Cancer Research 59: 4325-4333 (1999)).

  As long as the agent of the present invention does not cause an unfavorable interaction by combination with selenium lactoferrin, other active ingredients such as antiallergic or antihistamine components, decongestant components, local anesthetic components, vitamin components, and active amino acids Of amino acids (eg, valine, leucine, isoleucine, serine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, histidine, citrulline, ornithine, cystine, taurine, glycine) It may be. As such other active ingredients, various drugs known per se can be appropriately used.

  The dosage of the agent of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but when used in the form of eye drops or eye ointment as a therapeutic agent for presbyopia, for example, as the amount of selenium lactoferrin 2 to 200 μg / day can be administered once a day or divided into 2 to 4 times. It should be noted that the amount applied and the number of times can be appropriately increased or decreased depending on the degree of symptoms. When used as an eye drop, it can be used even when a contact lens such as a non-oxygen permeable hard contact lens, an oxygen permeable hard contact lens, or a soft contact lens is worn. In the case of other parenteral administration and oral administration, an equivalent amount can be administered.

  In one aspect, the present invention relates to a non-human disease associated with lens hardening comprising exposing a non-human animal to an atmosphere containing 50 to 250 ppm of tobacco smoke in a carbon monoxide concentration for a total of 30 hours or more. A method for producing a model animal is provided.

  The non-human animal used in the method for producing a non-human model animal of a disease involving lens sclerosis according to the present invention (hereinafter sometimes abbreviated as “model animal of the present invention”) is generally an experimental animal. If it is used, it will not specifically limit, For example, a vertebrate is mentioned, Preferably it is a warm-blooded animal (For example, a mouse, a rat, a guinea pig, a hamster, a rabbit, a ferret, a pig, a dog, a cat, a monkey, a chicken etc.) More preferably, it is a mammal, further preferably a rodent, particularly preferably a mouse or a rat.

  As the non-human animal used in the present invention, it is desirable to use a genetically and microbiologically controlled animal suitable for conducting an evaluation experiment using the obtained model animal of the present invention. That is, genetically, it is preferable to use an inbred line. For example, in the case of a mouse, strains such as C57BL / 6J, BALB / c, CBA, DBA / 2, and C3H / He are exemplified, and preferably C57BL / 6J, but not limited thereto. In the case of rats, examples include Wistar and Sprague-Dawley. From the microbiological viewpoint, it is preferable to use SPF or gnotobiate grades.

  The non-human animal used in the present invention may be an animal of any age. For example, in the case of a mouse, it is less than 1 year, preferably 4-10 weeks of age, and in the case of a rat, less than 1.5 years. Preferably, the rat is 1 to 3 months old. According to the model animal production method of the present invention, it is possible to easily cause lens hardening in a short period of time using a non-aged non-human animal. Therefore, in order to obtain a lens-cured animal, the non-human animal is bred for a long period of time. There is no need to do so, and a great effect can be obtained in terms of time and cost reduction.

In the method for producing a model animal of the present invention, lens hardening is induced by smoking treatment (exposure to tobacco smoke) on the non-human animal. The type of tobacco used in the smoking treatment is not particularly limited as long as it can induce lens hardening. In the smoking treatment, either mainstream smoke or sidestream smoke of tobacco can be used, but from the viewpoint that smoke can be easily collected, it is preferable to use mainstream smoke. In the method, the concentration of tobacco smoke provided to non-human animals in the air is preferably 50 to 250 ppm, for example, when expressed as a carbon monoxide concentration. For example, air containing cigarette smoke in which carbon monoxide has the above-mentioned concentration is obtained by using cigarette mainstream smoke (50 mL per suction) containing 14 mg of tar per pipe using a suction device such as a syringe. 10 to 10 times, preferably 7 to 9 times, and more preferably 8 times, and a volume of 300 to 500 mL, preferably 350 to 450 mL, more preferably 400 mL, is obtained by 0.06 to 0.1 m ^3. In the air, preferably 0.07 to 0.09 m ³ . In order to adjust the amount of smoke to be injected, it is preferable to use a closed breeding container, a breeding room or the like when smoking a non-human animal. For the survival of non-human animals, it is necessary to continuously send fresh air to the closed breeding environment and circulate the air, so that the concentration of tobacco smoke in the environment decreases with time. Therefore, although it is considered that the smoking effect due to the single operation of injecting the tobacco smoke lasts about 30 minutes, the duration of the smoking effect is the amount of fresh air fed per unit time, It may also vary depending on the type and number of non-human animals in the breeding environment.

  In the method for producing a model animal of the present invention, the hardening of the lens can be induced by treating a non-human animal for 30 hours or more, preferably 36 hours or more in air containing tobacco smoke at the above concentration. it can. The upper limit of the treatment time is not particularly limited, but for example, it is 90 hours or less in total, preferably 84 hours or less, more preferably 60 hours or less. As described above, since the duration of the smoking effect by a single smoke injection operation is limited, multiple smoke injections are required to induce lens hardening. Multiple injections of smoke may be performed at any interval. For example, the smoking treatment time for one smoke injection is usually 15 to 45 minutes, preferably 30 minutes. It can be carried out 4 to 8 times per day, preferably 6 times, continuously or at an appropriate interval (in this specification, within 30 minutes or before the carbon monoxide concentration in the atmosphere is less than 50 ppm). When the next tobacco smoke injection operation is performed, it is said that the smoking process is “continuous”, and after the carbon monoxide concentration in the atmosphere is less than 50 ppm for more than 30 minutes, the next tobacco smoke injection operation is performed. If done, the smoking treatment is said to be “intermittent”, in which case 30 minutes after the previous injection operation or when the carbon monoxide concentration is less than 50 ppm, from the later time to the next injection operation Is called "interval" ). The interval until the next injection operation is, for example, within 1 hour, preferably within 30 minutes. More preferably, the injection operation is continuous. The smoking treatment time per day is 2 to 4 hours, preferably 3 hours. The processing time per day throughout the smoking processing period may be constant or variable. Curing of the crystalline lens can be induced by repeating this operation until the smoking treatment time reaches a total of 30 hours or more.

  As described above, a disease involving lens sclerosis in which the smoking-treated animal of the present invention can be used as a model animal is any disease that exhibits a pathological condition that makes it difficult to adjust the focus of the eye due to the lens hardening and loss of elasticity. Examples include presbyopia, hyperopia, and cataracts, and preferable examples include presbyopia.

Furthermore, by using the model animal of the present invention, it is possible to screen a candidate substance for a therapeutic and / or prophylactic agent for a disease involving lens sclerosis, and the screening method includes the following steps.
(A) a step of administering a test substance to the model animal of the present invention (b) a step of evaluating a change in the disease state of a disease involving lens hardening in the animal

  The test substances that can be used in the screening method of the present invention are natural substances, organic compounds, inorganic compounds, proteins, peptides, nucleic acids, fermentation products, cell extracts, plant extracts, animal tissue extracts, cell culture supernatants. Including, but not limited to.

  Methods for administering the test substance include, but are not limited to, ocular administration, oral administration, intraportal administration, intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, and transdermal administration. .

  When the test substance is administered to the eye, for example, the component is the same except that the agent containing the test substance is contained in one eye of a non-human animal and the test substance is not contained in the other eye. Agents can be administered. According to the administration method, since the effect of the test substance can be examined in the same individual, it is exemplified as a preferred method.

  In addition, the test substance can be administered by expressing proteins, peptides, shRNA, miRNA and the like in a non-human animal body using transfection or a viral vector. Various transfection reagents (for example, DharmaFECT (registered trademark) 1 (Thermo Scientific), Lipofectamine (registered trademark) LTX (life technologies), etc.) are commercially available, and those skilled in the art can easily follow the manuals for using these reagents. Transfection can be performed. Examples of viral vectors include adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, Sendai virus vectors and the like, and those skilled in the art can prepare and use these virus vectors by known methods.

In the screening method of the present invention, the pathological change of a disease involving lens hardening is evaluated after administration of a test substance. In order to evaluate the pathological change, the hardness of the lens can be examined. The measurement of the hardness (elasticity) of the crystalline lens can be performed, for example, by the following method. First, a lens is extracted from an animal, and a value obtained by measuring the thickness of the lens in the axial direction using a caliper is set as an initial value. The lens is placed in a constant pressure thickness measuring instrument, and the thickness of the lens after deformation when a predetermined pressure is applied is measured. The value obtained by dividing the measured value when the pressure is applied is calculated, and the hardness of the crystalline lens can be inspected by comparing the value. The value shows a larger value as the crystalline lens is hardened. To improve the pathology of diseases involving lens sclerosis, for example, compare the hardness of the lens to which the test substance is administered with the lens to which the test substance is not administered, and judge whether there is a statistically significant difference. Can do. If the hardness of the lens to which the test substance is administered is significantly lower than the hardness of the lens to which the test substance is not administered, it can be determined that an improvement in the disease state caused by lens hardening is observed. A test substance having an effect can be a promising candidate substance for the treatment and / or prevention of a disease involving lens hardening.
In the screening method of the present invention, the pathological change of a disease involving lens hardening, which can be used as an indicator of a therapeutic effect, is not limited to the above-described change in lens hardness (elasticity), but is expressed in the model animal of the present invention. Any pathological change characteristic of the disease can be used. Examples of such other disease states include cataracts.

  As a statistical analysis method, a known statistical analysis method can be appropriately selected and used, and preferable statistical analysis methods include, for example, Student's t test, Dunnett, Tukey test, and the like. When comparing the hardness of the lens administered with the test substance and the hardness of the lens not administered with the test substance, it is determined that there is a significant difference when the p value is less than 0.05, and the p value is When it is 0.05 or more, it can be determined that there is no significant difference.

  Optimum conditions for the dose and period of the test substance can be appropriately set according to the type of test substance and the type, age, size, etc. of the non-human animal to be administered.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Example 1: Induction of lens hardening by smoking treatment
With reference to the method of Higuchi et al. (Free Radic. Biol. Med., 2011, Vol. 51, pages 2210-2216), the rats were treated for smoking by the following method. Male 8-week-old SD rats were placed in a smoking chamber (60 cm × 40 cm × 35 cm) prepared for experiments (up to 12 animals), and fresh air was fed into the chamber using an air pump. Tobacco (Seven Star (registered trademark)) was attached to a 50 mL syringe, mainstream smoke was sucked and blown into the chamber. This suction operation was repeated 8 times, and mainstream smoke was put in a total of 400 mL chamber and left for 30 minutes. The same operation was repeated 5 more times (6 times in total). This procedure exposes the rats to mainstream smoke for a total of 3 hours per day. Thereafter, the rats were returned to the breeding room. This mainstream smoke exposure operation was carried out for a maximum of 4 weeks.

Example 2: Measurement of lens hardness after smoking treatment The lens was extracted from the eyeball of a rat subjected to the smoking treatment, and the hardness of the lens was measured by the following method. First, the thickness of the crystalline lens in the axial direction was measured using a caliper (initial value a). Thereafter, the lens was placed on a constant pressure thickness measuring device, the probe was slowly lowered from above, the lens was deformed by a 10 g weight, and the thickness of the deformed lens was measured (b value). A value (b / a value) obtained by dividing the b value by the a value was calculated. The b / a value increases as the crystalline lens hardens.
FIG. 1 shows the relative hardness of a rat lens of a group not subjected to smoking treatment (NT) or a group subjected to smoking treatment for 1 week, 2 weeks or 4 weeks. The horizontal axis in FIG. 1 represents the mainstream smoke exposure period, and the vertical axis represents the relative value of the b / a value (NT is 1). The lenses of the rats treated with smoking treatment for 2 or 4 weeks were harder than the non-smoking treated and 1 week smoking treated rats. Therefore, it was shown that the lens can be cured by smoking treatment.

Example 3: Inhibition of lens hardening by selenium lactoferrin Selenium lactoferrin was obtained by the method described in Example 1 of WO2012 / 161112.
The selenium lactoferrin solution obtained by the above method was prepared so as to be 0.1 w / v% to prepare an eye drop. The rats were treated with smoking for 12 days by the method described in Example 1, and selenium lactoferrin was instilled by the following method.
(1) Apply 5 μL of 0.1% selenium lactoferrin eye drop to one eye and phosphate buffered saline to the other eye.
(2) Instillation is performed 4 times a day with a minimum interval of 2 hours. The first time is before the mainstream smoke exposure treatment, the second time is after the mainstream smoke exposure treatment, and the remaining two times thereafter.
(3) Instillation was performed only twice before the mainstream smoke exposure treatment and after the mainstream smoke exposure treatment only on the last day, and then the lens hardness was measured by the method described in Example 2.
The measurement result of the lens hardness is shown in FIG. The vertical axis in FIG. 2 indicates the relative value of the b / a value (NT is 1). Lens hardness increased with mainstream smoke exposure (FIG. 2, PBS), and this increase in hardness was significantly suppressed by administration of selenium lactoferrin (FIG. 2, 0.1% Se-lactoferrin).

  Since selenium lactoferrin has an excellent lens hardening inhibitory effect, effective treatment and / or prevention means for diseases associated with lens hardening such as presbyopia, for which no effective therapeutic and / or preventive drug has existed so far. Is provided. Furthermore, if the model animal of the present invention is used, a novel therapeutic and / or prophylactic agent for the disease can be searched.

Claims (7)

  A lens hardening inhibitor comprising selenium lactoferrin or a salt thereof as an active ingredient.   The agent according to claim 1, which is a therapeutic and / or prophylactic agent for a disease involving lens hardening.   The agent according to claim 2, wherein the disease involving lens hardening is presbyopia.   The agent according to any one of claims 1 to 3, which is an eye drop or an eye ointment.   A method for producing a non-human model animal for a disease involving lens hardening, which comprises exposing the non-human animal to an atmosphere containing 50 to 250 ppm of tobacco smoke at a carbon monoxide concentration of at least 30 hours in total. .   The method according to claim 5, wherein the disease involving lens hardening is presbyopia. (A) a step of administering a test substance to the non-human model animal produced by the method according to claim 5, and (b) a step of evaluating a change in a disease state of a disease involving lens sclerosis in the animal,
A method for screening candidate substances for therapeutic and / or prophylactic agents for the disease.