WO2010107069A1 - Amino acid-containing ophthalmic composition - Google Patents

Amino acid-containing ophthalmic composition Download PDF

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Publication number
WO2010107069A1
WO2010107069A1 PCT/JP2010/054571 JP2010054571W WO2010107069A1 WO 2010107069 A1 WO2010107069 A1 WO 2010107069A1 JP 2010054571 W JP2010054571 W JP 2010054571W WO 2010107069 A1 WO2010107069 A1 WO 2010107069A1
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Prior art keywords
amino acid
glutamine
ophthalmic composition
asparagine
arginine
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PCT/JP2010/054571
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French (fr)
Japanese (ja)
Inventor
茂 木下
千恵 外園
淳爾 羽室
明 岡野
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千寿製薬株式会社
味の素株式会社
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Priority to JP2011504870A priority Critical patent/JPWO2010107069A1/en
Publication of WO2010107069A1 publication Critical patent/WO2010107069A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an ophthalmic composition, and particularly to an ophthalmic composition useful for the prevention and treatment of ocular surface diseases associated with dry eye.
  • the lacrimal fluid covers the eyeball surface consisting of the cornea and conjunctiva to maintain the wetness of the cornea and prevent it from drying.
  • various kinds of sensations such as fatigue and foreign body sensation have occurred due to dryness of the keratoconjunctiva surface associated with tear reduction, dryness of the eyes when wearing contact lenses, or dryness of the eyes that occurs during the operation of OA equipment.
  • An increasing number of people complain of symptoms, ie dry eye symptoms. Dry eye is often accompanied by corneal epithelial damage or corneal epithelial fistula due to damage of corneal epithelial cells, and in severe cases, it may cause corneal ulcers and eye infections.
  • the first choice for dry eye treatment is artificial eye drops.
  • Various artificial tears have been proposed so far, and artificial tears mainly composed of salts such as sodium chloride, eye drops containing hydroxyethyl cellulose, chondroitin sulfate or hyaluronic acid, and the like are conventionally known.
  • these artificial tears alone do not improve symptoms.
  • dry eye is largely associated not only with dryness (decrease in tear volume) but also with abnormal tear composition (ie, tear quality).
  • eye drops in which specific amino acids are added to conventional eye drops have been developed (Patent Documents 1 to 3), but for dry eyes, particularly dry eyes associated with serious epithelial disorders, There is still no artificial tear that can provide a satisfactory therapeutic effect.
  • An object of the present invention is to provide an ophthalmic composition useful for the prevention and treatment of dry eye, particularly dry eye associated with severe epithelial disorder.
  • an ophthalmic composition comprising at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine.
  • the amino acid is 2 or 3 amino acids selected from alanine, asparagine and glutamine.
  • the amino acids are three kinds of alanine, asparagine and glutamine.
  • the ophthalmic composition according to the above [1], wherein the amino acids are arginine, asparagine and glutamine.
  • the present invention not only mild dry eye as a symptom, but also dry eye in which improvement of the symptom was not observed even when conventional artificial tears were frequently instilled, particularly dry eye associated with severe epithelial disorder. It has a therapeutic effect on. Furthermore, the preventive effect which prevents the onset of dry eye can be expected.
  • the present invention contains an ophthalmic composition containing at least one amino acid selected from alanine, asparagine, glutamine, and arginine (hereinafter sometimes abbreviated as the ophthalmic composition of the present invention).
  • an ophthalmic composition useful for the prevention and treatment of ocular surface diseases associated with dry eye is provided.
  • dry eye refers to keratoconjunctival epithelial disorder caused by a qualitative or quantitative abnormality in tears (layer).
  • Alanine, asparagine, glutamine and arginine, which are active ingredients of the ophthalmic composition of the present invention, may be free forms or pharmacologically acceptable salts.
  • a salt for example, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal, alkaline earth metal) is used, and an alkali metal salt ( Examples: sodium salts, potassium salts), alkaline earth metal salts (eg, calcium salts, magnesium salts) and the like are preferably exemplified.
  • a peptide known to exhibit an effect equivalent to that of an amino acid that is an active ingredient of the ophthalmic composition of the present invention on cells, such as L-alanylglutamine, can also be used.
  • Alanine, asparagine, glutamine and arginine (hereinafter also referred to as effective amino acids), which are active ingredients of the ophthalmic composition of the present invention, may be used alone or in combination of two or more. This is highly effective and preferable. As such a preferable combination, asparagine and alanine, asparagine and glutamine, asparagine and arginine, alanine and glutamine, alanine and arginine, a combination of glutamine and arginine, and asparagine and alanine in the case of three combinations.
  • Combinations of alanine and glutamine, asparagine and alanine and arginine, alanine and glutamine and arginine, arginine and asparagine and glutamine, and combinations of four types include combinations of asparagine, alanine, glutamine and arginine.
  • the effective amino acid combination of the ophthalmic composition of the present invention is a combination consisting of 2 or 3 amino acids selected from alanine, asparagine and glutamine.
  • Another preferred combination is a combination of alanine and glutamine, glutamine and arginine, alanine and glutamine and arginine, asparagine and glutamine and arginine, alanine, asparagine, glutamine and arginine.
  • the concentration of the effective amino acid in the ophthalmic composition of the present invention is not particularly limited as long as the effect of promoting the proliferation of the keratoconjunctival epithelial cells and / or the wound healing effect is obtained and the cytotoxicity does not occur. Is a total of 0.05 mM to 1000 mM, preferably 0.1 mM to 800 mM, more preferably 0.3 mM to 200 mM, and particularly preferably 0.5 mM to 100 mM. Each effective amino acid concentration is not particularly limited as long as a desired effect is exhibited.
  • each effective amino acid concentration can be appropriately selected from the range of 0.1 ⁇ M to 800 mM, preferably 0.5 ⁇ M to 100 mM, more preferably 1 ⁇ M to 600 ⁇ M, and particularly preferably 1 ⁇ M to 500 ⁇ M.
  • alanine for example, 0.5 ⁇ M to 600 mM, preferably 1 ⁇ M to 500 mM, more preferably 8 ⁇ M to 150 mM, especially 10 ⁇ M to 100 mM, and in the case of asparagine, for example, 1 ⁇ M to 300 mM, preferably 5 ⁇ M to 150 mM, more preferably 10 ⁇ M to 100 mM, in the case of arginine, for example, 0.1 ⁇ M to 200 mM, preferably 0.5 ⁇ M to 100 mM, more preferably 3 ⁇ M to 50 mM, in the case of glutamine, for example, 1 ⁇ M to 600 mM It is preferably 5 ⁇ M to 500 mM, more preferably 10 ⁇ M to 200 mM, especially 12 to 150 ⁇ M.
  • the ophthalmic composition of the present invention may be any preparation that can be administered to the local tissue of the eye, and examples thereof include eye drops, patches, ointments, lotions, creams, etc. It is an eye drop.
  • a substrate may be used as appropriate.
  • Base materials used in eye drops include phosphate buffer, Hanks buffer, physiological saline, perfusate (eg, BS Plus (Nippon Alcon Co., Ltd.), Opeguard Neo Kit (Senju Pharmaceutical Co., Ltd.)), artificial For example, lacrimal fluid.
  • BS Plus is used as a base material, it is possible to use only the diluted solution without adding the oxyglutathione solution.
  • the ophthalmic composition of the present invention may further contain another drug having an effect of preventing or treating keratoconjunctival epithelial disorder.
  • a concomitant drug include, but are not limited to, a corneal protective agent (eg, hyaluronic acid, chondroitin sulfate or a salt thereof), an immunosuppressive / anti-inflammatory agent (eg, cyclosporine, steroid) and the like.
  • the ophthalmic composition of the present invention includes any other active ingredient such as an antiallergic or antihistamine ingredient, a decongestant ingredient, a local anesthetic ingredient, a vitamin ingredient, and an amino acid ingredient other than an effective amino acid (eg, valine, Leucine, isoleucine, serine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, histidine, citrulline, ornithine, cystine, taurine, glycine) and the like.
  • an effective amino acid eg, valine, Leucine, isoleucine, serine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan
  • an effective amino acid eg, valine, Leucine, isoleucine, serine, threonine, methionine, proline,
  • the ophthalmic composition of the present invention may be appropriately mixed with various additives such as a buffer, an isotonic agent, a preservative, a solubilizer, a stabilizer, a chelating agent, a thickener, and a pH adjuster. it can.
  • the buffer examples include boric acid or a salt thereof (borax, etc.), citric acid or a salt thereof (sodium citrate, etc.), tartaric acid or a salt thereof (sodium tartrate, etc.), gluconic acid or a salt thereof (sodium gluconate, etc.) ), Acetic acid or a salt thereof (sodium acetate, etc.), phosphoric acid or a salt thereof (sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), various amino acids such as glutamic acid or epsilon aminocaproic acid, and a tris buffer, etc. Combinations are listed.
  • isotonic agent examples include sorbitol, mannitol, glycerin, propylene glycol, sodium chloride, potassium chloride and the like.
  • preservative examples include paraoxybenzoates, benzalkonium chloride, benzethonium chloride, benzyl alcohol, sorbic acid or a salt thereof, chlorhexidine gluconate, sodium dehydroacetate, cetylpyridinium chloride, alkyldiaminoethylglycine hydrochloride, chlorobutanol Etc.
  • solubilizer examples include polyvinyl pyrrolidone, polyethylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil 60, polyoxyl 40 stearate, polysorbate 80 (trade name: Tween 80), and the like.
  • the stabilizer examples include sodium edetate, sodium thiosulfate, ascorbic acid, cyclodextrin, condensed phosphoric acid or a salt thereof, sulfite, citric acid or a salt thereof, and dibutylhydroxytoluene.
  • chelating agent examples include sodium edetate, sodium citrate, condensed phosphoric acid or a salt thereof (such as condensed sodium phosphate).
  • thickener examples include methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose sodium, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, hyaluronic acid and the like.
  • 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.
  • the pH of the ophthalmic composition of the present invention is adjusted to 3 to 10, preferably 5 to 8.
  • the ophthalmic composition of the present invention exhibits excellent effects such as reduction of eye pain, difficulty in opening the eye, foreign body sensation, fatigue, etc., as well as improvement in visual acuity, conjunctival hyperemia, corneal epithelial state, and the like. Therefore, the ophthalmic composition of the present invention is useful as a prophylactic / therapeutic agent for ocular surface diseases, particularly keratoconjunctival epithelial disorders.
  • keratoconjunctival epithelial disorders include corneal epithelial disorders caused by desiccation (eg, tear reduction, dry eye, meibomian gland dysfunction, Sjogren's syndrome, dry keratoconjunctivitis, blepharitis, Stevens-Johnson syndrome, VDT) Dry eye syndrome such as dry eye associated with work, or keratoconjunctival epithelial disorder associated with dry eye, scarring keratoconjunctivitis, corneal epithelial fistula, corneal ulcer, blepharitis, pemphigoid, spring catarrh, allergy Conjunctivitis, etc.), corneal epithelial damage caused by ultraviolet rays (eg, keratitis, snow eyes, etc.), chemical trauma caused by chemicals, drugs contained in eye drops (eg, preservatives, eye anesthetics, prostaglandins, Corneal epithelial disorder (for example, punctate superficial keratitis,
  • the ophthalmic composition of the present invention can also be used as an ophthalmic composition for preventing or improving eye fatigue, eye irritation, blurred vision, itchy eyes, conjunctival hyperemia, and discomfort when wearing contact lenses. Can be used.
  • the ophthalmic composition of the present invention is used for preventing or treating the above-mentioned diseases or conditions in humans and animals other than humans (for example, mammals other than humans (domestic animals such as pigs, cows, horses, dogs, and pets)). Can be used.
  • the dosage of the ophthalmic composition of the present invention may vary depending on the dosage form, target disease, animal species to be administered, age, sex, weight, symptoms, etc., for example, adult (for example, 60 kg body weight) In this case, it is usually in the range of 0.1 ⁇ g to 2.0 g, preferably 0.5 ⁇ g to 1.8 g, more preferably 1.0 ⁇ g to 1.g in terms of the total amount of amino acids in the composition per day. The range is 5 g.
  • the daily dose of the ophthalmic composition of the present invention can be administered once or divided into several times.
  • the ophthalmic composition of the present invention is used 5 to 20 times, preferably 16 to 18 times per day (generally once an hour) for severe dry eye patients. ), 2 to 10 times, preferably 3 to 7 times a day for patients with mild dry eye.
  • the dose per administration is 1 to 3 drops (50 to 150 ⁇ L), preferably 1 to 2 drops (50 to 100 ⁇ L) per eye.
  • the administration period is not particularly limited.
  • amino acids and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below.
  • optical isomer with respect to an amino acid the L form is shown unless otherwise specified.
  • Gly Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys Argin P : Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine Orn: Ornithine Cit: Citrulline (Cys) 2 : Cystine Tau: Taurine
  • the target amino acids were glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, asparagine, glutamine, lysine, arginine, tryptophan, histidine, methionine, aspartic acid, glutamic acid, ornithine, citrulline. Cystine, taurine. Assuming that the total amount of amino acids to be measured in tears of each subject of healthy subjects and dry eye patients was 100, the composition ratio of each amino acid was calculated. The results are shown in Table 1. In addition, a 2-hour VDT operation was carried out with a resting tear fluid of a healthy person, and each amino acid concentration ( ⁇ M) in the tear fluid when a temporary dry eye state was obtained was measured. The results are shown in Table 2.
  • Zero medium powder in double distilled water 400mL [CaCl 2 (anhyd) 200mg , Fe (NO 3) 3 ⁇ 9H 2 O 0.1mg, KCl 400mg, MgSO 4 (anhyd) 97.67mg, NaCl 6400mg, NaHPO 4 ⁇ H 2 O 125 mg, Sodium pyruvate 110 mg, D-Capantheneate 4 mg, Choline Chloride 4 mg, Folic acid 4 mg, I-inositol 7.2 mg, Niacinide 4 mg, Riboflavin 4 mg, Riboflavin 4 NaHCO 3 3.7 g and Glucose 1 g were added and dissolved.
  • the medium was adjusted to pH 7.4 with HCl (hydrochloric acid) and diluted to 500 mL with double distilled water. Then, after sterilizing filtration with a 0.22 ⁇ m sterilizing filter, it was stored at 4 ° C.
  • HCl hydrochloric acid
  • Amino acid powder was appropriately added to the 2 ⁇ Zero medium and dissolved, and then sterilized with a 0.22 ⁇ m sterilizing filter and stored at 4 ° C.
  • 2 ⁇ Zero medium 50 mL is filled with Full medium powder [Glycine 3.0 mg, L-Aline 3.56 mg, L-Series 4 .2 mg, L-Throneine 9.5 mg, L-Cystein 2HCl 6.3 mg, L-Methionine 3.0 mg, L-Glutamine 58.4 mg, L-Asparagine 5.28 mg, L-Glutamate Acid-Asp.
  • amino acid-containing medium lacking some amino acids was also prepared by dissolving the necessary amino acid powder in 2 ⁇ Zero medium in the same manner as described above, or by aseptically adding a previously prepared amino acid solution.
  • For the supplemented medium containing essential amino acids and 1 to 4 non-essential amino acids dissolve the necessary amino acid powder in 2 ⁇ Zero medium in the same manner as above, or aseptically add a prepared amino acid solution. Adjusted.
  • the cultured cells were collected using 0.25% trypsin, seeded in a 96-well plate (Corning) at 2 ⁇ 10 4 cells / well, and EpiLife medium was used in a 5% CO 2 atmosphere.
  • the cells were cultured at 37 ° C. for 2 days. Thereafter, the medium was replaced with Zero medium, and cultured at 37 ° C. for 1 day in a 5% CO 2 atmosphere. Subsequently, the medium was changed again to each specimen medium, and the culture was continued for 3 days (5% CO 2 , 37 ° C.).
  • the amino acid composition of each specimen medium is shown in Table 3 for the deficient medium and Table 4 for the supplemented medium.
  • the essential amino acid medium contains glutamine and arginine, asparagine and alanine, asparagine and alanine and glutamine, asparagine and alanine and arginine, alanine and glutamine and arginine, arginine and asparagine and glutamine, and asparagine and alanine, glutamine and arginine.
  • the total concentration of amino acids contained in the medium is preferably less than about 18 mM.
  • cytotoxicity tends to be expressed more strongly than the effect of amino acid addition.
  • the cultured cells were collected using 0.25% trypsin, seeded in a 6-well plate (Corning) at 5 ⁇ 10 4 cells / well, and EpiLife medium was used in a 5% CO 2 atmosphere.
  • the cells were cultured at 37 ° C. for 2 weeks. Thereafter, the medium was changed to Zero medium, and cultured at 37 ° C. for 1 day in a 5% CO 2 atmosphere.
  • the confluent state cells were scraped linearly with the tip of a micropipette tip (1000 ⁇ L) to prepare a wound model. After wound preparation, the specimen medium was replaced and cultured for 48 hours. The recovery state of the wound site was examined using a phase contrast microscope.
  • wound width was measured using ImageJ image analysis software (NIH). A total of 6 measurements were made for each group.
  • amino acid-containing ophthalmic solution 2 As a result of observation from day 1 to day 7 of administration, no abnormality was observed in the cornea, iris and conjunctiva. Moreover, the blink immediately after administration was never observed. The total score of this group was 0, and the evaluation category of eye irritation corresponded to non-irritating substances.
  • amino acid-free ophthalmic solution As a result of observation from the first day of administration to the seventh day of administration, no abnormality was observed in the cornea, iris and conjunctiva. In addition, no blinks were observed immediately after administration. The total score of this group was 0, and the evaluation category of eye irritation corresponded to non-irritating substances. From the above, it can be determined that both the amino acid-containing ophthalmic solutions 1 and 2 have no eye irritation.
  • Example 2 Production test of ophthalmic composition of the present invention as an eye drop As shown in Table 9 below, an eye drop containing each amino acid is prepared.
  • Example 2 Clinical test using amino acid-containing eye drops
  • the amino acid-containing eye drops of each of the production examples described in Example 2 were administered to subjects, and the subjective symptoms or objective findings of the subjects after the administration were measured between subjects. Compare with. Specifically, the eye drop of any one of Production Examples 1, 2, 4, and 5 is administered to one eye of each subject, and the eye drop of Production Example 10 is administered to the other eye as a control. Eye drops are administered to healthy subjects and severe dry eye patients once or twice (50 to 100 ⁇ L) / eye once an hour. The number of administrations per day is 16-18. For patients with mild dry eye and VDT workers, 1-2 drops (50-100 ⁇ L) / eye is administered 4-6 times / day.
  • the above severe dry eye patient means a patient having a Schirmer test I method value of 5 mm or less and a Rose Bengal staining score (Van Bijsterveld score) of 9 points. It means a patient having a method I value of 5 mm or less and a Rose Bengal staining score (Van Bijsterveld score) of 6 to 8 points.
  • Mild dry eye patient means a dry eye patient with a Schirmer test I method value greater than 5 mm and a Rose Bengal staining score (Van Bijsterveld score) of 3-5 points.
  • Subjective symptoms Dryness, foreign body, eye pain, fatigue
  • Objective findings Improvement of visual acuity, presence of abnormalities of each conjunctiva by slit slit microscope, keratoconjunctival disorder caused by fluorescein staining Presence or absence (especially conjunctival punctuate staining (CPS) between ruptures), presence or absence of degenerated or damaged cells of the keratoconjunctival epithelium by rose bengal or Lissami green staining, BUT Time), observation of tear oil layer distribution (by dry eye observation device DR-1 TM (manufactured by Kowa))
  • Example 3 Clinical test using an eye drop containing an amino acid
  • the amino acid-containing eye drop prepared in Example 3 was administered to a subject, and the subjective symptoms or objective findings of the subject after the administration were evaluated.
  • amino acid-containing ophthalmic solution 3 or amino acid-containing ophthalmic solution 4 was administered once to 1-2 drops (50 to 100 ⁇ L) / eye and 6 times / day to each subject.
  • the subject has a normal tear volume with a Schirmer test I value of 5 mm or more, but the BUT (tear film breakage time) is significantly shorter than 5 seconds, and is there no keratoconjunctival epithelial disorder? It was a minor case and a BUT shortened dry eye patient with very strong subjective symptoms such as dryness.
  • SPK was improved from D2 to D1 at 8 weeks after administration in both the right eye of the subject 1 to which the amino acid-containing ophthalmic solution 3 was administered and the left eye of the subject 1 to which the amino acid-containing ophthalmic solution 4 was administered.
  • BUT was prolonged 8 weeks after administration in the left and right eyes of subject 2 and left and right eyes of subject 3 to which amino acid-containing ophthalmic solution 3 was administered.
  • the subjective symptom scores decreased in the left and right eyes of all subjects.
  • amino acid-containing ophthalmic solution 3 containing arginine and glutamine and the amino acid-containing ophthalmic solution 4 containing alanine, asparagine and glutamine are effective for the treatment of dry eye.
  • the therapeutic effect is acquired with respect to the dry eye in which the improvement of the symptom was not recognized even if the conventional artificial tears were instilled frequently, especially the dry eye accompanying a serious epithelial disorder. Specifically, it exhibits excellent effects in reducing subjective symptoms (eye pain, difficulty in opening the eye, foreign body sensation, fatigue, etc.) and improving objective findings (visual acuity, conjunctival hyperemia, corneal epithelial condition).
  • This application is based on Japanese Patent Application No. 2009-065284 filed in Japan (filing date: March 17, 2009), the contents of which are incorporated in full herein.

Abstract

Disclosed is an ophthalmic composition which is useful for prevention or treatment of dry eye conditions, especially for prevention or treatment of dry eye conditions associated with serious epithelial disorders. Specifically disclosed is an ophthalmic composition which is characterized by containing at least one amino acid selected from a group consisting of alanine, asparagine, glutamine and arginine. The ophthalmic composition preferably contains: two or three amino acids selected from among alanine, asparagine and glutamine; three amino acids, namely arginine, asparagine and glutamine; or two amino acids, namely arginine and glutamine. Also disclosed is an ophthalmic composition for prevention or treatment of ocular surface disorders, especially for prevention or treatment of corneal and conjunctival epithelial disorders.

Description

アミノ酸含有眼科用組成物Amino acid-containing ophthalmic composition
　本発明は、眼科用組成物、特にドライアイを伴う眼表面疾患の予防・治療に有用な眼科用組成物に関する。 The present invention relates to an ophthalmic composition, and particularly to an ophthalmic composition useful for the prevention and treatment of ocular surface diseases associated with dry eye.
　涙液は、角膜や結膜からなる眼球表面を覆って角結膜の湿潤性を保持し、乾燥を防いでいる。しかしながら、近年、涙液減少症に伴う角結膜表面の乾燥、コンタクトレンズ装用時の眼の乾燥、あるいはＯＡ機器の操作中に起きる眼の乾燥等により、疲労感、異物感を始めとした種々の症状、すなわち、ドライアイ症状を訴える人が増えている。ドライアイは、しばしば角膜上皮細胞の障害による角膜上皮障害や角膜上皮糜爛等を伴い、重篤な場合は角膜潰瘍や眼感染症を発症させることもある。 The lacrimal fluid covers the eyeball surface consisting of the cornea and conjunctiva to maintain the wetness of the cornea and prevent it from drying. However, in recent years, various kinds of sensations such as fatigue and foreign body sensation have occurred due to dryness of the keratoconjunctiva surface associated with tear reduction, dryness of the eyes when wearing contact lenses, or dryness of the eyes that occurs during the operation of OA equipment. An increasing number of people complain of symptoms, ie dry eye symptoms. Dry eye is often accompanied by corneal epithelial damage or corneal epithelial fistula due to damage of corneal epithelial cells, and in severe cases, it may cause corneal ulcers and eye infections.
　一般的に、ドライアイ治療の第一選択は人工涙液の点眼である。これまでに種々の人工涙液が提案されており、塩化ナトリウムなどの塩類を主眼とした人工涙液、ヒドロキシエチルセルロース、コンドロイチン硫酸又はヒアルロン酸を含む点眼剤等が従来知られている。しかし、これらの人工涙液の点眼のみでは症状が改善しないケースが多い。ドライアイは、乾燥（涙液量の減少）だけでなく涙液の組成（即ち、涙液の質）の異常が大きく関与していると考えられる。これに対して、従来型の点眼剤に特定のアミノ酸を添加した点眼剤も開発されているが（特許文献１～３）、ドライアイ、特に重篤な上皮障害に伴うドライアイに対して、満足な治療効果が得られる人工涙液は未だ無い。 In general, the first choice for dry eye treatment is artificial eye drops. Various artificial tears have been proposed so far, and artificial tears mainly composed of salts such as sodium chloride, eye drops containing hydroxyethyl cellulose, chondroitin sulfate or hyaluronic acid, and the like are conventionally known. However, in many cases, these artificial tears alone do not improve symptoms. It is considered that dry eye is largely associated not only with dryness (decrease in tear volume) but also with abnormal tear composition (ie, tear quality). In contrast, eye drops in which specific amino acids are added to conventional eye drops have been developed (Patent Documents 1 to 3), but for dry eyes, particularly dry eyes associated with serious epithelial disorders, There is still no artificial tear that can provide a satisfactory therapeutic effect.
国際公開第２００７／０４８５２３号パンフレットInternational Publication No. 2007/048523 Pamphlet 特開２００６－１０４１１４JP 2006-104114 A 国際公開第１９９６／１９２１１号パンフレットInternational Publication No. 1996/19211 Pamphlet
　本発明の課題は、ドライアイ、特に重篤な上皮障害に伴うドライアイの予防・治療に有用な眼科用組成物を提供することである。 An object of the present invention is to provide an ophthalmic composition useful for the prevention and treatment of dry eye, particularly dry eye associated with severe epithelial disorder.
　本発明者らは、ドライアイ患者群およびＶＤＴ（Ｖｉｓｕａｌ　Ｄｉｓｐｌａｙ　Ｔｅｒｍｉｎａｌ）作業により急性のドライアイ状態を惹起した群（ＶＤＴ作業群）の涙液中のアミノ酸組成を分析した結果、健常者の涙液と比較して、特定のアミノ酸が有意に低減していることを見出した。そこで本発明者らは、ヒト角膜上皮細胞を種々のアミノ酸組成の培地を用いて培養し、各アミノ酸の細胞増殖促進及び創傷治癒効果を試験した結果、前記特定のアミノ酸が顕著な角膜上皮障害治療効果を有することを確認して、本発明を完成するに至った。 As a result of analyzing the amino acid composition in the tear fluid of the dry eye patient group and the group that caused an acute dry eye state by the VDT (Visual Display Terminal) work (VDT work group), We found that certain amino acids were significantly reduced compared to. Accordingly, the present inventors have cultured human corneal epithelial cells using a medium having various amino acid compositions, and tested the promotion of cell growth and wound healing effect of each amino acid. It was confirmed that the present invention had an effect, and the present invention was completed.
　すなわち、本発明は以下の通りである。
［１］アラニン、アスパラギン、グルタミン及びアルギニンからなる群から選択される少なくとも１種のアミノ酸を含有することを特徴とする眼科用組成物。
［２］上記アミノ酸が、アラニン、アスパラギン及びグルタミンから選択される２又は３種のアミノ酸である上記［１］記載の眼科用組成物。
［３］上記アミノ酸が、アラニン、アスパラギン及びグルタミンの３種である上記［２］記載の眼科用組成物。
［４］上記アミノ酸が、アルギニン、アスパラギン及びグルタミンの３種である上記［１］記載の眼科用組成物。
［５］上記アミノ酸が、アルギニンとグルタミンとの組み合わせである上記［１］記載の眼科用組成物。
［６］眼表面疾患の予防・治療用である、上記［１］～［５］のいずれか１つに記載の眼科用組成物。
［７］眼表面疾患が、角結膜上皮障害である上記［６］記載の眼科用組成物。
［８］アラニン、アスパラギン、グルタミン及びアルギニンからなる群から選択される少なくとも１種のアミノ酸を含有することを特徴とする眼科用組成物の有効量を眼表面疾患を有する患者に投与することを特徴とする、眼表面疾患の予防・治療方法。
［９］眼表面疾患が、角結膜上皮障害である上記［８］記載の予防・治療方法。
［１０］眼表面疾患予防・治療剤の製造のためのアラニン、アスパラギン、グルタミン及びアルギニンからなる群から選択される少なくとも１種のアミノ酸を含有する組成物の使用。
［１１］眼表面疾患が、角結膜上皮障害である上記［１０］記載の使用。 That is, the present invention is as follows.
[1] An ophthalmic composition comprising at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine.
[2] The ophthalmic composition according to the above [1], wherein the amino acid is 2 or 3 amino acids selected from alanine, asparagine and glutamine.
[3] The ophthalmic composition according to the above [2], wherein the amino acids are three kinds of alanine, asparagine and glutamine.
[4] The ophthalmic composition according to the above [1], wherein the amino acids are arginine, asparagine and glutamine.
[5] The ophthalmic composition according to the above [1], wherein the amino acid is a combination of arginine and glutamine.
[6] The ophthalmic composition according to any one of [1] to [5] above, which is used for prevention / treatment of ocular surface diseases.
[7] The ophthalmic composition according to the above [6], wherein the ocular surface disease is keratoconjunctival epithelial disorder.
[8] An effective amount of an ophthalmic composition containing at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine is administered to a patient having an ocular surface disease And a method for preventing and treating ocular surface diseases.
[9] The prevention / treatment method according to the above [8], wherein the ocular surface disease is keratoconjunctival epithelial disorder.
[10] Use of a composition containing at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine for the manufacture of a prophylactic / therapeutic agent for ocular surface diseases.
[11] The use according to [10] above, wherein the ocular surface disease is keratoconjunctival epithelial disorder.
　本発明によれば、症状として中軽度のドライアイだけでなく、従来の人工涙液を頻回点眼しても症状の改善が認められなかったドライアイ、特に重篤な上皮障害に伴うドライアイに対して治療効果を有する。さらにドライアイ発症を未然に防止する予防的効果を期待できる。 According to the present invention, not only mild dry eye as a symptom, but also dry eye in which improvement of the symptom was not observed even when conventional artificial tears were frequently instilled, particularly dry eye associated with severe epithelial disorder. It has a therapeutic effect on. Furthermore, the preventive effect which prevents the onset of dry eye can be expected.
　以下において、本発明について詳細に説明する。
　本発明は、アラニン、アスパラギン、グルタミン及びアルギニンのうちの少なくとも１種のアミノ酸を含有することを特徴とする眼科用組成物（以下、本発明の眼科用組成物と略記する場合もある。）、特にドライアイを伴う眼表面疾患の予防・治療に有用な眼科用組成物を提供する。本明細書において、ドライアイとは、涙液（層）の質的又は量的な異常により、引き起こされた角結膜上皮障害を指す。 Hereinafter, the present invention will be described in detail.
The present invention contains an ophthalmic composition containing at least one amino acid selected from alanine, asparagine, glutamine, and arginine (hereinafter sometimes abbreviated as the ophthalmic composition of the present invention). In particular, an ophthalmic composition useful for the prevention and treatment of ocular surface diseases associated with dry eye is provided. As used herein, dry eye refers to keratoconjunctival epithelial disorder caused by a qualitative or quantitative abnormality in tears (layer).
　本発明の眼科用組成物の有効成分であるアラニン、アスパラギン、グルタミン及びアルギニンは、遊離体であってもよいし、薬理学的に許容される塩であってもよい。そのような塩としては、例えば、生理学的に許容される酸（例：無機酸、有機酸）や塩基（例：アルカリ金属、アルカリ土類金属）などとの塩が用いられ、アルカリ金属塩（例：ナトリウム塩、カリウム塩）、アルカリ土類金属塩（例：カルシウム塩、マグネシウム塩）などが好ましく例示される。本明細書においては、以後単にアミノ酸名で記載する場合、特に矛盾しない限りは、当該アミノ酸の塩をも包含する意味で用いられていると解すべきである。またＬ－アラニルグルタミンのように、細胞に対し本発明の眼科用組成物の有効成分であるアミノ酸と同等の効果を示すことが知られているペプチドを用いることもできる。 Alanine, asparagine, glutamine and arginine, which are active ingredients of the ophthalmic composition of the present invention, may be free forms or pharmacologically acceptable salts. As such a salt, for example, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal, alkaline earth metal) is used, and an alkali metal salt ( Examples: sodium salts, potassium salts), alkaline earth metal salts (eg, calcium salts, magnesium salts) and the like are preferably exemplified. In the present specification, when the amino acid names are simply described hereinafter, it should be understood that they are used to include salts of the amino acids unless otherwise contradicted. In addition, a peptide known to exhibit an effect equivalent to that of an amino acid that is an active ingredient of the ophthalmic composition of the present invention on cells, such as L-alanylglutamine, can also be used.
　本発明の眼科用組成物の有効成分であるアラニン、アスパラギン、グルタミン及びアルギニン（以下、有効アミノ酸ともいう）は、そのうちの１種のみを用いてもよいが、２または３種以上を組み合わせて使用すると効果が高く、好ましい。そのような好ましい組み合わせとしては、２種類の組み合わせの場合、アスパラギンとアラニン、アスパラギンとグルタミン、アスパラギンとアルギニン、アラニンとグルタミン、アラニンとアルギニン、グルタミンとアルギニンの組み合わせ、３種類の組み合わせの場合、アスパラギンとアラニンとグルタミン、アスパラギンとアラニンとアルギニン、アラニンとグルタミンとアルギニン、アルギニンとアスパラギンとグルタミンの組み合わせ、４種類の組み合わせの場合、アスパラギンとアラニンとグルタミンとアルギニンの組み合わせが挙げられる。好ましくは、本発明の眼科用組成物の有効アミノ酸の組み合わせとしては、アラニン、アスパラギン及びグルタミンから選択される２又は３種のアミノ酸からなる組み合わせである。また、別の好ましい組み合わせでは、アラニンとグルタミン、グルタミンとアルギニン、アラニンとグルタミンとアルギニン、アスパラギンとグルタミンとアルギニン、アラニンとアスパラギンとグルタミンとアルギニンの組み合わせである。 Alanine, asparagine, glutamine and arginine (hereinafter also referred to as effective amino acids), which are active ingredients of the ophthalmic composition of the present invention, may be used alone or in combination of two or more. This is highly effective and preferable. As such a preferable combination, asparagine and alanine, asparagine and glutamine, asparagine and arginine, alanine and glutamine, alanine and arginine, a combination of glutamine and arginine, and asparagine and alanine in the case of three combinations. Combinations of alanine and glutamine, asparagine and alanine and arginine, alanine and glutamine and arginine, arginine and asparagine and glutamine, and combinations of four types include combinations of asparagine, alanine, glutamine and arginine. Preferably, the effective amino acid combination of the ophthalmic composition of the present invention is a combination consisting of 2 or 3 amino acids selected from alanine, asparagine and glutamine. Another preferred combination is a combination of alanine and glutamine, glutamine and arginine, alanine and glutamine and arginine, asparagine and glutamine and arginine, alanine, asparagine, glutamine and arginine.
　本発明の眼科用組成物における有効アミノ酸の濃度は、角結膜上皮細胞の増殖促進及び／又は創傷治癒効果が得られ、かつ細胞毒性が出ない範囲であれば、特に制限されないが、通常有効アミノ酸の総和として、０．０５ｍＭ～１０００ｍＭ、好ましくは０．１ｍＭ～８００ｍＭ、さらに好ましくは０．３ｍＭ～２００ｍＭ、特に好ましくは、０．５ｍＭ～１００ｍＭである。各有効アミノ酸濃度は、所望の効果が発揮される限り特に限定されない。通常、各有効アミノ酸濃度は、０．１μＭ～８００ｍＭ、好ましくは０．５μＭ～１００ｍＭ、さらに好ましくは１μＭ～６００μＭ、特に好ましくは１μＭ～５００μＭの範囲から適宜選択することができる。アラニンの場合、例えば、０．５μＭ～６００ｍＭ、好ましくは１μＭ～５００ｍＭ、さらに好ましくは、特に好ましくは８μＭ～１５０ｍＭ、就中、１０μＭ～１００ｍＭであり、アスパラギンの場合、例えば、１μＭ～３００ｍＭ、好ましくは、５μＭ～１５０ｍＭ、より好ましくは、１０μＭ～１００ｍＭ、アルギニンの場合、例えば、０．１μＭ～２００ｍＭ、好ましくは０．５μＭ～１００ｍＭ、より好ましくは、３μＭ～５０ｍＭ、グルタミンの場合、例えば、１μＭ～６００ｍＭ、好ましくは５μＭ～５００ｍＭ、より好ましくは、１０μＭ～２００ｍＭ、就中、１２～１５０μＭである。 The concentration of the effective amino acid in the ophthalmic composition of the present invention is not particularly limited as long as the effect of promoting the proliferation of the keratoconjunctival epithelial cells and / or the wound healing effect is obtained and the cytotoxicity does not occur. Is a total of 0.05 mM to 1000 mM, preferably 0.1 mM to 800 mM, more preferably 0.3 mM to 200 mM, and particularly preferably 0.5 mM to 100 mM. Each effective amino acid concentration is not particularly limited as long as a desired effect is exhibited. Usually, each effective amino acid concentration can be appropriately selected from the range of 0.1 μM to 800 mM, preferably 0.5 μM to 100 mM, more preferably 1 μM to 600 μM, and particularly preferably 1 μM to 500 μM. In the case of alanine, for example, 0.5 μM to 600 mM, preferably 1 μM to 500 mM, more preferably 8 μM to 150 mM, especially 10 μM to 100 mM, and in the case of asparagine, for example, 1 μM to 300 mM, preferably 5 μM to 150 mM, more preferably 10 μM to 100 mM, in the case of arginine, for example, 0.1 μM to 200 mM, preferably 0.5 μM to 100 mM, more preferably 3 μM to 50 mM, in the case of glutamine, for example, 1 μM to 600 mM It is preferably 5 μM to 500 mM, more preferably 10 μM to 200 mM, especially 12 to 150 μM.
　本発明の眼科用組成物は、眼の局所組織に投与することができる製剤であればよく、例えば、点眼剤、貼付剤、軟膏剤、ローション剤、クリーム剤等が挙げられるが、好ましくは、点眼剤である。本発明の眼科用組成物が点眼剤である場合には、適宜基材が用いられてもよい。点眼剤に用いられる基材としては、リン酸緩衝液、ハンクスバッファー、生理食塩水、灌流液（例えば、ビーエスエスプラス（日本アルコン株式会社）、オペガードネオキット（千寿製薬株式会社））、人工涙液などが挙げられる。ビーエスエスプラスを基材として使用する場合には、オキシグルタチオン溶液を添加せずに、希釈液だけを使用することもできる。 The ophthalmic composition of the present invention may be any preparation that can be administered to the local tissue of the eye, and examples thereof include eye drops, patches, ointments, lotions, creams, etc. It is an eye drop. When the ophthalmic composition of the present invention is an eye drop, a substrate may be used as appropriate. Base materials used in eye drops include phosphate buffer, Hanks buffer, physiological saline, perfusate (eg, BS Plus (Nippon Alcon Co., Ltd.), Opeguard Neo Kit (Senju Pharmaceutical Co., Ltd.)), artificial For example, lacrimal fluid. When BS Plus is used as a base material, it is possible to use only the diluted solution without adding the oxyglutathione solution.
　本発明の眼科用組成物は、角結膜上皮障害の予防・治療効果を有する他の薬剤をさらに含有していてもよい。そのような併用薬剤としては、例えば、角膜保護剤（例：ヒアルロン酸、コンドロイチン硫酸又はその塩）、免疫抑制／抗炎症剤（例：シクロスポリン、ステロイド）等が挙げられるが、それらに限定されない。 The ophthalmic composition of the present invention may further contain another drug having an effect of preventing or treating keratoconjunctival epithelial disorder. Examples of such a concomitant drug include, but are not limited to, a corneal protective agent (eg, hyaluronic acid, chondroitin sulfate or a salt thereof), an immunosuppressive / anti-inflammatory agent (eg, cyclosporine, steroid) and the like.
　また、本発明の眼科用組成物は、任意の他の活性成分、例えば、抗アレルギーまたは抗ヒスタミン成分、充血除去成分、局所麻酔薬成分、ビタミン成分、有効アミノ酸以外のアミノ酸成分（例：バリン、ロイシン、イソロイシン、セリン、スレオニン、メチオニン、プロリン、フェニルアラニン、チロシン、トリプトファン、アスパラギン酸、グルタミン酸、リジン、ヒスチジン、シトルリン、オルニチン、シスチン、タウリン、グリシン）などをさらに含有していてもよい。そのような他の活性成分としては、自体公知の各種薬剤を適宜使用することができる。 In addition, the ophthalmic composition of the present invention includes any other active ingredient such as an antiallergic or antihistamine ingredient, a decongestant ingredient, a local anesthetic ingredient, a vitamin ingredient, and an amino acid ingredient other than an effective amino acid (eg, valine, Leucine, isoleucine, serine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, histidine, citrulline, ornithine, cystine, taurine, glycine) and the like. As such other active ingredients, various drugs known per se can be appropriately used.
　さらに本発明の眼科用組成物は、緩衝剤、等張化剤、保存剤、溶解補助剤、安定化剤、キレート剤、増粘剤、ｐＨ調整剤等の各種添加剤を適宜配合することができる。 Furthermore, the ophthalmic composition of the present invention may be appropriately mixed with various additives such as a buffer, an isotonic agent, a preservative, a solubilizer, a stabilizer, a chelating agent, a thickener, and a pH adjuster. it can.
　緩衝剤としては、例えば、ホウ酸またはその塩（ホウ砂等）、クエン酸またはその塩（クエン酸ナトリウム等）、酒石酸またはその塩（酒石酸ナトリウム等）、グルコン酸またはその塩（グルコン酸ナトリウム等）、酢酸またはその塩（酢酸ナトリウム等）、リン酸またはその塩（リン酸一水素ナトリウム、リン酸二水素ナトリウム等）、グルタミン酸やイプシロンアミノカプロン酸等の各種アミノ酸およびトリス緩衝剤等、またはそれらの組み合わせが挙げられる。 Examples of the buffer include boric acid or a salt thereof (borax, etc.), citric acid or a salt thereof (sodium citrate, etc.), tartaric acid or a salt thereof (sodium tartrate, etc.), gluconic acid or a salt thereof (sodium gluconate, etc.) ), Acetic acid or a salt thereof (sodium acetate, etc.), phosphoric acid or a salt thereof (sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), various amino acids such as glutamic acid or epsilon aminocaproic acid, and a tris buffer, etc. Combinations are listed.
　等張化剤としては、例えば、ソルビトール、マンニトール、グリセリン、プロピレングリコール、塩化ナトリウム、塩化カリウム等が挙げられる。 Examples of the isotonic agent include sorbitol, mannitol, glycerin, propylene glycol, sodium chloride, potassium chloride and the like.
　保存剤としては、例えば、パラオキシ安息香酸エステル類、塩化ベンザルコニウム、塩化ベンゼトニウム、ベンジルアルコール、ソルビン酸またはその塩、グルコン酸クロルヘキシジン、デヒドロ酢酸ナトリウム、塩化セチルピリジニウム、塩酸アルキルジアミノエチルグリシン、クロロブタノール等が挙げられる。 Examples of the preservative include paraoxybenzoates, benzalkonium chloride, benzethonium chloride, benzyl alcohol, sorbic acid or a salt thereof, chlorhexidine gluconate, sodium dehydroacetate, cetylpyridinium chloride, alkyldiaminoethylglycine hydrochloride, chlorobutanol Etc.
　溶解補助剤としては、例えば、ポリビニルピロリドン、ポリエチレングリコール、プロピレングリコール、ポリオキシエチレン硬化ヒマシ油６０、ステアリン酸ポリオキシル４０、ポリソルベート８０（商品名：Ｔｗｅｅｎ８０）等が挙げられる。 Examples of the solubilizer include polyvinyl pyrrolidone, polyethylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil 60, polyoxyl 40 stearate, polysorbate 80 (trade name: Tween 80), and the like.
　安定化剤としては、例えば、エデト酸ナトリウム、チオ硫酸ナトリウム、アスコルビン酸、シクロデキストリン、縮合リン酸またはその塩、亜硫酸塩、クエン酸またはその塩、ジブチルヒドロキシトルエン等が挙げられる。 Examples of the stabilizer include sodium edetate, sodium thiosulfate, ascorbic acid, cyclodextrin, condensed phosphoric acid or a salt thereof, sulfite, citric acid or a salt thereof, and dibutylhydroxytoluene.
　キレート剤としては、例えば、エデト酸ナトリウム、クエン酸ナトリウム、縮合リン酸またはその塩（縮合リン酸ナトリウム等）等が挙げられる。 Examples of the chelating agent include sodium edetate, sodium citrate, condensed phosphoric acid or a salt thereof (such as condensed sodium phosphate).
　増粘剤としては、例えば、メチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、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ナトリウム、ポリビニルピロリドン、ポリビニルアルコール、ポリエチレングリコール、ヒアルロン酸等が挙げられる。 Examples of the thickener include methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose sodium, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, hyaluronic acid and the like.
　ｐＨ調整剤としては、例えば、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、炭酸水素ナトリウム、ホウ酸またはその塩（ホウ砂）、塩酸、クエン酸またはその塩（クエン酸ナトリウム、クエン酸二水素ナトリウム等）、リン酸またはその塩（リン酸水素二ナトリウム、リン酸二水素カリウム等）、酢酸またはその塩（酢酸ナトリウム、酢酸アンモニウム等）、酒石酸またはその塩（酒石酸ナトリウム等）等が挙げられる。 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.
　本発明の眼科用組成物のｐＨは、３～１０、好ましくはｐＨ５～８となるように調整される。 The pH of the ophthalmic composition of the present invention is adjusted to 3 to 10, preferably 5 to 8.
　本発明の眼科用組成物は、眼痛、開眼困難、異物感、疲労感等の軽減、ならびに視力、結膜充血、角膜上皮の状態の改善などに優れた効果を発揮する。従って、本発明の眼科用組成物は、眼表面疾患、特に角結膜上皮障害の予防・治療剤として有用である。
　角結膜上皮障害としては、例えば、乾燥によって引き起こされる角膜上皮障害（例えば、涙液減少症、眼乾燥症、マイボーム腺機能不全、シェーグレン症候群、乾性角結膜炎、眼瞼炎、スティーブンス－ジョンソン症候群、ＶＤＴ作業に関連したドライアイ等のドライアイ症候群、または、ドライアイに伴う角結膜上皮障害、瘢痕性角結膜上皮症、角膜上皮糜爛、角膜潰瘍、眼瞼縁炎、眼類天疱瘡、春季カタル、アレルギー性結膜炎等）のほか、紫外線による角膜上皮障害（例えば、角膜炎、雪目等）、化学物質による化学外傷、点眼剤中に含まれる薬物（例えば、保存剤、点眼麻酔薬、プロスタグランジン、アミノグリコシド系抗生物質、β遮断薬、抗真菌薬、抗ウイルス薬等）起因性の角膜上皮障害（例えば、点状表層角膜炎、角膜潰瘍等）などが挙げられる。 The ophthalmic composition of the present invention exhibits excellent effects such as reduction of eye pain, difficulty in opening the eye, foreign body sensation, fatigue, etc., as well as improvement in visual acuity, conjunctival hyperemia, corneal epithelial state, and the like. Therefore, the ophthalmic composition of the present invention is useful as a prophylactic / therapeutic agent for ocular surface diseases, particularly keratoconjunctival epithelial disorders.
Examples of keratoconjunctival epithelial disorders include corneal epithelial disorders caused by desiccation (eg, tear reduction, dry eye, meibomian gland dysfunction, Sjogren's syndrome, dry keratoconjunctivitis, blepharitis, Stevens-Johnson syndrome, VDT) Dry eye syndrome such as dry eye associated with work, or keratoconjunctival epithelial disorder associated with dry eye, scarring keratoconjunctivitis, corneal epithelial fistula, corneal ulcer, blepharitis, pemphigoid, spring catarrh, allergy Conjunctivitis, etc.), corneal epithelial damage caused by ultraviolet rays (eg, keratitis, snow eyes, etc.), chemical trauma caused by chemicals, drugs contained in eye drops (eg, preservatives, eye anesthetics, prostaglandins, Corneal epithelial disorder (for example, punctate superficial keratitis, corneal ulcer) caused by aminoglycoside antibiotics, β-blockers, antifungals, antivirals, etc. ), And the like.
　本発明の眼科用組成物はさらに、目の疲れ、目のかわき、目のかすみ、目のかゆみ、結膜充血、コンタクトレンズ着用時の不快感等の予防または改善するための点眼用組成物としても用いることができる。 The ophthalmic composition of the present invention can also be used as an ophthalmic composition for preventing or improving eye fatigue, eye irritation, blurred vision, itchy eyes, conjunctival hyperemia, and discomfort when wearing contact lenses. Can be used.
　本発明の眼科用組成物は、ヒト、ヒト以外の動物〔例えば、ヒト以外の哺乳類（ブタ、ウシ、ウマ、イヌ等の家畜および愛玩動物）等〕における上記疾患または状態を予防・治療するために用いることができる。 The ophthalmic composition of the present invention is used for preventing or treating the above-mentioned diseases or conditions in humans and animals other than humans (for example, mammals other than humans (domestic animals such as pigs, cows, horses, dogs, and pets)). Can be used.
　本発明の眼科用組成物の投与量は、剤形、対象疾患、投与対象の動物種、年齢、性別、体重、症状などに依存して変化し得るが、例えば、成人（例えば、体重６０ｋｇ）の場合、１日あたり、組成物中のアミノ酸総量に換算して、通常０．１μｇ～２．０ｇの範囲であり、好ましくは０．５μｇ～１．８ｇ、さらに好ましくは１．０μｇ～１．５ｇの範囲である。 The dosage of the ophthalmic composition of the present invention may vary depending on the dosage form, target disease, animal species to be administered, age, sex, weight, symptoms, etc., for example, adult (for example, 60 kg body weight) In this case, it is usually in the range of 0.1 μg to 2.0 g, preferably 0.5 μg to 1.8 g, more preferably 1.0 μg to 1.g in terms of the total amount of amino acids in the composition per day. The range is 5 g.
　本発明の眼科用組成物の１日あたりの用量は、一度にもしくは数回に分けて投与することができる。例えば、点眼剤として使用する場合には、本発明の眼科用組成物を、重症ドライアイ患者に対しては１日あたり５～２０回、好ましくは１６～１８回（概ね、一時間に１回）、中軽症ドライアイ患者に対しては１日あたり２～１０回、好ましくは３～７回投与する。１回あたりの投与量としては、一つの眼あたり１～３滴（５０～１５０μＬ）、好ましくは１～２滴（５０～１００μＬ）投与することができる。また投与期間は特に限定されない。 The daily dose of the ophthalmic composition of the present invention can be administered once or divided into several times. For example, when used as an eye drop, the ophthalmic composition of the present invention is used 5 to 20 times, preferably 16 to 18 times per day (generally once an hour) for severe dry eye patients. ), 2 to 10 times, preferably 3 to 7 times a day for patients with mild dry eye. The dose per administration is 1 to 3 drops (50 to 150 μL), preferably 1 to 2 drops (50 to 100 μL) per eye. The administration period is not particularly limited.
　本明細書および図面において、アミノ酸などを略号で表示する場合、ＩＵＰＡＣ－ＩＵＢ　Ｃｏｍｍｉｓｓｉｏｎ　ｏｎ　Ｂｉｏｃｈｅｍｉｃａｌ　Ｎｏｍｅｎｃｌａｔｕｒｅによる略号あるいは当該分野における慣用略号に基づくものであり、その例を下記する。またアミノ酸に関し光学異性体があり得る場合は、特に明示しなければＬ体を示すものとする。 In the present specification and drawings, amino acids and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
　Ｇｌｙ　　　　：グリシン
　Ａｌａ　　　　：アラニン
　Ｖａｌ　　　　：バリン
　Ｌｅｕ　　　　：ロイシン
　Ｉｌｅ　　　　：イソロイシン
　Ｓｅｒ　　　　：セリン
　Ｔｈｒ　　　　：スレオニン
　Ｃｙｓ　　　　：システイン
　Ｍｅｔ　　　　：メチオニン
　Ｇｌｕ　　　　：グルタミン酸
　Ａｓｐ　　　　：アスパラギン酸
　Ｌｙｓ　　　　：リジン
　Ａｒｇ　　　　：アルギニン
　Ｈｉｓ　　　　：ヒスチジン
　Ｐｈｅ　　　　：フェニルアラニン
　Ｔｙｒ　　　　：チロシン
　Ｔｒｐ　　　　：トリプトファン
　Ｐｒｏ　　　　：プロリン
　Ａｓｎ　　　　：アスパラギン
　Ｇｌｎ　　　　：グルタミン
　Ｏｒｎ　　　　：オルニチン
　Ｃｉｔ　　　　：シトルリン
　(Ｃｙｓ)２　　 ：シスチン
　Ｔａｕ　　　　：タウリン Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys Argin P : Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine Orn: Ornithine Cit: Citrulline (Cys) 2 : Cystine Tau: Taurine
　以下に、実施例および試験例を挙げて、本発明を具体的に説明するが、本発明はこれらによって限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples and test examples, but the present invention is not limited thereto.
[参考例]　涙液中のアミノ酸含量の測定
　健常者とドライアイ患者の間、および健常者のＶＤＴ作業前とＶＤＴ作業後の間で、被験者の涙液中に含有されるアミノ酸量の比較検討を行った。
　健常者、ドライアイ患者および健常者のＶＤＴ作業前とＶＤＴ作業後（作業時間２時間）の各被験者から、毛細管を用いて眼から０．５μＬの涙液を採取した。得られた涙液を蒸留水で１０倍に希釈後、４倍量のアセトニトリルを添加し、振とう撹拌を行った。撹拌後、該混合液を１０，０００Ｇで１分間、高速遠心した。上清を採取後、凍結保存することでサンプルとした。解凍後のサンプル各５μＬに、内部標準物質（濃度既知のアミノ酸）５μＬ、ホウ酸緩衝液３０μＬを加えた。サンプル中のアミノ酸のアミノ基を標識するために、アミノ基誘導体化試薬（ｐ‐トリメチルアンモニウムアリニル‐Ｎ－ヒドロキシスクシンイミジカルバメートアイオダイド）１０μＬを加え、５５℃で１０分間保温した。その後、０．２％酢酸５０μＬを加えて反応を終結させた。高速液体クロマトグラフ‐タンデム質量分析計（ＡＰＩ４０００ＬＣ／ＭＳ／ＭＳ　Ｓｙｓｔｅｍ，Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）によって、涙液中のアミノ酸含量を分析した。測定の対象としたアミノ酸は、グリシン、アラニン、バリン、ロイシン、イソロイシン、フェニルアラニン、プロリン、セリン、スレオニン、チロシン、アスパラギン、グルタミン、リシン、アルギニン、トリプトファン、ヒスチジン、メチオニン、アスパラギン酸、グルタミン酸、オルニチン、シトルリン、シスチン、タウリンである。
　健常者およびドライアイ患者の各被験者の涙液中の測定対象となったアミノ酸総量を１００とした場合、各アミノ酸の構成比率を計算した。結果を表１に示す。
　また、健常者の安静時涙液と２時間ＶＤＴ作業を行い、仮性ドライアイ状態にしたときの涙液中のそれぞれの各アミノ酸濃度（μＭ）を測定した。結果を表２に示す。 [Reference Example] Measurement of amino acid content in tear fluid Comparison of the amount of amino acids contained in the tear fluid of subjects between healthy subjects and dry eye patients, and before and after VDT work of healthy subjects Went.
From each subject before and after VDT work (working time 2 hours) of healthy subjects, dry eye patients, and healthy subjects, 0.5 μL of tear fluid was collected from the eyes using capillaries. The obtained tear was diluted 10 times with distilled water, 4 times the amount of acetonitrile was added, and the mixture was shaken and stirred. After stirring, the mixture was centrifuged at 10,000 G for 1 minute. After collecting the supernatant, the sample was frozen and stored. To 5 μL of each sample after thawing, 5 μL of an internal standard substance (amino acid with a known concentration) and 30 μL of borate buffer were added. In order to label the amino group of the amino acid in the sample, 10 μL of an amino group derivatization reagent (p-trimethylammonium alinyl-N-hydroxysuccinimidibacarbide iodide) was added and incubated at 55 ° C. for 10 minutes. Thereafter, 50 μL of 0.2% acetic acid was added to terminate the reaction. Amino acid content in tears was analyzed by high performance liquid chromatograph-tandem mass spectrometer (API4000LC / MS / MS System, Applied Biosystems). The target amino acids were glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, asparagine, glutamine, lysine, arginine, tryptophan, histidine, methionine, aspartic acid, glutamic acid, ornithine, citrulline. Cystine, taurine.
Assuming that the total amount of amino acids to be measured in tears of each subject of healthy subjects and dry eye patients was 100, the composition ratio of each amino acid was calculated. The results are shown in Table 1.
In addition, a 2-hour VDT operation was carried out with a resting tear fluid of a healthy person, and each amino acid concentration (μM) in the tear fluid when a temporary dry eye state was obtained was measured. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　上記の測定結果から、ドライアイ患者の涙液では、健常者の涙液に比してＧｌｎ、Ａｒｇ、Ａｓｐの比率が有意に低下していることが分かった。さらに、ドライアイ患者の涙液では、健常者の涙液に比してＯｒｎ、Ｔａｕ等の比率が上昇していることも分かった。また、ＶＤＴ作業によるドライアイ状態の涙液では、安静時に比してＡｓｎ、Ａｌａ、Ｔａｕが有意に低下していることが分かった。 From the above measurement results, it was found that the ratio of Gln, Arg, Asp in the tears of dry eye patients was significantly lower than that of healthy subjects. Furthermore, it was also found that the ratio of Orn, Tau, etc. increased in tears from dry eye patients compared to tears from healthy subjects. It was also found that Asn, Ala, and Tau were significantly reduced in dry eye tears by VDT work compared to rest.
［２×Ｚｅｒｏ培地の調製］
　二段蒸留水４００ｍＬにＺｅｒｏ培地粉末[ ＣａＣｌ２(ａｎｈｙｄ) ２００ｍｇ，　Ｆｅ(ＮＯ３)３・９Ｈ２Ｏ　０．１ｍｇ，　ＫＣｌ　４００ｍｇ，　ＭｇＳＯ４(ａｎｈｙｄ) ９７．６７ｍｇ，　ＮａＣｌ　６４００ｍｇ，　ＮａＨＰＯ４・Ｈ２Ｏ　１２５ｍｇ，　Ｓｏｄｉｕｍ　ｐｙｒｕｖａｔｅ　１１０ｍｇ，　Ｄ－Ｃａ　ｐａｎｔｏｔｈｅｎａｔｅ　４ｍｇ，　Ｃｈｏｌｉｎｅ　Ｃｈｌｏｒｉｄｅ　４ｍｇ，　Ｆｏｌｉｃ　ａｃｉｄ　４ｍｇ，　Ｉ－ｉｎｏｓｉｔｏｌ　７．２ｍｇ，　Ｎｉａｃｉｎａｍｉｄｅ　４ｍｇ，　Ｒｉｂｏｆｌａｖｉｎ　０．４ｍｇ，　Ｔｈｉａｍｉｎｅ　ＨＣｌ　４ｍｇ，　Ｐｙｒｉｄｏｘｉｎｅ　ＨＣｌ　４ｍｇ]を溶解させ、さらにＮａＨＣＯ３　３．７ｇおよびＧｌｕｃｏｓｅ　１ｇを添加して溶解させた。培地をＨＣｌ（塩酸）でｐＨ７．４に調整し、二段蒸留水で５００ｍＬにまでメスアップした。その後、０．２２μｍ滅菌フィルターで滅菌濾過した後、４℃で保存した。
［アミノ酸含有培地の調製］
　上記の２×Ｚｅｒｏ培地に適宜アミノ酸粉末を添加して溶解させた後、０．２２μｍ滅菌フィルターにて滅菌処理し、４℃保存した。例えば、全２０種のアミノ酸を含有する培地（以下、Ｆｕｌｌ培地と表記）の場合、２×Ｚｅｒｏ培地（５０ｍＬ）にＦｕｌｌ培地粉末［Ｇｌｙｃｉｎｅ　３．０ｍｇ，　Ｌ-Ａｌａｎｉｎｅ　３．５６ｍｇ，　Ｌ-Ｓｅｒｉｎｅ　４．２ｍｇ，　Ｌ-Ｔｈｒｅｏｎｉｎｅ　９．５ｍｇ，　Ｌ-Ｃｙｓｔｉｎｅ　２ＨＣｌ　６．３ｍｇ，　Ｌ-Ｍｅｔｈｉｏｎｉｎｅ　３．０ｍｇ，　Ｌ-Ｇｌｕｔａｍｉｎｅ　５８．４ｍｇ，　Ｌ-Ａｓｐａｒａｇｉｎｅ　５．２８ｍｇ，　Ｌ-Ｇｌｕｔａｍｉｃ　Ａｃｉｄ ５．８８ｍｇ，　Ｌ-Ａｓｐａｒｔｉｃ　Ａｃｉｄ　５．３２ｍｇ，　Ｌ-Ｖａｌｉｎｅ　９．４ｍｇ，　Ｌ-Ｌｅｕｃｉｎｅ　１０．５ｍｇ，　Ｌ-Ｉｓｏｌｅｕｃｉｎｅ　１０．５ｍｇ，　Ｌ-Ｐｈｅｎｙｌａｌａｎｉｎｅ　６．６ｍｇ，　Ｌ-Ｔｙｒｏｓｉｎｅ・２Ｎａ・２Ｈ２Ｏ　１０．４ｍｇ，　Ｌ-Ｔｒｙｐｔｏｐｈａｎ　１６ｍｇ，　Ｌ-Ｌｙｓｉｎｅ-ＨＣｌ　１４．６ｍｇ，　Ｌ-Ａｒｇｉｎｉｎｅ-ＨＣｌ　８．４ｍｇ，　Ｌ-Ｈｉｓｔｉｄｉｎｅ　ＨＣｌ-Ｈ２Ｏ　４．２ｍｇ，　Ｌ-Ｐｒｏｌｉｎｅ　４．６ｍｇ］を溶解させ、滅菌水で１００ｍＬまでメスアップした後、０．２２μｍ滅菌フィルターにて滅菌処理し、４℃保存することで調製した。一部のアミノ酸が欠損したアミノ酸含有培地についても、上記と同様に必要なアミノ酸粉末を２×Ｚｅｒｏ培地に溶解させるか、あるいは、前もって調製したアミノ酸溶液を無菌的に添加することで調製した。必須アミノ酸と１～４種の非必須アミノ酸を含む添加培地についても、上記と同様に必要なアミノ酸粉末を２×Ｚｅｒｏ培地に溶解させるか、あるいは、前もって調整したアミノ酸溶液を無菌的に添加することで調整した。 [Preparation of 2 × Zero medium]
Zero medium powder in double distilled water 400mL [CaCl 2 (anhyd) 200mg , Fe (NO 3) 3 · 9H 2 O 0.1mg, KCl 400mg, MgSO 4 (anhyd) 97.67mg, NaCl 6400mg, NaHPO 4 · H 2 O 125 mg, Sodium pyruvate 110 mg, D-Capantheneate 4 mg, Choline Chloride 4 mg, Folic acid 4 mg, I-inositol 7.2 mg, Niacinide 4 mg, Riboflavin 4 mg, Riboflavin 4 NaHCO 3 3.7 g and Glucose 1 g were added and dissolved. The medium was adjusted to pH 7.4 with HCl (hydrochloric acid) and diluted to 500 mL with double distilled water. Then, after sterilizing filtration with a 0.22 μm sterilizing filter, it was stored at 4 ° C.
[Preparation of amino acid-containing medium]
Amino acid powder was appropriately added to the 2 × Zero medium and dissolved, and then sterilized with a 0.22 μm sterilizing filter and stored at 4 ° C. For example, in the case of a medium containing all 20 types of amino acids (hereinafter referred to as a Full medium), 2 × Zero medium (50 mL) is filled with Full medium powder [Glycine 3.0 mg, L-Aline 3.56 mg, L-Series 4 .2 mg, L-Throneine 9.5 mg, L-Cystein 2HCl 6.3 mg, L-Methionine 3.0 mg, L-Glutamine 58.4 mg, L-Asparagine 5.28 mg, L-Glutamate Acid-Asp. Acid 5.32mg, L-Valine 9.4mg, L-Leucine 10.5mg, L-isoleucine 10.5mg, L-Phenylalanine 6.6mg, L-Tyrosine · 2Na · 2H 2 O 10.4 g, L-Tryptophan 16mg, L -Lysine-HCl 14.6mg, L-Arginine-HCl 8.4mg, L-Histidine HCl-H 2 O 4.2mg, dissolved L-Proline 4.6mg], sterile water And then sterilized with a 0.22 μm sterilizing filter and stored at 4 ° C. An amino acid-containing medium lacking some amino acids was also prepared by dissolving the necessary amino acid powder in 2 × Zero medium in the same manner as described above, or by aseptically adding a previously prepared amino acid solution. For the supplemented medium containing essential amino acids and 1 to 4 non-essential amino acids, dissolve the necessary amino acid powder in 2 × Zero medium in the same manner as above, or aseptically add a prepared amino acid solution. Adjusted.
［実験例１］　ｉｎ　ｖｉｔｒｏにおけるヒト角膜上皮細胞増殖に対するアミノ酸の効果の検討－１
　５μｇ／ｍＬインスリン（ＧＩＢＣＯ）、０．１μｇ／ｍＬコレラトキシン（ＣＡＬＢＩＯＣＨＥＭ）および５μＬ／ｍＬペニシリン‐ストレプトマイシン溶液（ナカライテスク）を含むＤＭＥＭ／Ｆ１２（ＧＩＢＣＯ）を用いて、ＳＶ４０不死化ヒト角膜上皮細胞（ＨＣＥＴＣＯ）の予備培養を５％ＣＯ２雰囲気下、３７℃で２日間行った。予備培養後、該培養細胞を０．２５％トリプシンを用いて回収し、９６ウェルプレート（Ｃｏｒｎｉｎｇ）に２×１０４ｃｅｌｌｓ／ｗｅｌｌで播種し、ＥｐｉＬｉｆｅ培地を用いて、５％ＣＯ２雰囲気下、３７℃で２日間培養した。その後、Ｚｅｒｏ培地に培地交換し、５％ＣＯ２雰囲気下、３７℃で１日培養した。続いて、各検体培地に再度培地交換し、３日間培養を続けた（５％ＣＯ２、３７℃）。各検体培地のアミノ酸組成を欠損培地については表３、添加培地については表４に示す。 [Experimental Example 1] Examination of the effect of amino acids on proliferation of human corneal epithelial cells in vitro-1
SV40 immortalized human corneal epithelial cells (GIBCO) using DMEM / F12 (GIBCO) containing 5 μg / mL insulin (GIBCO), 0.1 μg / mL cholera toxin (CALBIOCHEM) and 5 μL / mL penicillin-streptomycin solution (Nacalai Tesque) HCETCO) was pre-cultured at 37 ° C. for 2 days in a 5% CO 2 atmosphere. After the preculture, the cultured cells were collected using 0.25% trypsin, seeded in a 96-well plate (Corning) at 2 × 10 4 cells / well, and EpiLife medium was used in a 5% CO 2 atmosphere. The cells were cultured at 37 ° C. for 2 days. Thereafter, the medium was replaced with Zero medium, and cultured at 37 ° C. for 1 day in a 5% CO 2 atmosphere. Subsequently, the medium was changed again to each specimen medium, and the culture was continued for 3 days (5% CO 2 , 37 ° C.). The amino acid composition of each specimen medium is shown in Table 3 for the deficient medium and Table 4 for the supplemented medium.
　培養３日目の時点において細胞増殖を観測した。具体的には、２．５％グルタルアルデヒドで１５分間処理することで細胞を固定し、０．５％メチレンブルーで１５分間処理することで細胞を染色した。メチレンブルーを抽出するため、０．３３Ｎ塩酸を２００μＬ／ｗｅｌｌで添加し、撹拌機上で撹拌した。６３０ｎｍの吸光度を測定することで細胞増殖を分析した。吸光度の測定結果を欠損培地については表３に、添加培地については表４にそれぞれ示す。なお、表３及び表４に示した吸光度は、各培地についてそれぞれ６ｗｅｌｌ（ｎ＝６）培養して吸光度を測定し、その平均値を示した。 Cell proliferation was observed at the third day of culture. Specifically, the cells were fixed by treatment with 2.5% glutaraldehyde for 15 minutes, and the cells were stained by treatment with 0.5% methylene blue for 15 minutes. In order to extract methylene blue, 0.33N hydrochloric acid was added at 200 μL / well and stirred on a stirrer. Cell proliferation was analyzed by measuring absorbance at 630 nm. The absorbance measurement results are shown in Table 3 for the deficient medium and Table 4 for the supplemented medium. In addition, the light absorbency shown in Table 3 and Table 4 measured the light absorbency after culturing 6well (n = 6) about each culture medium, respectively, and showed the average value.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
［実験例２］　ｉｎ　ｖｉｔｒｏにおけるヒト角膜上皮細胞増殖に対するアミノ酸の効果の検討－２
　検体培地の処方を変更した以外は、ｉｎ　ｖｉｔｒｏにおけるヒト角膜上皮細胞増殖に対するアミノ酸の効果の検討―１と同様の操作を行った。検体培地の処方及び吸光度の測定結果を表５に示す。 [Experimental Example 2] Examination of the effect of amino acids on human corneal epithelial cell proliferation in vitro-2
The same procedure as in Examination of the effect of amino acids on human corneal epithelial cell proliferation in vitro was performed except that the formulation of the sample medium was changed. Table 5 shows the formulation of the sample medium and the measurement results of the absorbance.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　上記実験例１及び実験例２の結果、ＡｒｇがＦｕｌｌ培地から欠損した場合に、その他のアミノ酸が欠損した場合と比較して著しくヒト角膜上皮細胞の増殖が抑制されていることが分かった。また、表５から必須アミノ酸培地にグルタミンとアルギニン、アスパラギンとアラニン、アスパラギンとアラニンとグルタミン、アスパラギンとアラニンとアルギニン、アラニンとグルタミンとアルギニン、アルギニンとアスパラギンとグルタミン、およびアスパラギンとアラニンとグルタミンとアルギニンの組み合わせで添加した場合、必須アミノ酸培地にＴａｕを添加したものと同程度以上の細胞増殖効果が得られることが分かった。なお、ｉｎ　ｖｉｔｒｏにおける系では、培地中に含まれるアミノ酸の濃度の総和は、約１８ｍＭ未満であるのが好ましい。約１８ｍＭよりアミノ酸の総濃度が高い培地中で細胞培養を行った場合には、アミノ酸添加の効果よりも、細胞毒性が強く発現する傾向にある。 As a result of Experimental Example 1 and Experimental Example 2, it was found that when Arg was deficient from the Full medium, the growth of human corneal epithelial cells was remarkably suppressed as compared with the case where other amino acids were deleted. From Table 5, the essential amino acid medium contains glutamine and arginine, asparagine and alanine, asparagine and alanine and glutamine, asparagine and alanine and arginine, alanine and glutamine and arginine, arginine and asparagine and glutamine, and asparagine and alanine, glutamine and arginine. When added in combination, it was found that a cell growth effect equal to or higher than that obtained by adding Tau to the essential amino acid medium was obtained. In an in vitro system, the total concentration of amino acids contained in the medium is preferably less than about 18 mM. When cell culture is performed in a medium having a total concentration of amino acids higher than about 18 mM, cytotoxicity tends to be expressed more strongly than the effect of amino acid addition.
［実験例３］　創傷治癒に対するアミノ酸の効果の検討
　５μｇ／ｍＬインスリン（ＧＩＢＣＯ）、０．１μｇ／ｍＬコレラトキシン（ＣＡＬＢＩＯＣＨＥＭ）および５μＬ／ｍＬペニシリン‐ストレプトマイシン溶液（ナカライテスク）を含むＤＭＥＭ／Ｆ１２（ＧＩＢＣＯ）を用いて、ＳＶ４０不死化ヒト角膜上皮細胞（ＨＣＥＴＣＯ）の予備培養を５％ＣＯ２雰囲気下、３７℃で２日間行った。予備培養後、該培養細胞を０．２５％トリプシンを用いて回収し、６ウェルプレート（Ｃｏｒｎｉｎｇ）に５×１０４ｃｅｌｌｓ／ｗｅｌｌで播種し、ＥｐｉＬｉｆｅ培地を用いて、５％ＣＯ２雰囲気下、３７℃で２週間培養した。その後、Ｚｅｒｏ培地に培地交換し、５％ＣＯ２雰囲気下、３７℃で１日間培養した。コンフルエント状態でマイクロピペットチップ（１０００μＬ）の先端にて細胞を直線状に擦過し、創傷モデルを作製した。創傷作製後、検体培地に交換し、４８時間培養を行った。創傷箇所の回復状態を位相差顕微鏡を用いて検討した。具体的には、創傷幅の測定をＩｍａｇｅＪイメージ解析ソフト（ＮＩＨ）を用いて行った。各群については合計６箇所の測定を行った。検体培地の処方及び、０ｈｒにおける創傷幅を１００とした４８時間後の創傷幅の割合（創傷治癒改善率）及び、４８時間後のＦｕｌｌ培地における創傷治癒改善率を１００とし、４８時間後のＺｅｒｏ培地における創傷治癒率を０とした場合の各培地における４８時間後の創傷治癒改善率の相対比（創傷治癒相対比）を表６に示す。 [Experimental Example 3] Examination of effect of amino acid on wound healing DMEM / F12 (GIBCO) containing 5 μg / mL insulin (GIBCO), 0.1 μg / mL cholera toxin (CALBIOCHEM) and 5 μL / mL penicillin-streptomycin solution (Nacalai Tesque) ) Was used for preculture of SV40 immortalized human corneal epithelial cells (HCETCO) at 37 ° C. for 2 days in a 5% CO 2 atmosphere. After the preculture, the cultured cells were collected using 0.25% trypsin, seeded in a 6-well plate (Corning) at 5 × 10 4 cells / well, and EpiLife medium was used in a 5% CO 2 atmosphere. The cells were cultured at 37 ° C. for 2 weeks. Thereafter, the medium was changed to Zero medium, and cultured at 37 ° C. for 1 day in a 5% CO 2 atmosphere. In the confluent state, cells were scraped linearly with the tip of a micropipette tip (1000 μL) to prepare a wound model. After wound preparation, the specimen medium was replaced and cultured for 48 hours. The recovery state of the wound site was examined using a phase contrast microscope. Specifically, wound width was measured using ImageJ image analysis software (NIH). A total of 6 measurements were made for each group. The prescription of the specimen medium, the ratio of the wound width after 48 hours (wound healing improvement rate) when the wound width at 0 hr was 100, and the wound healing improvement rate in Full medium after 48 hours was 100, and Zero after 48 hours Table 6 shows the relative ratios of wound healing improvement rates after 48 hours in each medium when the wound healing rate in the medium is 0 (relative ratio of wound healing).
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　その結果、Ｆｕｌｌ培地からアルギニンを欠損させた培地では、グルタミン及びグルタミン酸の２種を欠損させた培地に比べて創傷治癒率が顕著に低減していることが分かった。すなわち、角膜上皮損傷治癒に対してもアルギニンの寄与が高いことが示唆された。 As a result, it was found that the wound healing rate was significantly reduced in the medium lacking arginine from the Full medium as compared to the medium lacking two kinds of glutamine and glutamic acid. That is, it was suggested that arginine contributes to corneal epithelial damage healing.
［実施例１］　アミノ酸含有点眼液の調製
　各アミノ酸含有点眼液およびコントロールとして使用したアミノ酸非含有点眼液の処方を表７に示す。 [Example 1] Preparation of amino acid-containing ophthalmic solution Table 7 shows the formulation of each amino acid-containing ophthalmic solution and an amino acid-free ophthalmic solution used as a control.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
［試験例１］　アミノ酸含有点眼液の安全性確認試験
　Ｄｒａｉｚｅ法（Ｊ．Ｈ．Ｄｒａｉｚｅ，Ｇ．Ｗｏｏｄａｒｄ　ａｎｄ　Ｈ．Ｏ．Ｃａｌｖｅｒｙ(１９４４):ｍｅｔｈｏｄｓ　ｆｏｒ　ｔｈｅ　ｓｔｕｄｙ　ｏｆ　ｉｒｒｉｔａｔｉｏｎ　ａｎｄ　ｔｏｘｉｃｉｔｙ　ｏｆ　ｓｕｂｓｔａｎｃｅｓ　ａｐｐｌｉｅｄ　ｔｏｐｉｃａｌｌｙ　ｔｏ　ｔｈｅ　ｓｋｉｎ　ａｎｄ　ｍｕｃｏｕｓ　ｍｅｍｂｒａｎｅｓ．Ｊ．Ｐｈａｒｍａｃｏｌ．Ｅｘｐ．Ｔｈｅｒ．，８２，３７７－３９０）に従い、日本白色種雄ウサギ９羽を用い、実施例１で調製した各アミノ酸含有点眼液およびアミノ酸非含有点眼液を結膜嚢内へ７日間点眼投与し、その眼刺激性を検討した。
　点眼はそれぞれ左眼に対してのみ行い、右眼は無処置対照とした。点眼投与は１００μＬ／眼を1時間ごとに８回／日行った。点眼投与直後に約一秒間閉眼し、瞬目の有無を観察した。投与後の洗眼は実施しなかった。眼反応は、各投与日における最終投与後にＤｒａｉｚｅ法による判定基準に従ってスリットランプを用いて観察した。この操作を７日間繰り返した。また、点眼投与開始前および最終投与日の投与後には角膜障害度検査を行った。 [Test Example 1] Safety confirmation test of amino acid-containing ophthalmic solution Draize method (J. H. Draize, G. Woodard and H. O. Calvery (1944): methods for the study of toxicology of toxicology. In accordance with skin and mucous members. J. Pharmacol. Exp. Ther., 82, 377-390), each amino acid-containing ophthalmic solution and amino acid-free ophthalmic solution prepared in Example 1 were used as conjunctiva. Intraocular administration for 7 days was conducted, and the eye irritation was examined.
Instillation was performed only on the left eye, and the right eye served as an untreated control. Intraocular administration was performed 8 times / day at 100 μL / eye every hour. Immediately after instillation, the eyes were closed for about 1 second, and the presence or absence of blinks was observed. No eye wash was performed after administration. The ocular reaction was observed using a slit lamp according to the criteria of the Draize method after the final administration on each administration day. This operation was repeated for 7 days. In addition, a corneal disorder degree test was performed before the start of eye drop administration and after administration on the last administration day.
　以下の項目について評価を行った。
（１）一般状態
　各投与日の最終投与後に一日一回、全被験対象について一般状態を観察した。
（２）体重測定
　投与開始日および実験終了日（投与７日目）に電子天秤（ＰＭ１１－Ｋ，メトラー・メレド株式会社）を用いて全被験対象について測定した。
（３）眼反応の観察
　各投与日の最終投与後（８回目）にスリットランプを用いて両眼を表８に示す基準に従って観察した。角膜障害度検査として投与７日後の投与終了後、全被験対象について両眼をフルオレセインナトリウム（フローレス試験紙（登録商標））による角膜染色を実施し、角膜の損傷の有無をスリットランプにて観察した。 The following items were evaluated.
(1) General condition General condition was observed about all test subjects once a day after the last administration of each administration day.
(2) Body weight measurement All subjects were measured using an electronic balance (PM11-K, METTLER MELED Co., Ltd.) on the administration start date and the experiment end date (administration day 7).
(3) Observation of ocular reaction Both eyes were observed according to the criteria shown in Table 8 using a slit lamp after the final administration (8th time) on each administration day. After completion of administration 7 days after administration as a corneal disorder test, both eyes were subjected to corneal staining with fluorescein sodium (Flowless Test Paper (registered trademark)) for all subjects, and the presence or absence of corneal damage was observed with a slit lamp. .
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　以下に結果を示す。
［アミノ酸含有点眼液１］
　投与１日より投与７日目まで観察した結果、角膜、虹彩および結膜に異常は認められなかった。しかし、投与直後に行った瞬目の観察では、アミノ酸含有点眼液１投与群のうち、一個体のみで投与３日目の２、３回目および投与６日目の３回目に瞬目が観察された。他の個体では瞬目は観察されなかった。この群の合計評価点はいずれも０であり、眼刺激性の評価区分は無刺激物に該当した。
［アミノ酸含有点眼液２］
　投与１日より投与７日目まで観察した結果、角膜、虹彩および結膜に異常は認められなかった。また、投与直後の瞬目は一度も観察されなかった。この群の合計評点はいずれも０であり、眼刺激性の評価区分は無刺激物に該当した。
［アミノ酸非含有点眼液］
　投与１日目より投与７日目まで観察した結果、角膜、虹彩および結膜に異常は認められなかった。また、投与直後の瞬目は一度も認められなかった。この群の合計評点はいずれも０であり、眼刺激性の評価区分は無刺激物に該当した。
　以上より、アミノ酸配合点眼液１、および２はいずれも眼刺激性がないものと判断できた。 The results are shown below.
[Amino acid-containing ophthalmic solution 1]
As a result of observation from day 1 to day 7 of administration, no abnormality was observed in the cornea, iris and conjunctiva. However, in the observation of blinks performed immediately after administration, blinks were observed in the second and third times on the third day of administration and the third time on the sixth day of administration in only one individual in the amino acid-containing ophthalmic solution 1 administration group. It was. No blink was observed in other individuals. The total evaluation score of this group was 0, and the evaluation category of eye irritation corresponded to non-irritating substances.
[Amino acid-containing ophthalmic solution 2]
As a result of observation from day 1 to day 7 of administration, no abnormality was observed in the cornea, iris and conjunctiva. Moreover, the blink immediately after administration was never observed. The total score of this group was 0, and the evaluation category of eye irritation corresponded to non-irritating substances.
[Amino acid-free ophthalmic solution]
As a result of observation from the first day of administration to the seventh day of administration, no abnormality was observed in the cornea, iris and conjunctiva. In addition, no blinks were observed immediately after administration. The total score of this group was 0, and the evaluation category of eye irritation corresponded to non-irritating substances.
From the above, it can be determined that both the amino acid-containing ophthalmic solutions 1 and 2 have no eye irritation.
［実施例２］　点眼剤としての本発明の眼科用組成物の製造試験
　下表９に示す通り、各アミノ酸を配合する点眼剤を作製する。 [Example 2] Production test of ophthalmic composition of the present invention as an eye drop As shown in Table 9 below, an eye drop containing each amino acid is prepared.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
［試験例２］　アミノ酸を含有した点眼剤を用いた臨床試験
　実施例２に記載の各製造例のアミノ酸含有点眼剤を被験者に投与し、投与後の被験者の自覚症状または他覚所見について被験者間での比較を行う。具体的には、各被験者の片眼に製造例１、２、４および５のいずれかの点眼剤を投与し、対照として他眼に製造例１０の点眼剤を投与する。点眼投与は健常者および重症ドライアイ患者には、１回１～２滴（５０～１００μＬ）／眼を１時間ごとに行う。１日当たりの投与回数は１６～１８回である。中軽症ドライアイ患者およびＶＤＴ作業者には、１回１～２滴（５０～１００μＬ）／眼を４～６回／日投与する。なお、上記重症ドライアイ患者は、シルマー試験Ｉ法の値が５ｍｍ以下であり、且つローズベンガル染色スコア（Ｖａｎ　Ｂｉｊｓｔｅｒｖｅｌｄのスコア）が９点である患者を意味し、中症ドライアイ患者はシルマー試験Ｉ法の値が５ｍｍ以下であり、且つローズベンガル染色スコア（Ｖａｎ　Ｂｉｊｓｔｅｒｖｅｌｄのスコア）が６～８点である患者を意味する。軽症ドライアイ患者は、シルマー試験Ｉ法の値が５ｍｍより多く、且つローズベンガル染色スコア（Ｖａｎ　Ｂｉｊｓｔｅｒｖｅｌｄのスコア）が３～５点であるドライアイ患者を意味する。 [Test Example 2] Clinical test using amino acid-containing eye drops The amino acid-containing eye drops of each of the production examples described in Example 2 were administered to subjects, and the subjective symptoms or objective findings of the subjects after the administration were measured between subjects. Compare with. Specifically, the eye drop of any one of Production Examples 1, 2, 4, and 5 is administered to one eye of each subject, and the eye drop of Production Example 10 is administered to the other eye as a control. Eye drops are administered to healthy subjects and severe dry eye patients once or twice (50 to 100 μL) / eye once an hour. The number of administrations per day is 16-18. For patients with mild dry eye and VDT workers, 1-2 drops (50-100 μL) / eye is administered 4-6 times / day. The above severe dry eye patient means a patient having a Schirmer test I method value of 5 mm or less and a Rose Bengal staining score (Van Bijsterveld score) of 9 points. It means a patient having a method I value of 5 mm or less and a Rose Bengal staining score (Van Bijsterveld score) of 6 to 8 points. Mild dry eye patient means a dry eye patient with a Schirmer test I method value greater than 5 mm and a Rose Bengal staining score (Van Bijsterveld score) of 3-5 points.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　投与開始してから１週間後、２週間後、及び４週間後の３点で下記項目について評価を行う。
（１）自覚症状
　乾燥感、異物感、眼痛、疲労感の有無
（２）他覚所見
　視力の改善の有無、細隙燈顕微鏡による各結膜異常所見の有無、フルオレセイン染色による角結上皮障害の有無（特に、瞼裂間の結膜の点状染色（ｃｏｎｊｕｎｃｔｉｖａｌ　ｐｕｎｃｔｕａｔｅ　ｓｔａｉｎｉｎｇ　:　ＣＰＳ））、ローズベンガル、あるいは、リサミグリーン染色による角結膜上皮の変性若しくは傷害された細胞の有無、ＢＵＴ（涙膜破壊時間）、涙液油層の分布の観察（ドライアイ観察装置ＤＲ－１ＴＭ（興和社製）による） The following items are evaluated at 3 points, 1 week, 2 weeks and 4 weeks after the start of administration.
(1) Subjective symptoms: Dryness, foreign body, eye pain, fatigue (2) Objective findings: Improvement of visual acuity, presence of abnormalities of each conjunctiva by slit slit microscope, keratoconjunctival disorder caused by fluorescein staining Presence or absence (especially conjunctival punctuate staining (CPS) between ruptures), presence or absence of degenerated or damaged cells of the keratoconjunctival epithelium by rose bengal or Lissami green staining, BUT Time), observation of tear oil layer distribution (by dry eye observation device DR-1 TM (manufactured by Kowa))
［実施例３］　アミノ酸含有点眼液の調製
　下表１１に示した処方のアミノ酸含有点眼液を調製した。 [Example 3] Preparation of amino acid-containing ophthalmic solution An amino acid-containing ophthalmic solution having the formulation shown in Table 11 below was prepared.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
［試験例３］　アミノ酸を含有した点眼剤を用いた臨床試験
　実施例３で調製したアミノ酸含有点眼液を被験者に投与し、投与後の被験者の自覚症状または他覚所見の評価を行った。具体的には、各被験者にアミノ酸含有点眼液３またはアミノ酸含有点眼液４を１回１～２滴（５０～１００μＬ）／眼、６回／日点眼投与した。被験者は、シルマー試験Ｉ法の値が５ｍｍ以上と涙液量は正常であるが、ＢＵＴ（涙液膜破壊時間）が５秒以下と顕著な短縮を示し、且つ角結膜上皮障害はないか、あっても軽微な症例で、眼乾燥感などの自覚症状が非常に強いＢＵＴ短縮型ドライアイ患者であった。
　投与開始してから８週間後に下記項目について評価を行った。
（１）自覚症状
乾燥感、頭痛、眼の奥の痛み、異物感、眼が開けられない、疲れ目、充血、目やに、痒み、眩しい、流涙、かすみ、目が重い、不快感
スコア　０：気にならない
　　　　１：少し気になる
　　　　２：気になる
　　　　３：非常に気になる
（２）他覚所見
　（ｉ）ＳＰＫ（Ｓｕｐｅｒｆｉｃｉａｌ　Ｐｕｎｃｔａｔｅ　Ｋｅｒａｔｉｔｉｓ；点状表層角膜炎）のＡＤ分類 [Test Example 3] Clinical test using an eye drop containing an amino acid The amino acid-containing eye drop prepared in Example 3 was administered to a subject, and the subjective symptoms or objective findings of the subject after the administration were evaluated. Specifically, amino acid-containing ophthalmic solution 3 or amino acid-containing ophthalmic solution 4 was administered once to 1-2 drops (50 to 100 μL) / eye and 6 times / day to each subject. The subject has a normal tear volume with a Schirmer test I value of 5 mm or more, but the BUT (tear film breakage time) is significantly shorter than 5 seconds, and is there no keratoconjunctival epithelial disorder? It was a minor case and a BUT shortened dry eye patient with very strong subjective symptoms such as dryness.
The following items were evaluated 8 weeks after the start of administration.
(1) Subjective symptom dryness, headache, back pain, foreign body sensation, unable to open eyes, tired eyes, redness, itching, dazzling, lacrimation, blurred vision, heavy eyes, discomfort score 0: I am not worried 1: I am worried a little 2: I am worried 3: Very worried (2) Objective findings (i) AD classification of SPK (Superficial Punctate Keratitis)
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
（ｉｉ）ＢＵＴ（ＢｒｅａｋＵｐ　Ｔｉｍｅ；涙液膜破壊時間）
結果を表１３に示す。 (Ii) BUT (BreakUp Time)
The results are shown in Table 13.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　表１３に示すように、アミノ酸含有点眼液３を投与した被験者１の右眼およびアミノ酸含有点眼液４を投与した被験者１の左眼ともに、投与８週間後にＳＰＫがＤ２からＤ１に改善されていた。また、アミノ酸含有点眼液３を投与した被験者２の左右眼および被験者３の左右眼において、投与８週間後にＢＵＴが延長した。さらに、すべての被験者の左右眼において、自覚症状のスコアが減少した。以上の結果から、アルギニン、グルタミンを含むアミノ酸含有点眼液３およびアラニン、アスパラギン、グルタミンを含むアミノ酸含有点眼液４はドライアイの治療に有効であることが分かる。 As shown in Table 13, SPK was improved from D2 to D1 at 8 weeks after administration in both the right eye of the subject 1 to which the amino acid-containing ophthalmic solution 3 was administered and the left eye of the subject 1 to which the amino acid-containing ophthalmic solution 4 was administered. . In addition, BUT was prolonged 8 weeks after administration in the left and right eyes of subject 2 and left and right eyes of subject 3 to which amino acid-containing ophthalmic solution 3 was administered. Furthermore, the subjective symptom scores decreased in the left and right eyes of all subjects. From the above results, it can be seen that the amino acid-containing ophthalmic solution 3 containing arginine and glutamine and the amino acid-containing ophthalmic solution 4 containing alanine, asparagine and glutamine are effective for the treatment of dry eye.
　本発明によれば、従来の人工涙液を頻回点眼しても症状の改善が認められなかったドライアイ、特に重篤な上皮障害に伴うドライアイに対して治療効果が得られる。具体的には、自覚症状（眼痛、開眼困難、異物感、疲労感等）の軽減、ならびに他覚所見（視力、結膜充血、角膜上皮の状態）の改善に優れた効果を発揮する。
　本出願は、日本で出願された特願２００９－０６５２８４（出願日：平成２１年３月１７日）を基礎としており、その内容はすべて本明細書に包含されるものとする。 ADVANTAGE OF THE INVENTION According to this invention, the therapeutic effect is acquired with respect to the dry eye in which the improvement of the symptom was not recognized even if the conventional artificial tears were instilled frequently, especially the dry eye accompanying a serious epithelial disorder. Specifically, it exhibits excellent effects in reducing subjective symptoms (eye pain, difficulty in opening the eye, foreign body sensation, fatigue, etc.) and improving objective findings (visual acuity, conjunctival hyperemia, corneal epithelial condition).
This application is based on Japanese Patent Application No. 2009-065284 filed in Japan (filing date: March 17, 2009), the contents of which are incorporated in full herein.

Claims (11)

  1. 　アラニン、アスパラギン、グルタミン及びアルギニンからなる群から選択される少なくとも１種のアミノ酸を含有することを特徴とする眼科用組成物。 An ophthalmic composition comprising at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine.
  2. 　上記アミノ酸が、アラニン、アスパラギン及びグルタミンから選択される２又は３種のアミノ酸である請求項１記載の眼科用組成物。 The ophthalmic composition according to claim 1, wherein the amino acid is 2 or 3 amino acids selected from alanine, asparagine and glutamine.
  3. 　上記アミノ酸が、アラニン、アスパラギン及びグルタミンの３種である請求項２記載の眼科用組成物。 3. The ophthalmic composition according to claim 2, wherein the amino acids are three kinds of alanine, asparagine and glutamine.
  4. 　上記アミノ酸が、アルギニン、アスパラギン及びグルタミンの３種である請求項１記載の眼科用組成物。 The ophthalmic composition according to claim 1, wherein the amino acids are arginine, asparagine and glutamine.
  5. 　上記アミノ酸が、アルギニンとグルタミンとの組み合わせである請求項１記載の眼科用組成物。 The ophthalmic composition according to claim 1, wherein the amino acid is a combination of arginine and glutamine.
  6. 　眼表面疾患の予防・治療用である、請求項１～５のいずれか１項に記載の眼科用組成物。 The ophthalmic composition according to any one of claims 1 to 5, which is used for prevention and treatment of ocular surface diseases.
  7. 　眼表面疾患が、角結膜上皮障害である請求項６記載の眼科用組成物。 The ophthalmic composition according to claim 6, wherein the ocular surface disease is keratoconjunctival epithelial disorder.
  8. 　アラニン、アスパラギン、グルタミン及びアルギニンからなる群から選択される少なくとも１種のアミノ酸を含有する組成物の有効量を眼表面疾患を有する患者に投与することを特徴とする、眼表面疾患の予防・治療方法。 Prevention and treatment of ocular surface disease, comprising administering an effective amount of a composition containing at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine to a patient having ocular surface disease Method.
  9. 　眼表面疾患が、角結膜上皮障害である請求項８記載の予防・治療方法。 The method for prevention and treatment according to claim 8, wherein the ocular surface disease is keratoconjunctival epithelial disorder.
  10. 　眼表面疾患予防・治療剤の製造のためのアラニン、アスパラギン、グルタミン及びアルギニンからなる群から選択される少なくとも１種のアミノ酸を含有する組成物の使用。 Use of a composition containing at least one amino acid selected from the group consisting of alanine, asparagine, glutamine and arginine for the manufacture of an agent for preventing or treating ocular surface diseases.
  11. 　眼表面疾患が、角結膜上皮障害である請求項１０記載の使用。 The use according to claim 10, wherein the ocular surface disease is keratoconjunctival epithelial disorder.
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