HK1200111B

HK1200111B - Prophylactic, ameliorating or therapeutic agent for retinal diseases

Info

Publication number: HK1200111B
Application number: HK15100636.3A
Authority: HK
Inventors: Kurose Takahiro; Miyano Takayuki; Kato Mariyo; Takai Yoshihiro
Original assignee: 日本乐敦制药株式会社
Priority date: 2012-02-27
Filing date: 2013-02-25
Publication date: 2017-09-29

Description

Preventive, ameliorating or therapeutic agent for retinal diseases

Technical Field

The present invention relates to a prophylactic, ameliorating or therapeutic agent for a retinal disease.

Background

Teprenone (wei de w) was a mixture containing 5E,9E,13E geranylgeranylacetone and 5Z,9E,13E geranylgeranylacetone in a weight ratio of 3: 2. Teprenone is widely used as a therapeutic agent for peptic ulcer for oral administration.

In addition, a scheme for using teprenone in the field of ophthalmology has also been proposed. For example, patent document 1 teaches the use of teprenone as an active ingredient of a preventive or therapeutic agent for dry eye, asthenopia or dry eye. Further, patent document 2 discloses a clear eye drop containing teprenone, phospholipids, a synthetic surfactant, and water.

Furthermore, it is also known that geranylgeranylacetone (wei-defense corporation) in which cis-trans isomer ratio is not defined is useful as an active ingredient of a therapeutic agent for retinal diseases.

For example, patent document 3 teaches the following method: the expression or activity of a heat shock protein in an eye tissue is increased by administering geranylgeranylacetone to a patient with an eye disease such as diabetic retinopathy or glaucoma, and the eye disease is ameliorated by supplementing stem cells to the eye tissue.

Further, non-patent document 1 teaches: when geranylgeranylacetone is administered intraperitoneally to an animal introduced with retinal detachment, expression of heat shock protein 70 is induced, resulting in a significant reduction in apoptosis of the opsocytes.

Further, non-patent document 2 teaches: when geranylgeranylacetone is administered intraperitoneally to glaucoma model rats, the expression of heat shock protein 72 is induced, retinal ganglion cell death is reduced, and optic nerve injury is improved.

Further, non-patent document 3 teaches: when geranylgeranylacetone is orally administered to mice that have damaged visual cells due to light irradiation, thioredoxin and heat shock protein 72 in the retinal pigment epithelium are induced. Furthermore, it is taught that thioredoxin release from the retinal pigment epithelium plays an important role in maintaining the visual cells, and geranylgeranylacetone plays a role in protecting the visual cells from photodamage.

Further, non-patent document 4 teaches: when geranylgeranylacetone was orally administered to mice with retinal damage due to ischemia, the survival number of retinal nerves was significantly increased, and geranylgeranylacetone was useful for treating retinal damage diseases accompanied by ischemia.

Further, non-patent document 5 teaches: when geranylgeranylacetone is orally administered to multiple sclerosis model mice, the visual function can be improved, the number of injured nerve axons of optic nerve is reduced, and the reduction of the number of cells of ganglia is inhibited.

Teprenone sold by the sanitary company contains 5E,9E,13E geranylgeranylacetone and 5Z,9E,13E geranylgeranylacetone in a weight ratio of 3:2 (WO2004/047822, Japanese Kokai publication Hei 9-169639, Japanese patent No. 4621326, Japanese Kokai publication No. 2006-89393, Japanese pharmacopoeia revision 16, Schweishu addition document). Accordingly, geranylgeranylacetone described in patent document 3 and non-patent documents 1 to 5 contains 5E,9E,13E geranylgeranylacetone and 5Z,9E,13E geranylgeranylacetone in a weight ratio of 3: 2. In addition, teprenone sold by companies other than Celastrum, Inc. also contains 5E,9E,13E geranylgeranylacetone and 5Z,9E,13E geranylgeranylacetone (for example, reagent MSDS (202-.

However, the effect of improving retinal diseases by teprenone containing 5E,9E,13E geranylgeranylacetone and 5Z,9E,13E geranylgeranylacetone in a weight ratio of 3:2 is not sufficient in practical use.

Documents of the prior art

Patent document

Patent document 1: japanese unexamined patent publication No. 8-133967

Patent document 2: japanese patent laid-open No. 2000-319170

Patent document 3: japanese patent laid-open No. 2009-507770

Non-patent document

Non-patent document 1: the American Journal of Pathology, Vol.178, No.3, March2011,1080-1090

Non-patent document 2: investigative Ophthalmology & Visual Science, May 2003, Vol.44, No.5,1982-

Non-patent document 3: the Journal of Neuroscience, March2, 2005,25(9),2396-

Non-patent document 4: molecular division, 2007,13,1601-1607

Non-patent document 5: neuroscience Letters,462,2009, 281-285-

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a prophylactic, ameliorating or therapeutic agent for retinal diseases that is sufficiently effective in practical use.

Means for solving the problems

The present inventors have made extensive studies to solve the above problems and have obtained the following unexpected findings.

(i) Geranylgeranylacetone has a protective effect on retinal cells and is extremely effective for the prevention, amelioration or treatment of retinal diseases. This effect is higher in the case of 5E,9E,13E geranylgeranylacetone (hereinafter sometimes referred to as "all-trans body") and 5Z,9E,13E geranylgeranylacetone (hereinafter sometimes referred to as "5Z mono-cis body"), and lower in the case of a mixture of both.

(ii) The protective effect of retinal cells of the all-trans form is particularly excellent compared to teprenone, which is a mixture containing the all-trans form and 5Z mono-cis form in a weight ratio of 3: 2.

(iii) When the ratio of all-trans isomer in the mixture of all-trans isomer and 5Z monocistronic isomer is 80 wt% or more, the effect of protecting retinal cells becomes particularly high.

(iv) When the ratio of 5Z monocistronic form in the mixture of all-trans form and 5Z monocistronic form is made very high, an excellent retinal cell protective effect is exhibited.

The present invention has been made based on the above findings, and provides a prophylactic, ameliorating or therapeutic agent for a retinal disease described below.

An agent which is a preventive, ameliorating or therapeutic agent for a retinal disease containing geranylgeranylacetone,

the geranylgeranyl acetone is used as a raw material,

(a) is a mixture of 5E,9E,13E geranylgeranylacetone and 5Z,9E,13E geranylgeranylacetone and the mixture contains greater than 80% by weight of 5E,9E,13E geranylgeranylacetone;

(b) consisting of 5E,9E,13E geranylgeranylacetone only; or

(c) Consisting of only 5Z,9E,13E geranylgeranylacetone.

The pharmaceutical agent according to claim 1, which is an ophthalmic preparation.

The pharmaceutical preparation according to claim 3, wherein the geranylgeranylacetone is contained in an amount of 0.00001 to 10% by weight based on the total amount of the preparation.

The pharmaceutical agent according to item 4, which is an oral agent.

The pharmaceutical preparation according to item 5 or 4, wherein geranylgeranylacetone is contained in an amount of 0.001 to 80% by weight relative to the total amount of the preparation.

The pharmaceutical agent according to any one of claims 1 to 5, wherein the retinal disease is a disease selected from the group consisting of glaucoma, retinitis pigmentosa, age-related macular degeneration and diabetic retinopathy.

The pharmaceutical agent according to any one of claims 1 to 6, which has a pH of 6 to 8.

The pharmaceutical agent according to any one of claims 1 to 7, further comprising a phosphate buffer.

An agent which is a retinal cell protective agent containing geranylgeranylacetone as an active ingredient,

the geranylgeranyl acetone is used as a raw material,

(b) consisting of 5E,9E,13E geranylgeranylacetone only; or

(c) Consisting of only 5Z,9E,13E geranylgeranylacetone.

The pharmaceutical agent according to claim 9, which is an ophthalmic preparation.

The pharmaceutical preparation according to claim 10, wherein geranylgeranylacetone is contained in an amount of 0.00001 to 10% by weight based on the total amount of the preparation.

The pharmaceutical agent according to claim 12, which is an oral agent.

The pharmaceutical preparation according to claim 12, wherein the geranylgeranylacetone is contained in an amount of 0.001 to 80% by weight based on the total amount of the preparation.

The agent according to any one of claims 9 to 13, wherein the retinal cell is a retinal ganglion cell or a retinal pigment epithelial cell.

The pharmaceutical agent according to any one of claims 9 to 14, which has a pH of 6 to 8.

The pharmaceutical agent according to any one of claims 9 to 13, further comprising a phosphate buffer.

An agent which is an inhibitor of degeneration, injury or death of retinal cells containing geranylgeranylacetone as an active ingredient,

the geranylgeranyl acetone is used as a raw material,

(b) consisting of 5E,9E,13E geranylgeranylacetone only; or

(c) Consisting of only 5Z,9E,13E geranylgeranylacetone.

The pharmaceutical agent of claim 18, which is an ophthalmic preparation.

The pharmaceutical preparation according to claim 18, wherein the geranylgeranylacetone is contained in an amount of 0.00001 to 10% by weight based on the total amount of the preparation.

The pharmaceutical agent of claim 17, which is an oral agent.

The pharmaceutical preparation according to item 21, wherein geranylgeranylacetone is contained in an amount of 0.001 to 80% by weight based on the total amount of the preparation. .

The agent according to any one of claims 17 to 21, wherein the retinal cell is a retinal ganglion cell or a retinal pigment epithelial cell.

The pharmaceutical agent according to any one of claims 17 to 22, which has a pH of 6 to 8.

The pharmaceutical agent according to any one of claims 17 to 23, further comprising a phosphate buffer.

A composition for preventing, ameliorating or treating a retinal disease, which contains geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

The use of geranylgeranylacetone for producing a prophylactic, ameliorating or therapeutic agent for a retinal disease, which is geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

The use of geranylgeranylacetone as a preventive, ameliorating or therapeutic agent for a retinal disease, which is geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Item 28. a method for preventing, ameliorating or treating a retinal disease, which comprises the step of administering geranylgeranylacetone of the following (a), (b) or (c) to a patient with a retinal disease in an amount effective to prevent, ameliorate or treat the retinal disease,

(b) consisting of only 5E,9E,13E geranylgeranylacetone.

A composition for protecting retinal cells, which contains geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Item 31. use of geranylgeranylacetone as a retinal cell protective agent, which is geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

The use of geranylgeranylacetone in the manufacture of a retinal cytoprotective agent, said geranylgeranylacetone being a geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Item 32A method for protecting retinal cells, which comprises the step of administering geranylgeranylacetone of the following (a), (b) or (c) to a patient with a retinal disease to protect retinal cells,

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

A composition for inhibiting degeneration, injury or death of retinal cells, comprising geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Use of geranylgeranylacetone for the manufacture of an inhibitor of degeneration, injury or death of retinal cells, said geranylgeranylacetone being a geranylgeranylacetone of the following (a), (b) or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Use of geranylgeranylacetone as an inhibitor of degeneration, injury or death of retinal cells, which is geranylgeranylacetone of the following (a), (b), or (c):

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Item 36A method for inhibiting degeneration, damage or death of retinal cells, comprising the step of administering to a patient with a retinal disease an effective amount of geranylgeranylacetone of (a), (b) or (c) below to inhibit degeneration, damage or death of retinal cells,

(b) consisting of 5E,9E,13E geranylgeranylacetone only;

(c) consisting of only 5Z,9E,13E geranylgeranylacetone.

Effects of the invention

The agent of the present invention comprising geranylgeranylacetone (hereinafter sometimes simply referred to as "GGA") having an all-trans body ratio of 80% by weight or more protects various retinal cells from degeneration, injury, or death, significantly promoting their survival. Therefore, a significant effect is exhibited in the prevention, amelioration or treatment of various retinal diseases.

GGA having an all-trans ratio of 80% by weight or more exhibits a protective effect on retinal cells in a small amount, and therefore, the agent of the present invention may not contain GGA at a high concentration. In general, ophthalmic preparations are used at high concentrations because of low migration of components into the eyeball. Therefore, the agent of the present invention can reduce the GGA concentration, which is an excellent advantage as an ophthalmic preparation.

In addition, the conventional therapeutic agent for retinal diseases indirectly protects retinal cells by, for example, reducing intraocular pressure to suppress retinal nerve cell death or the like caused by an increase in intraocular pressure, whereas the agent of the present invention directly suppresses retinal cell death, and therefore, can fundamentally prevent, improve or treat retinal diseases, and is extremely useful as a therapeutic agent for retinal diseases.

In addition, GGA is a drug which is widely used and has established safety, and therefore, the agent of the present invention is safe.

The agent of the present invention can be prepared into a preparation which can be easily used by a patient at home, such as an eye drop or an oral preparation, and is therefore useful as an agent for preventing, ameliorating or treating severe retinal diseases.

In addition, a liquid formulation containing teprenone, which is a mixture containing all-trans and 5Z monocistronic isomers in a weight ratio of 3:2, is likely to produce white turbidity when stored at low temperatures. Therefore, the preparation is clouded particularly when it is distributed and stored in cold regions, and the commercial value is low.

In this regard, the pharmaceutical agent of the present invention containing GGA having an all-trans isomer ratio of 80% by weight or more is less likely to cause cloudiness even when stored at low temperatures. Therefore, the product can be circulated in any area, and the commodity value is high.

Further, the ophthalmic composition of the present invention containing GGA having an all-trans body ratio of 80% by weight or more can suppress irritation to the eye.

GGA, which is a single cis-isomer or a mixture of all-trans isomers and single cis-isomers and has a very high ratio of single cis-isomer, also has a protective effect on retinal cells and is extremely effective for the prevention, amelioration or treatment of retinal diseases. The effect is particularly superior to teprenone, which is a mixture containing all-trans and 5Z mono-cis in a weight ratio of 3: 2.

Drawings

FIG. 1 is a graph showing the effect of GGA in protecting cells from hypoxia, low glucose-induced cell death in a blood-deficient sample.

FIG. 2 is a graph showing the neurite outgrowth-inducing effect by GGA in rat RGCs.

FIG. 3 is a photograph showing the neurite outgrowth-inducing effect by GGA in rat RGCs.

FIG. 4 is a graph showing the effect of GGA in protecting cells from oxidative stress.

FIG. 5 is a graph showing the effect of GGA in inhibiting IL-8 production by TNF-. alpha..

FIG. 6 is a graph showing the neuroprotective effect of all-trans and 5Z monocarbitus on the eyes of NMDA-induced glaucoma model rats.

FIG. 7 is a graph showing the neuroprotective effect of the all-trans body on the eyes of NMDA-induced glaucoma model rats.

FIG. 8 is a graph showing that the thickness of the inner reticular layer of the retina of an NMDA-induced glaucoma model rat is increased by the all-trans body.

FIG. 9 is a graph showing the neuroprotective effect of the all-trans body on the eyes of NMDA-induced glaucoma model rats.

FIG. 10 is a graph showing the effect of an ophthalmic composition containing GGA in suppressing white turbidity during low-temperature storage.

Detailed Description

The present invention will be described in detail below.

The preventive, ameliorating or therapeutic agent for a retinal disease of the present invention contains GGA as an active ingredient.

The GGA is an all-trans isomer, a single-cis isomer, a mixture of an all-trans isomer and a single-cis isomer containing 80 wt% or more of the all-trans isomer, or a mixture of an all-trans isomer and a single-cis isomer having a very high ratio of the single-cis isomer.

Geranylgeranylacetone

(1) Class of geometric isomers

GGA has 8 geometric isomers. The specific types are as follows:

(5E,9E,13E) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5E,9E,13EGGA) (all-trans form),

(5Z,9E,13E) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5Z,9E,13EGGA) (5Z monoccis-form),

(5Z,9Z,13E) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5Z,9Z,13EGGA) (13E mono-trans form),

(5Z,9Z,13Z) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5Z,9Z,13ZGGA) (all-cis form),

(5E,9Z,13E) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5E,9Z,13EGGA) (9Z monocarbox-cis),

(5E,9Z,13Z) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5E,9Z,13ZGGA) (5E mono-trans form),

(5E,9E,13Z) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5E,9E,13ZGGA) (13Z monocsyn), and

(5Z,9E,13Z) -6,10,14, 18-tetramethyl-5, 9,13, 17-nonadecatetraen-2-one (5Z,9E,13ZGGA) (9E single trans).

In the present invention, GGA is composed of only an all-trans isomer, only a single cis isomer, or a mixture of an all-trans isomer and a single cis isomer. The monocistronic form may be any of 5Z monocistronic form, 9Z monocistronic form and 13Z monocistronic form. Further, a combination of 2 or more of these monocins may be used.

The monocistronic form is preferably a 5Z monocistronic form.

When GGA is a mixture of an all-trans isomer and a single-cis isomer (particularly 5Z single-cis isomer), the ratio of the all-trans isomer is 80 wt% or more, preferably 82 wt% or more, more preferably 84 wt% or more, still more preferably 86 wt% or more, still more preferably 88 wt% or more, still more preferably 90 wt% or more, still more preferably 92 wt% or more, still more preferably 94 wt% or more, still more preferably 96 wt% or more, and still more preferably 98 wt% or more. Particularly preferably, the compound is composed of only the all-trans isomer. When the content is within the above range, a significant effect is exhibited in the prevention, improvement or treatment of a retinal disease, and cloudiness is not easily generated in the case of low-temperature storage.

In addition, when the GGA is a mixture of all-trans and a single-cis (particularly 5Z single-cis), GGA having a very high ratio of a single-cis (particularly 5Z single-cis) also exhibits a significant effect in the prevention, amelioration or treatment of a retinal disease, and is therefore preferable.

(2) All-trans and 5Z monocistronic forms

5E,9E,13E geranylgeranylacetone (all-trans) is a compound represented by the following structural formula.

All-trans forms are available, for example, from Rionon corporation.

The all-trans isomer can also be obtained by separating commercially available teprenone (kamura et al, Wako pure chemical industries, Ltd., Yangtze Tang) from 5Z monocistronic isomer by silica gel column chromatography using a mobile phase of n-hexane and ethyl acetate at a ratio of 9:1, for example. The separation of the 5Z monocistronic form from the all-trans form of commercially available teprenone can also be performed, for example, by the Shenhu Natural products chemical company.

5Z,9E,13E geranylgeranylacetone (5Z monocistronic form) can also be obtained by isolation of commercially available teprenone. The 5Z monocarbitude is a compound shown in the following structural formula.

Furthermore, the all-trans isomer can be synthesized by the method described in Bull. Korean chem.Soc.,2009, Vol.30, No.9,215-217, for example. This document describes, for example, a method shown in the following synthetic route.

Specifically, in the above reaction formula, geranyl linalool 1, compound 2, and aluminum isopropoxide are mixed, and the mixture is slowly heated to 130 ℃ to cause a reaction. After the reaction was complete, residual compound 2 was removed and the reaction mixture was diluted with 5% sodium carbonate to quench the residual aluminum propoxide. Thus, an all-trans form was obtained. Further, all-trans compounds can be purified by silica gel column chromatography using methylene chloride as an eluent.

(3) Mixtures of all-trans and 5Z monocistronic forms

The mixture of all-trans and 5Z monocistronic forms can be obtained by adding all-trans or 5Z monocistronic forms to commercially available teprenone.

Preparation

The dosage form of the medicament of the present invention is not particularly limited, and pharmaceutically known dosage forms such as ophthalmic preparations, oral preparations, injections (e.g., intravenous, subcutaneous, and intramuscular injections), percutaneous absorption preparations, suppositories, and inhalants can be used without limitation. Among them, ophthalmic drugs, oral agents, or transdermal absorbents are preferable, and ophthalmic drugs are more preferable, from the viewpoint of excellent preventive, ameliorating, or therapeutic effects on retinal diseases and a dosage form that is easy to use for patients.

In the case of any dosage form, the preparation may contain a pharmaceutically acceptable base or carrier, a pharmaceutically acceptable additive, and/or a physiologically or pharmacologically active ingredient other than GGA in addition to GGA.

(1) Ophthalmic drug

The nature of the ophthalmic drug is not particularly limited, and may be any of liquid, flowable, gel, semisolid, and solid forms, for example.

The kind of the ophthalmic drug is not particularly limited. Examples thereof include eye drops, eye washes, corneal contact lens wearing solutions, corneal contact lens solutions (washing solutions, preservation solutions, disinfectant solutions, multifunctional care solutions, and packaged care solutions), storage agents for removed ocular tissues such as cornea for transplantation, perfusion solutions at the time of surgery, ocular ointments (water-soluble ocular ointments and oil-soluble ocular ointments), and intraocular injections (for example, intravitreal injections).

The ophthalmic preparation in a liquid, flowable, gel, semisolid, or solid form other than the solid form may be an aqueous composition, or an oily composition such as an ointment.

Methods for the preparation of ophthalmic formulations are well known. GGA can be prepared by mixing GGA with a pharmaceutically acceptable base or carrier, a pharmaceutically acceptable ophthalmic preparation additive to be added as needed, and other active ingredients (a physiologically active ingredient or a pharmacologically active ingredient other than GGA).

< base agent or Carrier >

Examples of the base or carrier include: water; aqueous solvents such as polar solvents; a polyol; a vegetable oil; oily base agents, and the like. Examples of the base or carrier of the intraocular injection include distilled water for injection and physiological saline.

The base or carrier may be used singly or in combination of 2 or more.

< additive >

Examples of the additives include surfactants, perfumes and cooling agents, preservatives, bactericides and antibacterial agents, pH adjusters, isotonizing agents, chelating agents, buffers, stabilizers, antioxidants, thickeners, and the like. The intraocular injection may contain a solubilizing agent, a suspending agent, an isotonic agent, a buffer, a painless agent, a stabilizer, a preservative, and the like.

The additives may be used singly in 1 kind or in combination of 2 or more kinds.

Specific examples of the additives are shown below.

Surfactant (b): for example, nonionic surfactants such as polyoxyethylene (hereinafter sometimes also referred TO as "POE") -polyoxypropylene (hereinafter sometimes also referred TO as "POP") block copolymers (e.g., Poloxamer 407, Poloxamer 235, Poloxamer 188), POE-POP block copolymer adducts of ethylenediamine (e.g., Poloxamer), POE sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 60, polysorbate 80(TO-10, etc.)), POE hardened castor oils (e.g., POE (60) hardened castor oil (HCO-60, etc.)), POE castor oil, POE alkyl ethers (e.g., polyoxyethylene (9) lauryl ether, polyoxyethylene (20) polyoxypropylene (4) cetyl ether), and polyethylene glycol stearate;

glycine type amphoteric surfactants (e.g., alkyldiaminoethylglycine, alkylpolyaminoethyl glycine) and betaine type amphoteric surfactants (e.g., lauryl dimethylaminoacetic acid betaine, imidazoline)Betaine) and the likeA sex agent; and

cationic surfactants such as quaternary alkylammonium salts (e.g., benzalkonium chloride and benzethonium chloride), and the like.

In addition, the numbers in parentheses indicate the number of moles added.

Flavor or cooling agent: for example, camphor, borneol, terpenes (which may be any of d-, l-or dl-isomer), peppermint water, eucalyptus oil, bergamot oil, anethol, eugenol, geraniol, menthol, limonene, peppermint oil, and essential oils such as rose oil.

Preservatives, bactericides, or antimicrobials: examples of the inorganic salt include, for example, poridium chloride, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, sodium dehydroacetate, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, hydroxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhexamethylene biguanide or hydrochloride thereof, and the like), and Glokill (Rhodia corporation).

pH regulator: for example, hydrochloric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, phosphoric acid, and the like.

Isotonic agent: for example, sodium hydrogen sulfite, sodium sulfite, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, potassium acetate, sodium hydrogencarbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogenphosphate, sodium dihydrogenphosphate, potassium dihydrogenphosphate, glycerin, propylene glycol and the like.

Chelating agent: for example, ascorbic acid, tetrasodium ethylenediaminetetraacetate, sodium ethylenediaminetetraacetate, citric acid, and the like.

Buffering agent: for example, phosphate buffers; citric acid buffers such as citric acid and sodium citrate; acetic acid buffers such as acetic acid, potassium acetate, sodium acetate; carbonic acid buffers such as sodium bicarbonate and sodium carbonate; boric acid buffers such as boric acid and borax; amino acid buffers such as taurine, aspartic acid and salts thereof (e.g., potassium salt), and aminocaproic acid.

Among them, it is preferable to use a phosphate buffer to adjust the pH, thereby suppressing adsorption of GGA to the vessel wall and further suppressing a decrease in the GGA content in the ophthalmic drug. Further, the following effects can be obtained: the haze during low-temperature storage is suppressed, the adsorption of GGA to a corneal contact lens is suppressed, and the stability to heat and light is improved.

The phosphate buffer may be used alone in 1 kind or in combination of 2 or more kinds.

The phosphate buffer is not particularly limited, and examples thereof include: phosphoric acid; alkali metal phosphates such as disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and tripotassium phosphate; alkaline earth metal phosphate salts such as calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, magnesium phosphate, dimagnesium phosphate (magnesium hydrogen phosphate), and trimagnesium phosphate; ammonium phosphate salts such as diammonium hydrogen phosphate and ammonium dihydrogen phosphate, and the like. The phosphate buffer may be either an anhydride or a hydrate.

Among these, at least one selected from the group consisting of phosphoric acid and alkali metal salts of phosphoric acid is preferably used, and at least one selected from the group consisting of phosphoric acid and sodium salts of phosphoric acid is more preferably used.

Preferred combinations of phosphate buffers include: a combination of phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate and trisodium phosphate; a combination of phosphoric acid, disodium hydrogen phosphate and sodium dihydrogen phosphate; a combination of phosphoric acid, disodium hydrogen phosphate and trisodium phosphate; a combination of phosphoric acid, sodium dihydrogen phosphate and trisodium phosphate; a combination of disodium hydrogen phosphate, sodium dihydrogen phosphate and trisodium phosphate; a combination of phosphoric acid and disodium hydrogen phosphate; a combination of phosphoric acid and sodium dihydrogen phosphate; a combination of phosphoric acid and trisodium phosphate; a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate; a combination of disodium hydrogen phosphate and trisodium phosphate; a combination of monosodium phosphate and trisodium phosphate.

Among them, preferred are: a combination of phosphoric acid, disodium hydrogen phosphate and sodium dihydrogen phosphate; a combination of phosphoric acid and disodium hydrogen phosphate; a combination of phosphoric acid and sodium dihydrogen phosphate; a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate, and more preferably a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate.

The content of the phosphate buffer is preferably 0.001 wt% or more, more preferably 0.005 wt% or more, further preferably 0.01 wt% or more, and further preferably 0.05 wt% or more, in terms of an anhydride, relative to the total amount of the ophthalmic drug. When the amount is within the above range, the stabilization effect of GGA, the effect of suppressing low-temperature white turbidity, and the effect of suppressing adsorption of GGA to the vessel wall and the corneal contact lens, which are brought about by the addition of a phosphate buffer, can be sufficiently obtained.

The content of the phosphate buffer in the ophthalmic drug is preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 5% by weight or less, and even more preferably 3% by weight or less, in terms of an anhydride, relative to the total amount of the ophthalmic drug. When the amount is within the above range, eye irritation is small.

The content of the phosphate buffer may be about 0.001 wt% to about 10 wt%, about 0.001 wt% to about 7 wt%, about 0.001 wt% to about 5 wt%, about 0.001 wt% to about 3 wt%, about 0.005 wt% to about 10 wt%, about 0.005 wt% to about 7 wt%, about 0.005 wt% to about 5 wt%, about 0.005 wt% to about 3 wt%, about 0.01 wt% to about 10 wt%, about 0.01 wt% to about 7 wt%, about 0.01 wt% to about 5 wt%, about 0.01 wt% to about 3 wt%, about 0.05 wt% to about 10 wt%, about 0.05 wt% to about 7 wt%, about 0.05 wt% to about 5 wt%, about 0.05 wt% to about 3 wt%, based on the total amount of the ophthalmic drug, in terms of anhydrous substance.

The content of the phosphate buffer is preferably 0.0005 parts by weight or more, more preferably 0.001 parts by weight or more, further preferably 0.005 parts by weight or more, and further preferably 0.01 parts by weight or more, in terms of an anhydride, relative to 1 part by weight of GGA. When the amount is within the above range, the stabilization effect of GGA, the effect of suppressing low-temperature white turbidity, and the effect of suppressing adsorption of GGA to the vessel wall and the corneal contact lens, which are brought about by the addition of a phosphate buffer, can be sufficiently obtained.

The content of the phosphate buffer is preferably 5000 parts by weight or less, more preferably 1000 parts by weight or less, further preferably 500 parts by weight or less, and further preferably 200 parts by weight or less in terms of anhydride per 1 part by weight of GGA. When the amount is within the above range, eye irritation is small.

The content of the phosphoric acid buffer may be, in terms of anhydride, about 0.0005 to about 5000 parts by weight, about 0.0005 to about 1000 parts by weight, about 0.0005 to about 500 parts by weight, about 0.0005 to about 200 parts by weight, about 0.001 to about 5000 parts by weight, about 0.001 to about 1000 parts by weight, about 0.001 to about 500 parts by weight, about 0.001 to about 200 parts by weight, about 0.005 to about 5000 parts by weight, about 0.005 to about 1000 parts by weight, about 0.005 to about 500 parts by weight, about 0.005 to about 200 parts by weight, about 0.01 to about 5000 parts by weight, about 0.01 to about 1000 parts by weight, about 0.01 to about 500 parts by weight, about 0.01 to about 200 parts by weight, based on 1 part by weight of GGA.

A stabilizer: tromethamine, sodium formaldehyde sulfoxylate (sodium formaldehyde sulfoxylate), tocopherol, sodium metabisulfite, monoethanolamine, aluminum monostearate, glyceryl monostearate, and the like.

Antioxidant: ascorbic acid, ascorbic acid derivatives (e.g., disodium ascorbate-2-sulfate, sodium ascorbate, magnesium ascorbate-2-phosphate, and sodium ascorbate-2-phosphate), sodium bisulfite, sodium sulfite, and sodium thiosulfate.

The ophthalmic drug may contain a fat-soluble antioxidant, whereby adsorption of the ophthalmic drug to the wall of the container can be suppressed, and further, reduction of the GGA content in the composition can be suppressed. Furthermore, the adsorption of GGA to a corneal contact lens can be suppressed, and the stability of GGA against heat and light can be improved.

Examples of fat-soluble antioxidants include: butyl-containing phenols such as Butylhydroxytoluene (BHT) and Butylhydroxyanisole (BHA); nordihydroguaiaretic acid (NDGA); ascorbyl esters such as ascorbyl palmitate, ascorbyl stearate, aminopropanol ascorbyl phosphate, ascorbyl tocopherol phosphate, ascorbyl triphosphate, ascorbyl palmitate phosphate; tocopherols such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, -tocopherol; tocopherol derivatives such as tocopherol acetate, tocopherol nicotinate, tocopherol succinate; gallic acid esters such as ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate; propyl gallate; 3-butyl-4-hydroxyquinolin-2-one; vegetable oils such as soybean oil, rapeseed oil, olive oil, sesame oil and the like; carotenoids such as lutein and astaxanthin; polyphenols such as anthocyanins, catechins, tannic acid, and curcumin; retinol, retinol esters (retinol acetate, retinol propionate, retinol butyrate, retinol caprylate, retinol laurate, retinol stearate, retinol myristate, retinol oleate, retinol linoleate, retinol palmitate, etc.), retinal, retinaldehyde esters (retinaldehyde acetate, retinaldehyde propionate, retinaldehyde palmitate, etc.), retinoic acid, retinoates (methyl retinoic acid, ethyl retinoic acid, retinol retinoic acid, tocopherol retinate, etc.), vitamin A such as dehydroretinol, dehydroretinal, dehydroretinoic acid, provitamin A (alpha-carotene, beta-carotene, gamma-carotene, lycopene, zeaxanthin, beta-cryptoxanthin, echinenone, etc.), vitamin A, etc.; CoQ10, and the like. These compounds are commercially available.

Among them, preferred are compounds containing butylphenol, NDGA, ascorbic acid ester, tocopherol derivative, gallic acid ester, propyl gallate, 3-butyl-4-hydroxyquinolin-2-one, vegetable oil, and vitamins A. Among them, preferred are compounds containing butylphenol, tocopherol derivatives, vegetable oils, and retinoids, more preferred are compounds containing butylphenol, vegetable oils, retinol, and retinol esters, and still more preferred are BHT, BHA, sesame oil, and retinol palmitate.

The fat-soluble antioxidants may be used alone in 1 kind or in combination of 2 or more kinds.

The content of the fat-soluble antioxidant in the ophthalmic drug is preferably 0.00001 wt% or more, more preferably 0.00005 wt% or more, still more preferably 0.0001 wt% or more, and still more preferably 0.0005 wt% or more, based on the total amount of the ophthalmic drug. When the amount is within the above range, the effect of suppressing adsorption of GGA to the container wall (the effect of suppressing decrease in the content of GGA), the effect of suppressing adsorption of GGA to a corneal contact lens, and the effect of improving the stability of GGA against heat and light, which are caused by the addition of a fat-soluble antioxidant, can be sufficiently obtained.

The content of the fat-soluble antioxidant in the ophthalmic drug is preferably 10% by weight or less, more preferably 5% by weight or less, still more preferably 2% by weight or less, and still more preferably 1% by weight or less, based on the total amount of the composition. When the amount is within the above range, eye irritation is small.

As the content of the fat-soluble antioxidant in the ophthalmic drug, about 0.00001 to about 10 wt%, about 0.00001 to about 5 wt%, about 0.00001 to about 2 wt%, about 0.00001 to about 1 wt%, about 0.00005 to about 10 wt%, about 0.00005 to about 5 wt%, about 0.00005 to about 2 wt%, about 0.00005 to about 1 wt%, about 0.0001 to about 10 wt%, about 0.0001 to about 5 wt%, about 0.0001 to about 2 wt%, about 0.0001 to about 1 wt%, about 0.0005 to about 10 wt%, about 0.0005 to about 5 wt%, about 0.0001 to about 2 wt%, about 0.0005 to about 1 wt%, may be cited, relative to the total amount of the ophthalmic drug.

The content of the fat-soluble antioxidant in the ophthalmic drug is preferably 0.0001 part by weight or more, more preferably 0.001 part by weight or more, still more preferably 0.005 part by weight or more, and still more preferably 0.01 part by weight or more, based on 1 part by weight of GGA. When the amount is within the above range, the effect of suppressing adsorption of GGA to the container wall (the effect of suppressing decrease in the content of GGA), the effect of suppressing adsorption of GGA to a corneal contact lens, and the effect of improving the stability of GGA against heat and light, which are caused by the addition of a fat-soluble antioxidant, can be sufficiently obtained.

The content of the fat-soluble antioxidant in the ophthalmic drug is preferably 100 parts by weight or less, more preferably 50 parts by weight or less, still more preferably 10 parts by weight or less, and still more preferably 5 parts by weight or less, based on 1 part by weight of GGA. When the amount is within the above range, eye irritation is small.

The content of the fat-soluble antioxidant in the ophthalmic preparation may be, for example, about 0.0001 to about 100 parts by weight, about 0.0001 to about 50 parts by weight, about 0.0001 to about 10 parts by weight, about 0.0001 to about 5 parts by weight, about 0.001 to about 100 parts by weight, about 0.001 to about 50 parts by weight, about 0.001 to about 10 parts by weight, about 0.001 to about 5 parts by weight, about 0.005 to about 100 parts by weight, about 0.005 to about 50 parts by weight, about 0.005 to about 10 parts by weight, about 0.005 to about 5 parts by weight, about 0.01 to about 100 parts by weight, about 0.01 to about 50 parts by weight, about 0.01 to about 10 parts by weight, or about 0.01 to about 5 parts by weight, based on 1 part by weight of GGA.

Thickening agent: cellulose-based high molecular compounds such as guar gum, hydroxypropyl guar gum, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and sodium carboxymethyl cellulose, gum arabic, karaya gum, xanthan gum, agar, alginic acid, α -cyclodextrin, dextrin, dextran, heparin, heparan, heparin sulfate, heparan sulfate, hyaluronic acid, hyaluronate (sodium salt, etc.), sodium chondroitin sulfate, starch, chitin and its derivative, chitosan and its derivative, carrageenan, sorbitol, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene-based high molecular compounds such as polyvinyl methacrylate, alkali metal salts (sodium salt, potassium salt, etc.) of polyacrylic acid, amine salts (monoethanolamine salt, diethanolamine salt, triethanolamine salt, etc.) of polyacrylic acid, carboxyvinyl polymers such as ammonium salt of polyacrylic acid, carboxyvinyl polymers such as sodium salt of polyacrylic acid, etc, Casein, gelatin, collagen, pectin, elastin, ceramide, liquid paraffin, glycerol, polyethylene glycol (macrogol), polyethyleneimine alginate (sodium salt, etc.), alginic acid ester (propylene glycol ester, etc.), tragacanth gum powder, triisopropanolamine, and the like.

< other preventive, ameliorative or therapeutic agent for retinal diseases >

The ophthalmic drug preferably contains, in addition to GGA, a component for preventing or treating retinal diseases by a different mechanism of action from GGA. That is, the ophthalmic drug preferably contains GGA in combination with other ingredients as an active ingredient for preventing, ameliorating or treating retinal diseases. The components for preventing or treating retinal diseases by a different mechanism of action from GGA may be used alone in 1 kind or in combination of 2 or more kinds.

Such a combination is not limited, and examples thereof include: combinations of GGA and prostaglandin F2 α derivatives such as combinations of GGA and prostaglandins (GGA and latanoprost, GGA and travoprost, GGA and tafluprost, etc.), combinations of GGA and prostamides (GGA and bimatoprost, etc.), combinations of GGA and prostaglandins (GGA and isopropyl unoprostone); combinations of GGA and a beta blocker (GGA and timolol maleate, GGA and gelled timolol, GGA and carteolol hydrochloride, GGA and gelled carteolol hydrochloride, etc.), combinations of GGA and a beta 1 blocker (GGA and betaxolol hydrochloride, etc.), combinations of GGA and an alpha beta blocker (GGA and levobunolol hydrochloride, GGA and niprolol, GGA and bunazosin hydrochloride, etc.), combinations of GGA and an alpha 2 blocker (GGA and brimonidine tartrate), and the like; GGA in combination with pilocarpine hydrochloride, GGA in combination with a GGA such as distigmine bromide, and a parasympathetic agonist; combinations of GGA with epinephrine, GGA with epinephrine bitartrate, GGA with GGA such as dipivefrin hydrochloride, and a sympathetic agonist; GGA in combination with dorzolamide hydrochloride, GGA in combination with a carbonate dehydrogenase inhibitor, such as brinzolamide; GGA in combination with SNJ-1656, GGA in combination with a specific inhibitor of ROCK (Rho-associated coil-forming protein kinase), such as GGA and K-115; GGA in combination with a calcium antagonist, such as lomerizine hydrochloride; GGA in combination with a GGA such as DE-117 and an EP2 agonist; GGA in combination with an adenosine A2a receptor agonist, such as OPA-6566; combinations of GGA with VEGF aptamers (GGA with pegaptanib sodium), combinations of GGA with VEGF inhibitors (GGA with ranibizumab, GGA with bevacizumab), combinations of GGA with age-related macular degeneration therapeutics, and the like.

Among them, a combination of GGA and a prostaglandin F2 α derivative and a combination of GGA and a sympatholytic agent (particularly, a combination of GGA and a β -blocker) are preferable from the viewpoint of providing a very high effect of preventing, ameliorating, and treating retinal diseases.

< other pharmacologically active ingredient or physiologically active ingredient >

In addition, a pharmacologically active ingredient or a physiologically active ingredient other than the preventive, ameliorating or therapeutic ingredient for a retinal disease may be blended in the ophthalmic drug. Such pharmacologically active ingredients or physiologically active ingredients may be used alone in 1 kind or in combination in 2 or more kinds.

Examples of such pharmacologically active ingredients or physiologically active ingredients include, in addition to neurotrophic factors, decongestants, eye muscle modulators, anti-inflammatory agents or astringents, antihistamines or antiallergic agents, vitamins, amino acids, antibacterial agents or antibacterial agents, saccharides, high-molecular compounds, cellulose or derivatives thereof, and local anesthetics. Specific examples of these agents are shown below.

Neurotrophic factors: neurotrophic factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and the like.

In addition, since the serum contains a trophic factor typified by a neurotrophic factor, the serum collected from a patient may be added to prepare a preparation for the patient.

Decongestant composition: for example, the α -adrenergic agonists include epinephrine, epinephrine hydrochloride, ephedrine hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, naphazoline hydrochloride, phenylephrine hydrochloride, methylephedrine hydrochloride, epinephrine bitartrate, and naphazoline nitrate. They may be any of d-, l-or dl-isomer.

Eye muscle regulating agent component: examples of the cholinesterase inhibitors having an active site similar to acetylcholine include neostigmine methylsulfate, tropicamide, cumarin and atropine sulfate.

Anti-inflammatory or astringent ingredients: for example, zinc sulfate, zinc lactate, allantoin, -aminocaproic acid, indomethacin, lysozyme chloride, silver nitrate, pranoprofen,sodium sulfonate, dipotassium glycyrrhizinate, diammonium glycyrrhizinate, diclofenac sodium, bromfenac sodium, berberine chloride, berberine sulfate and the like.

Antihistamine or antiallergic ingredients: for example, there are azafirlust, salts such as diphenhydramine or hydrochloride thereof, chlorpheniramine maleate, ketotifen fumarate, levocabastine or hydrochloride thereof, salts such as amlexanox, ibudilast, tazakast, tranilast, oxamide, suplatast or tosylate thereof, sodium cromoglycate, and pemirolast potassium.

Vitamins: for example, retinol acetate, retinol palmitate, pyridoxine hydrochloride, flavin adenine dinucleotide sodium, pyridoxal phosphate, cyanocobalamin, panthenol, calcium pantothenate, sodium pantothenate, ascorbic acid, tocopherol acetate, tocopherol nicotinate, tocopherol succinate, tocopherol calcium succinate, and ubiquinone derivatives, etc.

Amino acids: for example, aminoethylsulfonic acid (taurine), glutamic acid, creatinine, sodium aspartate, potassium aspartate, magnesium potassium aspartate mixtures, glutamic acid, sodium glutamate, magnesium glutamate, -aminocaproic acid, glycine, alanine, arginine, lysine, γ -aminobutyric acid, γ -aminopentanoic acid, and sodium chondroitin sulfate, and the like. They may be any of d-, l-or dl-isomer.

Antibacterial or bactericidal drug component: for example, alkylpolyaminoethylglycine, chloramphenicol, sulfamethoxazoleAzole and sulfadiazineAzole and sulfamethoxazoleAzole sodium and sulfadiazineAzole diethanolamine and sulfenyl isopropylAzole monoethanol amine and sulfamethoxazoleSodium azole, sulfadimidine sodium, ofloxacin, norfloxacin, levofloxacin, lomefloxacin hydrochloride, acyclovir and the like.

Saccharides: examples of the monosaccharide and the disaccharide include glucose, maltose, trehalose, sucrose, cyclodextrin, xylitol, sorbitol, mannitol, and the like.

High molecular compound: for example, alginic acid, sodium alginate, dextrin, dextran, pectin, hyaluronic acid, chondroitin sulfate, polyvinyl alcohol (fully saponified or partially saponified), polyvinylpyrrolidone, carboxyvinyl polymer, polyethylene glycol (macrogol), and pharmaceutically acceptable salts thereof.

Cellulose or a derivative thereof: for example, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, carboxyethyl cellulose, nitrocellulose and the like.

Local anesthetic ingredients: for example, chlorobutanol, procaine hydrochloride, lidocaine hydrochloride, and the like.

< content of GGA >

When the ophthalmic preparation is a liquid, flowable, gel, semisolid, or the like other than a solid preparation, the content of GGA in the ophthalmic composition is preferably 0.00001 wt% or more, more preferably 0.0001 wt% or more, and further preferably 0.001 wt% or more, based on the total amount of the composition. The content may be 0.01 wt% or more, may be 0.1 wt% or more, and may be 1 wt% or more. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

When the ophthalmic preparation is a liquid, flowable, gel, semisolid, or the like other than a solid preparation, for example, the content of GGA in the ophthalmic composition is preferably 10 wt% or less, more preferably 5 wt% or less, and still more preferably 3 wt% or less, relative to the total amount of the composition. When the content is within the above range, a sufficient preventive, ameliorating or therapeutic effect on a retinal disease can be obtained, and a clearer preparation which is less likely to cause fogging can be formed.

When the ophthalmic preparation is a liquid, flowable, gel, or semisolid preparation other than a solid preparation, the GGA content in the ophthalmic composition may be about 0.00001 to about 10 wt%, about 0.00001 to about 5 wt%, about 0.00001 to about 3 wt%, about 0.0001 to about 10 wt%, about 0.0001 to about 5 wt%, about 0.0001 to about 3 wt%, about 0.001 to about 10 wt%, about 0.001 to about 3 wt%, about 0.01 to about 10 wt%, about 0.01 to about 3 wt%, about 0.1 to about 10 wt%, about 0.1 to about 5 wt%, about 0.1 to about 1 wt%, about 3.1 to about 10 wt%, about 0.1 to about 5 wt%, about 0.1 to about 1 wt%, about 10 wt%, or the like, based on the total amount of the composition, About 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%.

The content of GGA in a solid preparation such as a sustained-release intraocular implant preparation or a sustained-release contact lens preparation containing GGA in a contact lens will be described later.

pH

When the ophthalmic drug is a composition containing moisture, the pH of the ophthalmic drug is preferably 4 or more, more preferably 5.5 or more, further preferably 6 or more, and further preferably 6.5 or more. When the amount is within the above range, GGA is a preparation having good stability to heat and light.

Further, the pH of the ophthalmic drug is preferably 9 or less, more preferably 8.5 or less, further preferably 8 or less, and further preferably 7.5 or less. When the amount is within the above range, irritation to the eyes can be suppressed.

Sustained release intraocular implant

Further, as the ophthalmic drug, a sustained-release intraocular implant can be cited. Various methods for the preparation of sustained release intraocular implants are known. Examples thereof include: a matrix preparation obtained by mixing and molding GGA and a carrier containing a high-molecular substance; a preparation obtained by coating a core containing GGA with a polymer film; and a capsule preparation in which GGA is encapsulated in a microcapsule containing a polymer.

The polymer used in the sustained-release intraocular implant can be used without limitation, and examples thereof include hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, pullulan, gelatin, collagen, atelocollagen, hyaluronic acid, casein, agar, gum arabic, dextrin, ethyl cellulose, methyl cellulose, chitin, chitosan, mannan, carboxymethyl ethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol, sodium alginate, polyvinyl alcohol, cellulose acetate, polyvinyl pyrrolidone, polysiloxane, polyvinyl acetal diethylaminoacetate, albumin, and lactic acid-glycolic acid copolymer.

The polymer may be used alone in 1 kind or in combination of 2 or more kinds.

The sustained-release intraocular implant preferably contains GGA and other components for preventing, ameliorating or treating retinal diseases. Examples of such combinations include the combinations exemplified above. The sustained-release intraocular implant may further contain other pharmacologically active ingredients or physiologically active ingredients. For example, the above-exemplified components can be used.

The content of GGA in the sustained-release intraocular implant is preferably 0.001mg or more, more preferably 0.01mg or more, and still more preferably 0.1mg or more, relative to the total amount of the preparation. Further, it is preferably 1000mg or less, more preferably 100mg or less, and further preferably 10mg or less. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

The content of GGA in the sustained-release intraocular implant may be, for example, about 0.001mg to about 1000mg, about 0.001mg to about 100mg, about 0.001mg to about 10mg, about 0.01mg to about 1000mg, about 0.01mg to about 100mg, about 0.01mg to about 10mg, about 0.1mg to about 1000mg, about 0.1mg to about 100mg, or about 0.1mg to about 10mg relative to the total amount of the preparation.

Sustained release corneal contact lens formulation

Further, as the ophthalmic drug, a sustained-release contact lens preparation in which the contact lens itself contains GGA can be cited. Such sustained-release preparations can be prepared, for example, by immersing a corneal contact lens in a solution for a corneal contact lens containing GGA, such as a washing solution, a preservation solution, a disinfectant solution, a multifunctional care solution, a pack care solution, and the like. Alternatively, the GGA can be impregnated with a raw material for producing a contact lens, for example, a constituent monomer of a contact lens polymer (hydroxyethyl methacrylate, methyl methacrylate, vinyl pyrrolidone, divinylbenzene, methacrylic acid, ethylene glycol dimethacrylate, benzoin methyl ether or the like), a coloring agent, or an ultraviolet absorber, and then the contact lens can be produced using the impregnated material.

The content of GGA in the sustained-release corneal contact lens preparation is preferably 0.001mg or more, more preferably 0.01mg or more, and still more preferably 0.1mg or more, based on the total amount of the preparation. Further, it is preferably 1000mg or less, more preferably 100mg or less, and further preferably 10mg or less. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

The content of GGA in the sustained-release corneal contact lens preparation may be about 0.001mg to about 1000mg, about 0.001mg to about 100mg, about 0.001mg to about 10mg, about 0.01mg to about 1000mg, about 0.01mg to about 100mg, about 0.01mg to about 10mg, about 0.1mg to about 1000mg, about 0.1mg to about 100mg, or about 0.1mg to about 10mg, based on the total amount of the preparation.

The sustained-release corneal contact lens preparation preferably contains GGA and other components for preventing, ameliorating or treating retinal diseases. Examples of such combinations include the combinations exemplified above. The sustained-release contact lens preparation may further contain a pharmacologically active ingredient or a physiologically active ingredient other than GGA. For example, the above-exemplified components can be used.

The dosage form of the ophthalmic drug is preferably an eye drop, an intraocular injection, an eye ointment, or an eye lotion, and more preferably an eye drop, from the viewpoint of good migration to the affected part.

(2) Oral preparation

GGA can be made into oral preparation or oral administration preparation. The oral agent is not limited, and examples thereof include: solid preparations such as tablets (including sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, troches, chewable agents, troches and the like; liquid preparations such as syrups, emulsions, and suspensions.

The solid preparation can be prepared by mixing GGA with a pharmaceutically acceptable carrier, and if necessary, a pharmaceutically acceptable additive for oral administration and a pharmacologically active ingredient or physiologically active ingredient other than GGA by a known method described in, for example, japanese pharmacopoeia.

The pharmaceutically acceptable carrier is not limited, and examples thereof include: excipients such as lactose, white sugar, D-mannitol, D-sorbitol, starch, gelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light silicic anhydride, synthetic aluminum silicate, and magnesium metasilicate silicate; a binder such as gelatinized starch, sucrose, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, white sugar, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and polyvinylpyrrolidone; disintegrants such as lactose, white sugar, starch, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light silicic anhydride, and low-substitution hydroxypropyl cellulose; stabilizers such as citric anhydride, sodium laurate, and glycerin.

Further, the liquid preparation can also be prepared by a known method described in, for example, japanese pharmacopoeia. For example, GGA is dissolved or dispersed in water, ethanol, glycerol, simple syrup, a mixture thereof, or the like.

The oral preparation may contain additives for oral preparations such as sweeteners, preservatives, lubricants, diluents, buffers, flavoring agents, coloring agents and antioxidants.

The oral agent preferably contains GGA in combination with a component for preventing or treating retinal diseases with a different mechanism of action from the agent of the present invention. Such a combination is not limited, and examples thereof include: GGA in combination with acetazolamide, GGA in methazolamide, GGA in combination with a carbonate dehydrogenase inhibitor, such as diclofenamide; GGA with concentrated glycerol, GGA with a combination of GGA such as isosorbide with a high osmotic pressure drug, and the like.

Among them, a combination of GGA and a carbonate dehydrogenase inhibitor is preferable, a combination of GGA and acetazolamide and a combination of GGA and methazolamide are more preferable, and a combination of GGA and methazolamide is further preferable.

The components for preventing or treating retinal diseases by a different mechanism of action from GGA may be used alone in 1 kind or in combination of 2 or more kinds.

The oral agent may contain a pharmacologically active ingredient or a physiologically active ingredient other than the preventive, ameliorative or therapeutic ingredient for retinal diseases.

In particular, as well-known pharmacologically active ingredients or physiologically active ingredients, for example, neurotrophic factors, decongestant ingredients, eye muscle regulating agent ingredients, anti-inflammatory agent ingredients or astringent ingredients, antihistamine ingredients or antiallergic ingredients, vitamins, amino acids, antibacterial agent ingredients or bactericidal agent ingredients, and the like can be cited.

In the case of a solid preparation, the GGA content in the oral preparation is preferably 0.001 wt% or more, more preferably 0.01 wt% or more, and still more preferably 0.1 wt% or more, relative to the total amount of the composition. Further, it is preferably 80% by weight or less, more preferably 60% by weight or less, and further preferably 50% by weight or less.

In the case of a solid preparation, the GGA content in the oral preparation may be about 0.001 wt% to about 80 wt%, about 0.001 wt% to about 60 wt%, about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 80 wt%, about 0.01 wt% to about 60 wt%, about 0.01 wt% to about 50 wt%, about 0.1 wt% to about 80 wt%, about 0.1 wt% to about 60 wt%, or about 0.1 wt% to about 50 wt%, based on the total amount of the preparation.

In the case of a liquid preparation, the content is preferably 0.001% by weight or more, more preferably 0.01% by weight or more, and still more preferably 0.1% by weight or more, based on the total amount of the composition. Further, it is preferably 80% by weight or less, more preferably 60% by weight or less, and further preferably 50% by weight or less.

In the case of a liquid preparation, the GGA content in the oral preparation may be about 0.001 wt% to about 80 wt%, about 0.001 wt% to about 60 wt%, about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 80 wt%, about 0.01 wt% to about 60 wt%, about 0.01 wt% to about 50 wt%, about 0.1 wt% to about 80 wt%, about 0.1 wt% to about 60 wt%, or about 0.1 wt% to about 50 wt%, based on the total amount of the preparation.

When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

In the oral preparation, the carrier, the additive and the pharmacologically active ingredient or physiologically active ingredient other than GGA may be used singly in 1 kind or in combination of 2 or more kinds.

(3) Injection preparation

The injectable preparation can be prepared by dissolving or dispersing GGA in distilled water for injection, physiological saline, or the like and using a known method described in, for example, japanese pharmacopoeia. The injection may contain pharmaceutically acceptable carriers such as a solubilizing agent, a suspending agent, an isotonizing agent, a buffer, a painless agent, a stabilizer and a preservative, or may further contain pharmaceutically acceptable additives for injection.

The injection preferably contains a component for preventing or treating retinal diseases on the basis of GGA by a different mechanism of action from GGA. That is, the injection preferably contains GGA in combination with other components as an effective component for preventing, ameliorating or treating retinal diseases. The components for preventing or treating retinal diseases by a different mechanism of action from GGA may be used alone in 1 kind or in combination of 2 or more kinds.

Such a combination is not limited, and examples thereof include: combinations of GGA and prostaglandin F2 α derivatives such as combinations of GGA and prostaglandins (GGA and latanoprost, GGA and travoprost, GGA and tafluprost, etc.), combinations of GGA and prostamides (GGA and bimatoprost, etc.), combinations of GGA and prostaglandins (GGA and isopropyl unoprostone); combinations of GGA and a beta blocker (GGA and timolol maleate, GGA and gelled timolol, GGA and carteolol hydrochloride, GGA and gelled carteolol hydrochloride, etc.), combinations of GGA and a beta 1 blocker (GGA and betaxolol hydrochloride, etc.), combinations of GGA and an alpha beta blocker (GGA and levobunolol hydrochloride, GGA and niprolol, GGA and bunazosin hydrochloride, etc.), combinations of GGA and an alpha 2 blocker (GGA and brimonidine tartrate), and the like; GGA in combination with pilocarpine hydrochloride, GGA in combination with a GGA such as distigmine bromide, and a parasympathetic agonist; combinations of GGA with epinephrine, GGA with epinephrine bitartrate, GGA with a GGA such as dipivefrin hydrochloride, and a sympathetic agonist; GGA in combination with dorzolamide hydrochloride, GGA in combination with a carbonate dehydrogenase inhibitor, such as brinzolamide; GGA in combination with SNJ-1656, GGA in combination with a specific inhibitor of ROCK (Rho-associated coil formed protein kinase), such as GGA and K-115; GGA in combination with a calcium antagonist, such as lomerizine hydrochloride; GGA in combination with a GGA such as DE-117 and an EP2 agonist; GGA in combination with an adenosine A2a receptor agonist, such as OPA-6566; combinations of GGA with VEGF aptamers (GGA with pegaptanib sodium), combinations of GGA with VEGF inhibitors (GGA with ranibizumab, GGA with bevacizumab), combinations of GGA with age-related macular degeneration therapeutics, and the like.

The injection may contain a pharmacologically active ingredient or a physiologically active ingredient other than the components for preventing, ameliorating or treating retinal diseases. Examples of such pharmacologically active ingredients or physiologically active ingredients include neurotrophic factors, decongestant ingredients, eye muscle regulating agent ingredients, anti-inflammatory agent ingredients or astringent ingredients, antihistamine ingredients or antiallergic ingredients, vitamins, amino acids, antibacterial agent ingredients or bactericidal agent ingredients.

The GGA content in the injection is preferably 0.001 wt% or more, more preferably 0.01 wt% or more, and still more preferably 0.1 wt% or more, based on the total amount of the preparation. Further, it is preferably 80% by weight or less, more preferably 60% by weight or less, and further preferably 50% by weight or less. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

The GGA content in the injection may be about 0.001 wt% to about 80 wt%, about 0.001 wt% to about 60 wt%, about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 80 wt%, about 0.01 wt% to about 60 wt%, about 0.01 wt% to about 50 wt%, about 0.1 wt% to about 80 wt%, about 0.1 wt% to about 60 wt%, about 0.1 wt% to about 50 wt%, relative to the total amount of the preparation.

In the injection, 1 kind of additive and pharmacologically active ingredient or physiologically active ingredient other than GGA can be used alone or 2 or more kinds can be used in combination.

(4) Percutaneous absorption agent

Examples of the percutaneous absorption agent include a coating agent obtained by mixing GGA with a base (pharmaceutically acceptable base) and components other than GGA, which are generally used in pharmaceutical external preparations.

Examples of the base include: polymers such as sodium alginate, gelatin, corn starch, tragacanth, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, xanthan gum, carrageenan, mannan, agarose, dextrin, carboxymethyl starch, polyvinyl alcohol, sodium polyacrylate, methoxyethylene-maleic anhydride copolymer, polyvinyl ether, polyvinylpyrrolidone, carboxyvinyl polymer, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, pullulan; hydrocarbons such as white petrolatum, yellow petrolatum, paraffin, ceresin, and microcrystalline wax; gelled hydrocarbons (for example, the trade name is Plastibase, manufactured by Bristol Myers Squibb); higher fatty acids such as stearic acid; higher alcohols such as cetyl alcohol, octyldodecanol, and stearyl alcohol; polyethylene glycol (e.g., polyethylene glycol 4000, etc.); polyhydric alcohols such as propylene glycol, glycerin, dipropylene glycol, 1, 3-butylene glycol, and concentrated glycerin; fatty acid esters such as monooleate and stearin. Further, pharmaceutically acceptable additives for percutaneous absorption agents such as a solubilizing agent, an inorganic filler, a humectant, a preservative, a thickener, an antioxidant and a refreshing agent may be added.

The percutaneous absorption agent may be a patch (cataplasm) in which a coating agent layer containing GGA and a support for supporting the coating agent layer are integrated. The preparation method of the cataplasm is well known, and for example, the cataplasm can be prepared by a known method described in japanese pharmacopoeia.

The cataplasm may be obtained by laminating a plurality of coating agent layers so that the concentration of GGA increases layer by layer to ensure the sustained release of GGA.

Further, as the percutaneous absorbent, a coating agent containing emulsified particles containing GGA may be mentioned. Such a coating agent can be obtained by stirring and mixing GGA with a surfactant (e.g., a phospholipid or a nonionic surfactant), water, and an oily base. Examples of the oily base include the above-exemplified hydrocarbons, higher fatty acids, higher alcohols, polyhydric alcohols, and fatty acid esters.

Further, examples of the percutaneous absorption agent include a preparation in which a GGA suspension is dispersed as fine particles in a hydrophobic polymer. The hydrophobic polymer is not limited, and examples thereof include polylactic acid.

The percutaneous absorption agent preferably contains a component for preventing or treating retinal diseases in a different mechanism of action from GGA, on the basis of GGA. That is, the percutaneous absorption agent preferably contains GGA in combination with other components as an active ingredient for preventing, ameliorating or treating a retinal disease. The components for preventing or treating retinal diseases by a different mechanism of action from GGA may be used alone in 1 kind or in combination of 2 or more kinds.

The transdermal absorption preparation may contain a pharmacologically active ingredient or a physiologically active ingredient other than the components for preventing, improving or treating retinal diseases. Examples of such pharmacologically active ingredients or physiologically active ingredients include decongestant ingredients, eye muscle controlling agent ingredients, anti-inflammatory agent ingredients or astringent ingredients, antihistamine ingredients or antiallergic agent ingredients, vitamins, amino acids, antibacterial agent ingredients or bactericidal agent ingredients.

The GGA content in the transdermal patch is preferably 0.001 wt% or more, more preferably 0.01 wt% or more, and still more preferably 0.1 wt% or more, based on the total amount of the preparation. Further, it is preferably 80% by weight or less, more preferably 60% by weight or less, and further preferably 50% by weight or less. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

The GGA content in the transdermal patch may be about 0.001 wt% to about 80 wt%, about 0.001 wt% to about 60 wt%, about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 80 wt%, about 0.01 wt% to about 60 wt%, about 0.01 wt% to about 50 wt%, about 0.1 wt% to about 80 wt%, about 0.1 wt% to about 60 wt%, or about 0.1 wt% to about 50 wt%, based on the total amount of the preparation.

In the transdermal absorption preparation, 1 additive and a pharmacologically active ingredient or a physiologically active ingredient other than GGA may be used alone or in combination of 2 or more.

(5) Suppository

Suppositories can be prepared by combining GGA with a pharmaceutically acceptable base such as the following and using a known method described in japanese pharmacopoeia: acrylic polymers such as carbopol and polycarbophil; cellulose polymers such as hydroxypropyl cellulose and hydroxypropylmethyl cellulose; natural polymers such as sodium alginate and chitosan; and fatty acid waxes, and the like.

The following pharmaceutically acceptable additives for suppositories may be blended: preservatives such as sodium benzoate, potassium sorbate and parabens; pH adjusters such as hydrochloric acid, citric acid and sodium hydroxide; stabilizers such as methionine, and the like.

The suppository preferably contains a component for preventing or treating retinal diseases on the basis of GGA by a different mechanism of action from GGA. That is, the suppository preferably contains GGA in combination with other ingredients as an effective ingredient for preventing, improving or treating retinal diseases. The components for preventing or treating retinal diseases by a different mechanism of action from GGA may be used alone in 1 kind or in combination of 2 or more kinds.

The suppository may contain a pharmacologically active ingredient or a physiologically active ingredient other than the components for preventing, improving or treating retinal diseases. Examples of such pharmacologically active ingredients or physiologically active ingredients include decongestant ingredients, eye muscle controlling agent ingredients, anti-inflammatory agent ingredients or astringent ingredients, antihistamine ingredients or antiallergic agent ingredients, vitamins, amino acids, antibacterial agent ingredients or bactericidal agent ingredients.

The GGA content in the suppository is preferably 0.001 wt% or more, more preferably 0.01 wt% or more, and further preferably 0.1 wt% or more, based on the total amount of the preparation. Further, it is preferably 80% by weight or less, more preferably 60% by weight or less, and further preferably 50% by weight or less. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

The GGA content in the suppository may be about 0.001 wt% to about 80 wt%, about 0.001 wt% to about 60 wt%, about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 80 wt%, about 0.01 wt% to about 60 wt%, about 0.01 wt% to about 50 wt%, about 0.1 wt% to about 80 wt%, about 0.1 wt% to about 60 wt%, about 0.1 wt% to about 50 wt%, based on the total amount of the preparation.

In the suppository, the additive and the pharmacologically active ingredient or physiologically active ingredient other than GGA may be used alone in 1 kind or in combination of 2 or more kinds.

(6) Inhalant

Examples of the inhalant include powder inhalants, liquid inhalants, and aerosol. Their preparation is well known.

Powder inhalants can be prepared, for example, by finely pulverizing GGA by a conventional method and mixing with an excipient such as lactose as needed. The powder inhalant can be administered by using an inhalation device such as Spinhaler (registered trade name).

The liquid inhalant can be prepared by adding GGA and, if necessary, pharmaceutically acceptable additives for liquid inhalants to a pharmaceutically acceptable carrier such as purified water or distilled water for injection, and dissolving the mixture with stirring. Examples of pharmaceutically acceptable additives for liquid inhalants include: isotonic agents such as sodium chloride; buffers such as boric acid buffers and phosphoric acid buffers; preservatives such as benzalkonium chloride; thickeners such as carboxyvinyl polymers, and the like. The liquid inhalant may be administered using an inhalation device such as a Nebulizer (registered trade name).

The aerosol can be prepared, for example, by finely pulverizing GGA by a conventional method, adding a dispersant as needed, and filling the dispersion in a spray container together with a propellant under coolingTo prepare the compound. Examples of the propellant include: liquefied hydrofluoroalkane (HFA134a (1,1,1, 2-tetrafluoroethane; CH)₂FCF₃) HFA227(1,1,1,2,3,3, 3-heptafluoropropane: CF (compact flash)₃-CHF-CF₃) Etc.) and the like. Examples of the dispersant include medium-chain fatty acid triglycerides such as Miglyol 812 (trademark: manufactured by Dynamit Nobel Co.), soybean lecithin, and the like.

The inhalant preferably contains a component for preventing or treating retinal diseases on the basis of GGA with a different mechanism of action from GGA. That is, the inhalant preferably contains GGA in combination with other components as an effective component for preventing, ameliorating or treating retinal diseases. The components for preventing or treating retinal diseases by a different mechanism of action from GGA may be used alone in 1 kind or in combination of 2 or more kinds.

Among them, a combination of GGA and a prostaglandin F2 α derivative and a combination of GGA and a sympatholytic agent (particularly, a combination of GGA and a β -blocker) are preferable from the viewpoint of providing a very high effect of preventing, ameliorating or treating a retinal disease.

The inhalant may contain a pharmacologically active ingredient or a physiologically active ingredient other than the preventive, ameliorating or therapeutic ingredient for a retinal disease. Examples of such pharmacologically active ingredients or physiologically active ingredients include decongestant ingredients, eye muscle controlling agent ingredients, anti-inflammatory agent ingredients or astringent ingredients, antihistamine ingredients or antiallergic agent ingredients, vitamins, amino acids, antibacterial agent ingredients or bactericidal agent ingredients.

The GGA content in the inhalant is preferably 0.001 wt% or more, more preferably 0.01 wt% or more, and still more preferably 0.1 wt% or more, based on the total amount of the preparation. Further, it is preferably 80% by weight or less, more preferably 60% by weight or less, and further preferably 50% by weight or less. When the amount is within the above range, the effect of preventing, ameliorating or treating retinal diseases can be sufficiently obtained.

The GGA content in the inhalant may be about 0.001 wt% to about 80 wt%, about 0.001 wt% to about 60 wt%, about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 80 wt%, about 0.01 wt% to about 60 wt%, about 0.01 wt% to about 50 wt%, about 0.1 wt% to about 80 wt%, about 0.1 wt% to about 60 wt%, about 0.1 wt% to about 50 wt%, relative to the total amount of the preparation.

In the inhalant, 1 additive, and a pharmacologically active ingredient or physiologically active ingredient other than GGA may be used alone or in combination of 2 or more.

Reagent kit

The pharmaceutical agent of the present invention may be a pharmaceutical agent composed of a single-dose composition containing all the components, or a kit separately having a composition containing GGA and a composition containing a pharmacologically active ingredient or a physiologically active ingredient other than GGA. Further, it may be a kit having a composition containing a specific additive and a composition containing GGA separately. In the case of a kit, the respective compositions may be filled in different containers, or may be in-use preparation-type compositions filled in containers that can be mixed at the time of use. In the case of a kit, any type of two-or three-dose form may be used.

In the case where the pharmaceutical agent of the present invention is a kit comprising a composition containing GGA and a composition containing other components, in the case of a kit in which each composition is filled in a different container and in the case of a kit of a preparative type at the time of use, the GGA content of each preparation described above is a ratio to the total amount of each composition after mixing.

Disease of the subject

The retinal disease to which the present invention is directed may be any disease that causes degeneration, damage, or cell death of cells constituting the retina, or a disease caused by degeneration, damage, or cell death of cells constituting the retina, and examples thereof include glaucoma, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, retinal detachment, diabetic maculopathy, hypertensive retinopathy, retinal vascular occlusion (retinal artery occlusion; retinal vein occlusion such as central venous occlusion, and the like), retinal arteriosclerosis, retinal fissure, retinal round fissure, macular round hole, fundus hemorrhage, posterior vitreous detachment, pigmented venous atrophy, cycloidal retinal atrophy, choroideremia, crystalline retinopathy, Ichthyophthalmia, corneal dystrophy, pyramidal dystrophy, central areolar choroidal dystrophy, dobesian cellular retinal dystrophy, vitelliform macular dystrophy, cystoid macular edema, recessive macular dystrophy, starger's disease, retinoschisis, central serous retinal choroidopathy (central retinopathy), glabellar cord degeneration seventh type, familial exudative vitreoretinopathy, hyperchromic cone syndrome, retinal vascular streaks, autosomal dominant atrophy, autosomal dominant drusen, familial drusen, acute zonal latent outer retinopathy, cancer-related retinopathy, photodamage, ischemic retinopathy, inflammation-induced retinal degeneration diseases, and the like.

Among them, glaucoma, retinitis pigmentosa, age-related macular degeneration, and diabetic retinopathy are preferred diseases, and glaucoma is a more preferred disease.

In addition, the present invention can be directed to a disease in which any cell constituting the retina is damaged or a disease caused by damage of any cell constituting the retina. Examples of the retinal constituent cells include retinal ganglion cells, amacrine cells, horizontal cells, muller glia cells, bipolar cells, retinal visual cells (cones, rods), retinal pigment epithelial cells, and the like. It is particularly useful for diseases in which damage to retinal ganglion cells or retinal pigment epithelial cells is observed or caused by damage to these cells.

In addition, the present invention is also directed to a disease in which any of the layers constituting the retina, that is, the inner limiting membrane, the nerve fiber layer, the ganglion cell layer, the inner reticular membrane, the inner granular layer, the outer reticular layer, the outer granular layer, the outer limiting membrane, the visual cell layer, and the retinal pigment epithelium layer is damaged, or a disease caused by damage to any of these layers. In particular, a disease of injury of the ganglion cell layer, inner granular layer or outer granular layer is preferably used.

The patient to be treated by the present invention is a patient of the above-mentioned retinal disease.

As described above, the agent of the present invention prevents, ameliorates or treats retinal diseases by allowing GGA to protect retinal cells, i.e., promote the survival of retinal cells, or inhibit the degeneration, damage or death of retinal cells.

Accordingly, the present invention comprises a retinal cell protective agent, a retinal cell survival promoter, or an inhibitor of degeneration, injury or death of retinal cells, which contains GGA,

(a) is a mixture of all-trans and single-cis forms (especially 5Z single-cis form), and the mixture contains more than 80 wt% of all-trans form;

(b) consisting of all-trans bodies only;

(c) consisting of only the 5Z single cis form; or

(d) Is a mixture of all-trans and single-cis forms, especially 5Z single-cis form, and the ratio of the single-cis form in this mixture is very high.

Among these agents, the kinds of retinal cells suitable are as described above. The components in the preparation, the amounts used thereof, the properties of the preparation, the dosage forms, and the like are as described for the prophylactic, ameliorating, or therapeutic agent for retinal diseases of the present invention.

In the present invention, "prevention" includes avoidance, delay or reduction of the onset of disease, and "improvement" and "treatment" include alleviation of symptoms, inhibition of symptom development and cure or recovery.

Application method

< ophthalmic preparation >

When the drug of the present invention is an eye drop, the eye drop containing GGA at the above-mentioned concentration may be applied to the eye drop, for example, in a manner of about 1 to about 2 drops per time, about 1 to about 5 times per day, and preferably about 1 to about 3 times per day.

When the agent of the present invention is an eyewash, the eyewash containing GGA at the above-mentioned concentration may be used, for example, in an amount of about 1mL to about 20mL per time, about 1 to about 10 times per day, and preferably about 1 to about 5 times per day.

When the pharmaceutical agent of the present invention is an eye ointment, the eye ointment containing GGA at the above-mentioned concentration may be applied to the eyes, for example, in an amount of about 0.001g to about 5g per time, about 1 to about 5 times per day, and preferably about 1 to about 3 times per day.

When the drug of the present invention is an intraocular injection, the injection containing GGA at the above-mentioned concentration may be injected about 0.005mL to about 1mL each time, about 1 to about 3 times for 1 to 14 days, and preferably 1 time.

In the case where the drug of the present invention is a solution for a corneal contact lens (a washing solution, a preservation solution, a disinfectant solution, a multi-purpose care solution, a pack care solution), a preservative for an excised eye tissue such as a cornea for transplant, or a perfusion solution for use in surgery, the GGA-containing composition may be used in an amount corresponding to the usual usage of these preparations.

When the agent of the present invention is a sustained-release contact lens preparation, the contact lens containing GGA in the above amount may be replaced with new one, for example, about 1 to about 3 times, preferably 1 time, for 1 to 14 days.

When the agent of the present invention is a sustained-release intraocular implant, a new implant may be implanted as needed about 1 to about 14 days after the implant containing GGA in the above amount is implanted.

When the pharmaceutical agent of the present invention is an ophthalmic composition, the daily dose of GGA is preferably 50ng or more, more preferably 500ng or more, and still more preferably 5 μ g or more. The daily dose of GGA is preferably 50mg or less, more preferably 20mg or less, and still more preferably 10mg or less.

When the pharmaceutical agent of the present invention is an ophthalmic composition, the daily dose of GGA may be about 50ng to about 50mg, about 50ng to about 20mg, about 50ng to about 10mg, about 500ng to about 50mg, about 500ng to about 20mg, about 500ng to about 10mg, about 5. mu.g to about 50mg, about 5. mu.g to about 20mg, or about 5. mu.g to about 10 mg.

< other preparations >

When the pharmaceutical agent of the present invention is a dosage form other than an ophthalmic preparation, the daily dose of GGA is preferably about 0.1mg or more, more preferably about 1mg or more, and still more preferably about 5mg or more. Further, it is preferably about 5000mg or less, more preferably about 1000mg or less, and further preferably about 500mg or less.

When the pharmaceutical agent of the present invention is a dosage form other than an ophthalmic preparation, the daily dose of GGA is preferably about 0.1mg to about 5000mg, more preferably about 1mg to about 1000mg, and still more preferably about 5mg to about 500 mg. The daily dose may be administered, for example, about 1 to 5 times per day, preferably about 1 to 3 times per day.

The administration time varies depending on the type, stage, age, body weight, sex, administration route, etc., and can be appropriately selected, for example, from about 1 day to 30 years. For example, in the case of retinal diseases such as glaucoma, retinitis pigmentosa, age-related macular degeneration, and diabetic retinopathy, the retinal diseases may be prevented, improved, or treated in a short administration period of about 1 to 20 years, particularly about 1 to 10 years. When the ophthalmic composition of the present invention utilizes a retinal protective effect to inhibit the progression of retinal diseases, continuous administration may be employed.

The invention comprises the following steps:

(i) a method for preventing, ameliorating or treating a retinal disease, which comprises the step of administering a GGA of the following (a), (b), (c) or (d) to a patient with a retinal disease in an amount effective to prevent, ameliorate or treat the retinal disease;

(ii) a method for inhibiting degeneration, damage or death of retinal cells, comprising the step of administering an effective amount of GGA of (a), (b), (c) or (d) below to a patient with a retinal disease to inhibit degeneration, damage or death of retinal cells; and

(iii) a method for protecting retinal cells, which comprises the step of administering an effective amount of GGA of the following (a), (b), (c) or (d) to a patient with a retinal disease to protect retinal cells,

(a) is a mixture of all-trans and single-cis forms (especially 5Z single-cis form) and contains more than 80% by weight of GGA of all-trans form;

(b) GGA consisting of all-trans bodies only;

(c) GGA consisting of only 5Z single cis-bodies;

(d) is a mixture of all-trans and single-cis forms (especially 5Z single-cis form) and the ratio of single-cis form in this mixture is very high.

GGA may be administered in the form of each of the preparations of the present invention described above. The administration method varies depending on the dosage form, and there can be exemplified: eye drop, eye wash, application to the eye, spray to the eye, implantation to the eye, wearing of corneal contact lens, injection (injection into the eye such as vitreous body, venous injection, subcutaneous injection, intramuscular injection), oral administration, transdermal, insertion into the rectum, inhalation, etc. In the case of an ophthalmic drug, it is sufficient to administer the drug to the eye.

The types of the target disease, the target patient, and the retinal cell are as described in the section for describing the drug of the present invention.

Examples

The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

(1) Preparation of geranylgeranylacetone

Commercially available teprenone (all-trans form: 5Z mono-cis form in a weight ratio of 3:2) (wako pure chemical industries) was purchased, and the all-trans form was purified by silica gel column chromatography.

As specific conditions, silica gel (PSQ60B, manufactured by fuji silicon chemical) was filled in a glass tube, and fractionated and purified by a mobile phase (n-hexane: ethyl acetate: 9: 1). After fractionation, each fraction was concentrated and dried under reduced pressure, and the purification degree and structure of the all-trans form were confirmed by GC and 1H-NMR (solvent: deuterated chloroform, internal standard: tetramethylsilane), respectively (yield about 20%).

< GC measurement Condition >

A chromatographic column: DB-1(J & Wscientific, 0.53 mm. times.30 m, film thickness 1.5 μm)

Column temperature: 200 ℃ → 5 ℃/min → 300 ℃ (10 min)

Temperature of the gasification chamber: 280 deg.C

Detector temperature: 280 deg.C

Carrier gas: helium gas

Hydrogen pressure: 60kPa

Air pressure: 50kPa

Make-up gas pressure: 75kPa (nitrogen)

Total flow rate: 41 mL/min

Flow rate of the chromatographic column: 6.52 mL/min

Linear velocity: 58.3 cm/sec

The split ratio is as follows: 5:1

Injection amount: mu.L of a 0.1g/100mL (ethanol solution) sample was injected

GGA was prepared in each weight ratio (all-trans form: 5Z mono-cis form: weight ratio 7:3, 8:2, 9:1, etc.) by mixing commercially available teprenone and all-trans form purified in the above manner at an arbitrary ratio. Since stability by mixing could not be confirmed, the preparation was performed at the time of use.

(2) Protecting retinal nerve cells from the effects of hypoxia, low glucose-induced ischemia-like cell death Evaluation of

The progression of visual field damage in glaucoma is associated with the death of optic ganglion cells (RGCs) due to ischemia near the optic nerve (heliolog (filia pharmacol. jpn.)128, 255-258 (2006)). The effect of GGA on protecting cells from hypoxic, low-glucose-induced ischemia-like cell death was tested using (JNeurosci Res.2000 May 15; 60(4):495-503.) PC12 as a representative neural cell line established from rat adrenal medullary pheochromocytoma and used as a functional evaluation model cell for RGC.

(evaluation method)

The test substance was prepared in the following manner. That is, the test substance was 4 GGAs containing all-trans forms and 5Z monocistronic forms at weight ratios of 10:0, 8:2, 6:4 and 0: 10. 100mg of GGA and 0.25mg of DL-alpha-tocopherol acetate (Wako pure chemical industries, Ltd.) as an antioxidant were weighed and dissolved in 789mg of 100% ethanol, and components other than GGA were prepared in the same manner, and the obtained product was used as a base. GGA at 10:0, 8:2 and 6:4 dissolved in 789mg of 100% ethanol was diluted to a concentration corrected so that 10% (v/v) horse serum (DS Pharma biomedicalal) and 5% (v/v) fetal bovine serum (first chemical) added to the high glucose concentration (4.5g/L) Dulbecco's Modified Eagle Medium (DMEM) substantially contained 30. mu.M all-trans form, and 0:10 GGA containing only 5Z monocins was diluted to 30. mu.M. The base was diluted at the same dilution rate as that used for the preparation of GGA in which the weight ratio of all-trans form to 5Z single-cis form was 6: 4.

PC12 (available from DS Pharma Biomedical) was added at 2.0 × 10 per well⁴Individual cell method cells were seeded in 100. mu.L each in a 96-well microplate (IWAKI) coated with collagen IV, and usedDMEM at 37 deg.C with 5% CO₂Was cultured under the conditions of (1) for 48 hours.

After 48 hours of culture, the cell culture supernatant was removed and replaced with pre-prepared DMEM containing GGA at 37 ℃ with 5% CO₂Cultured for 2 hours under the conditions of (1). After 2 hours of culture, the medium was replaced with DMEM with low glucose concentration (1.0g/L) supplemented with 2% horse serum and 1% fetal bovine serum at 37 ℃ with 5% CO₂Under hypoxic conditions, Anaeropack 5% (Mitsubishi gas chemistry) was used and changed to 0% O₂And cultured for 8 hours. DMEM with high glucose concentration (4.5g/L) supplemented with 2% (v/v) horse serum and 1% (v/v) fetal bovine serum was used at 37 ℃ and 5% CO₂The cells cultured for 8 hours at the usual oxygen concentration were used as an untreated group.

(test results)

After 8 hours of culture, 100. mu.L of a mixture of Cell Titer-glo (Promega) and PBS, which are living Cell detection reagents, was added to each well, and the amount of luminescence produced by the reaction with ATP in living cells was measured by a photometer (GloMax; manufactured by Promega). The effect of GGA in protecting cells from oxidative stress caused by hydrogen peroxide was evaluated by calculating the cell survival rate based on the actually measured luminescence amount and according to the following formula.

Cell survival (%). 100 × [ (light emission of base agent or GGA-treated group)/(light emission of untreated group) ]

The results are shown in FIG. 1. As can be seen from fig. 1, the GGA-treated group showed significantly higher cell survival rates than the base-treated group at any one weight ratio. Further, GGAs having weight ratios of all-trans to 5Z monoccis of 10:0, 8:2 and 0:10 all had significantly higher cytoprotective effects than GGAs of 6:4 (based on n ═ 10, P <0.05, P < 0.01, Tukey-kramer test, in addition, no significant differences were observed between 8:2, 10:0 and 0: 10).

(3) Neurite extension induction using culture system of Retinal Ganglion Cells (RGCs) derived from rat Evaluation of Effect

The progression of visual field damage in glaucoma is associated with the death of optic ganglion cells (RGCs) due to ischemia near the optic nerve (heliolog (Folia pharmacol. jpn.)128, 255-258 (2006)). Therefore, it is widely used as one of the tools for studying optic nerve diseases such as glaucomaDerived from ratThe neurite outgrowth-inducing effect of GGA was tested by the retinal nerve culture system (Current protocols in Neuroscience 3.22.1-3.22.10, October 2010).

(evaluation method)

4-day-old Wistar rats (manufactured by SLC Japan) were euthanized by cervical dislocation, and the eyeballs were removed. The removed eyeball was immersed in 70% ethanol for 10 seconds, transferred to hank's balanced salt solution containing 100U/mL of penicillin and 100. mu.g/mL of streptomycin, and the cornea, iris, crystalline lens and vitreous body were removed under a solid microscope with surgical scissors and forceps, and retinal tissue was removed. The removed retinal tissue was transferred to a centrifuge tube containing 5mL of a basic medium for nerve cell culture (Neurobasal, Invitrogen) containing 100U/mL of penicillin, 100. mu.g/mL of streptomycin, and an additive for nerve cell culture (B27)^TMSupplement, Invitrogen), 1. mu.L-cysteine (Cogeneration Biotech) and 15U/mL papain (Sigma Aldrich) were incubated at 37 ℃ for 30 minutes. After 30 minutes, the supernatant was removed and the suspension was treated with a solution containing 100U/mL of penicillin, 100. mu.g/mL of streptomycin, and B27^TM-Supplement Neurobasal washing 2 times after washing, 2mL Neurobasal was added, the tissue was made into small cell masses by pipetting with dry heat sterilized pasteur pipette (Hilgenberg), and transferred to 50mL Neurobasal prepared in advance, centrifuged at 900 × g for 5 minutes, after supernatant was removed, the cells were suspended again with 6mL Neurobasal to prepare cell suspension, the cell suspension was passed through a 40 μm nylon mesh cell strainer (japanese BD), after removing the aggregated cell masses, the cells were seeded on a 6-well plate coated with poly-D-lysine/laminin (japanese BD) and 5% CO was added at 37 ℃₂Under the conditions ofAnd (5) culturing.

The test substance was an all-trans form and 2 GGAs containing an all-trans form and a 5Z monocistronic form at a weight ratio of 6:4, 100mg of each GGA, 0.25mg of acetic acid DL- α -tocopherol (Wako pure chemical industries), which is an antioxidant, were weighed and dissolved in 789mg of 100% ethanol, the components other than GGA were prepared in the same manner, the resultant was used as a base, 10:0 and 6:4 GGA dissolved in 789mg of 100% ethanol was diluted at a concentration corrected so as to substantially contain 3. mu.M of an all-trans form, the base was diluted at the same dilution ratio as that used for the preparation of 6:4 GGA, and added to a cell culture supernatant 2 hours after inoculation of cells, and then incubated at 37 ℃ with 5% CO₂Was cultured under the conditions of (1) for 48 hours.

(results)

After culturing for 48 hours, the cell culture supernatant was removed, and the cells were fixed at room temperature for 30 minutes using 4% paraformaldehyde-phosphate buffer (Wako pure chemical industries) and 100% methanol (Wako pure chemical industries). After washing the cells with phosphate buffer (PBS, manufactured by KohjinBio), the cells were blocked in PBS containing 2% (w/v) bovine serum albumin (Sigma Aldrich) and 0.05% (v/v) Tween20(Sigma Aldrich) for 30 minutes at room temperature. After 30 minutes, a 1000-fold dilution of β III tubulin antibody (Promega) was prepared using PBS, 1mL of which was added to each well, followed by incubation at room temperature for 2 hours. After 2 hours, the antibody dilution was removed, washed 3 times with PBS, and then a 1000-fold dilution of Alexa Fluor 488 Goat Anti-mouse antibody (Invitrogen) was prepared using PBS, and 1mL of the solution was added to each well, followed by incubation at room temperature for 1 hour. After 1 hour, the antibody diluent was removed, and after 3 washes with PBS, 3mL of PBS was added to each well, and any 4 spots of each well were observed (excitation wavelength: 475nm, fluorescence wavelength: 535nm) using a cytometer (In Cell Analyzer1000, GE Healthcare Bioscience), and the average value of the neurite lengths (. mu.m) of the fluorescence-stained RGCs was calculated.

The results are shown in FIG. 2. As can be seen from FIG. 2, the GGA-treated group having a weight ratio of all-trans to 5Z single-cis of 10:0 showed a more significant neurite-inducing effect than the GGA-treated group and the base agent-treated group having a weight ratio of all-trans to 5Z single-cis of 6:4 (based on n-4, P <0.05, P < 0.01, Tukey-kramer test).

In addition, a representative observation image of the fluorescence-stained rat RGC is shown in fig. 3. It was found that the 10:0 GGA-treated group had a more significant neurite-inducing effect than the 6:4 GGA-treated group.

(4) Evaluation of Effect of protecting retinal pigment epithelial cells from oxidative stress caused by Hydrogen peroxide

The relationship between oxidative stress and ophthalmic diseases has been widely reported, and in addition to glaucoma and cataract, the relationship between oxidative stress and retinal diseases caused by diabetes, hypertension, hyperlipidemia, etc., age-related macular degeneration, retinopathy of prematurity, retinal vascular occlusion, retinal photodamage, etc. in the retina has been pointed out (Nian eye Diwill 112, 22-29 (2008)). In the retina, the retinal pigment epithelium is in an environment that readily produces reactive oxygen species (Invest Opthalmol VisSci.2006 July 47(7): 3164-. The effect of GGA on protecting cells from oxidative stress caused by hydrogen peroxide was tested using human-derived retinal pigment epithelial cell line ARPE-19.

(evaluation method)

3 GGAs containing all-trans and 5Z monoccis in the weight ratios of 10:0, 8:2 and 6:4 were prepared as test substances in the following manner. That is, 100mg of GGA and 0.25mg of DL-alpha-tocopherol acetate (Wako pure chemical industries, Ltd.) as an antioxidant were weighed and dissolved in 789mg of 100% ethanol, and components other than GGA were prepared in the same manner, and the obtained product was used as a base. GGA of 10:0, 8:2 and 6:4 dissolved in 100% ethanol was diluted to a concentration adjusted so that 280. mu.M of all-trans form was substantially contained in a specific mixed liquid medium (DMEM/F-12, manufactured by Invitrogen) such as Dulbecco's modified Eagle medium/Ham's F12 supplemented with 10% (v/v) fetal bovine serum (first chemical). The base was diluted at the same dilution rate as in the case of 6:4 GGA. The diluted solution was used as a test solution.

ARPE-19 (from ATCC) was added at 1.5 × 10 per well⁴Individual cell formats 100. mu.L of each cell was seeded in 96 well microtiter plates (CORNING) using DMEM/F-12 as described above at 37 ℃ with 5% CO₂Was cultured under the conditions of (1) for 48 hours.

After culturing for 48 hours, the cell culture supernatant was removed and replaced with a previously prepared test solution at 37 ℃ with 5% CO₂Was cultured for 14 hours under the conditions of (1). Immediately before the end of the culture, hydrogen peroxide for precision analysis (Wako pure chemical industries, Ltd.) was added to DMEM/F-12, and DMEM/F-12 supplemented with 750. mu.M hydrogen peroxide was prepared. After 14 hours of culture, the cell culture supernatant was removed, and 200. mu.L each of phosphate buffer solutions (PBS, manufactured by Kohjin Bio) was added thereto. PBS was immediately removed and replaced with pre-prepared DMEM/F-12 supplemented with hydrogen peroxide at 37 deg.C with 5% CO₂Cultured for 2 hours under the conditions of (1). The untreated group was replaced with DMEM/F-12 containing no hydrogen peroxide.

(results)

After 2 hours of culture, the cell culture supernatant was removed, 200. mu.L of each PBS was added, and the PBS was immediately removed. To each well, 100. mu.L of a mixture of Cell Titer-glo (Promega) and PBS, which are living Cell detection reagents, was added in equal amounts, and the amount of luminescence generated by the reaction with ATP in living cells was measured by a luminometer (GloMax; manufactured by Promega). The effect of GGA in protecting cells from oxidative stress caused by hydrogen peroxide was evaluated by calculating the cell survival rate based on the actually measured luminescence amount and according to the following formula.

The results are shown in FIG. 4. As can be seen from FIG. 4, the GGA-treated group showed higher cell viability than the base-treated group at any weight ratio. Furthermore, GGAs at 10:0 and 8:2 all had significantly higher cell viability than the group treated with the base agent (based on n-3, P <0.05, P <0.001, Tukey-kramer test).

(5) Evaluation of Effect of inhibiting IL-8 production from retinal pigment epithelial cells

Drusen are known to accumulate under the retinal pigment epithelium in age-related macular degeneration, where they attract macrophages. TNF-alpha is secreted by macrophages, and when TNF-alpha acts on retinal pigment epithelium or peripheral tissues, the cells further produce various cytokines to cause inflammation (Mol Vis.200814: 2292-303.). Interleukin-8 (IL-8) is involved in the migration of neutrophils, a representative cytokine that amplifies inflammation. The effect of GGA in inhibiting IL-8 production by TNF-. alpha.was examined using human-derived retinal pigment epithelial cell line ARPE-19.

(evaluation method)

All-trans form, 2 GGAs containing all-trans form and 5Z monoccis form in a weight ratio of 6:4 were prepared as test substances. That is, 100mg of GGA and 0.25mg of acetic acid DL-alpha-tocopherol as an antioxidant were weighed and dissolved in 789mg of 100% ethanol, and components other than GGA were prepared in the same manner, and the obtained product was used as a base. GGA dissolved in 789mg of 100% ethanol and having a weight ratio of all-trans to 5Z single-cis of 6:4 was diluted at a concentration corrected so that 50. mu.M of all-trans was substantially contained in DMEM/F-12. The base was diluted at the same dilution rate as that used for the preparation of GGA in which the weight ratio of all-trans form to 5Z single-cis form was 6: 4. The diluted solution was used as a test solution.

ARPE-19 was added at 2.5 × 10 per well⁴Individual cell method 100. mu.L of each cell was seeded in a 96-well microplate (CORNING) and 10% (v/v) fetal bovine serum-added DMEM/F-12 at 37 ℃ and 5% CO₂Cultured under the conditions of (1) for 24 hours.

After 24 hours of culture, the cell culture supernatant was removed, and 200. mu.L of a previously prepared test solution was added to each well, followed by incubation at 37 ℃ with 5% CO₂Was cultured for 16 hours under the conditions of (1). For the untreated group, the same appliesDMEM/F-12 was added and the culture was continued, immediately before the end of the culture, recombinant human TNF- α (R) was added using DMEM/F-12&D Systems) was prepared at 10ng/ml after 16 hours of culture, 2. mu.L of each well of DMEM/F-12 containing TNF- α prepared in advance was added to each well of the test solution at 37 ℃ with 5% CO₂For the untreated group, TNF- α was not added, and the culture was performed in the same manner.

(results)

After 4 hours of culture, 150. mu.L each of the cell culture supernatants was collected and stored at-80 ℃. The remaining cell culture supernatant was removed, 200. mu.L of PBS was added to each, and PBS was immediately removed. A mixture of 100. mu.L of a viable Cell detection reagent, Cell Titer-Glo, and PBS in equal amounts was added to each well, and the amount of luminescence generated by reaction with ATP in viable cells was measured by a photometer. Based on the actually measured luminescence amount, the cell survival rate was calculated as follows.

Cell survival (%) 100 × [ (luminescence of GGA-treated group)/(luminescence of base-treated group) ]

The cell culture supernatants were returned to room temperature and IL-8 concentration was quantified using a human CXCL8/IL-8 Quantikine ELISA kit (R & DSystems). The procedure was performed according to the instructions attached to the kit, and the absorbance measured was divided by the cell viability to correct it. The absorbance was measured using a microplate reader (VersaMax, manufactured by molecular instruments) having a measurement wavelength of 450nm and a calibration wavelength of 540nm (temperature in the apparatus was 20 to 25 ℃). The IL-8 concentration corresponding to the corrected measurement value was calculated, and the IL-8 concentration of the untreated group as a background value was subtracted, and the obtained value was defined as the IL-8 concentration of each treatment group.

The results are shown in FIG. 5. As can be seen from fig. 5, the GGA (all-trans) treated group having a weight ratio of all-trans to 5Z single-cis of 10:0 showed a more significant effect of inhibiting IL-8 production than the GGA treated group having a weight ratio of all-trans to 5Z single-cis of 6:4 (based on n ═ 3,. P <0.05, Tukey-kramer test).

(6) Protection ofEvaluation of Effect of retinal ganglion cells on protection from NMDA-induced nerve injury

In recent years, NMDA (N-methyl-D-aspartic acid), which is an analog of glutamic acid, has been reported in large numbers as one of causative substances of neurodegenerative diseases including alzheimer's disease. In the field of ophthalmology, NMDA is thought to be associated with optic nerve damage observed in glaucoma (Brain Research Bulletin,81(2010) 349-358). Therefore, this time, the neuroprotective effect of GGA was evaluated using NMDA-induced glaucoma model rats.

Test method

After administering all-trans, 5Z monocistronic, or teprenone to Sprague-dawley (sd) rats in advance by oral administration (test example 1), intravitreal administration (test example 2), and eye drop administration (test example 3), 5 μ L of NMDA was administered into the vitreous body, thereby inducing nerve damage. In test example 2, a commercially available eye drop for glaucoma treatment (trade name) as a positive control was administered into the vitreous body 1 time and 5 days. In each test example, a base agent containing no GGA or AIPHAGAN was administered as a control in the same manner.

The usage and amount of test examples 1 to 3 are shown in table 1, and the composition of the base used in each test is shown in table 2.

[ Table 1]

[ Table 2]

g/100mL	Test example 1	Test example 2	Test example 3
				Arabic gum	5.000	-	-
α -tocopherol	0.200	-	-
				Boric acid	-	1.300	1.300
Borax	-	0.400	0.400
				Polysorbate 80	-	2.000	2.000
POE hardened Castor oil 60	-	2.000	2.000
				POE Castor oil	-	0.100	0.100
Dibutylhydroxytoluene	-	0.005	0.005
				Purified water	Proper amount of	Proper amount of	Proper amount of

After 3 days of NMDA administration, the eyeball was removed, fixed with Half Karnovsky fixative for 24 hours, embedded with paraffin, and cut into thin slices to prepare pathological tissue sections stained with hematoxylin-eosin (HE). The tissue sections were observed with an optical microscope, the thickness (μm) of the inner reticular layer (IPL) of the retina was measured, and the neuroprotective effect of the test agent was evaluated using the thickness of the inner reticular layer (IPL) of the retina as an index.

Results

The results of test example 1 are shown in FIG. 6. As can be seen from fig. 6, all-trans and 5Z monoccis exhibit more significant neuroprotective effects on NMDA-induced nerve damage than the base dose when administered orally (based on p <0.05, 0.01, dunnett's multiple comparisons). On the other hand, teprenone (all-trans form: 5Z mono-cis form in a weight ratio of 6:4) did not show significant neuroprotective effect.

The results of test example 2 are shown in FIG. 7. As can be seen from fig. 7, all-trans and 5Z monoccis exhibit more significant neuroprotective effects on NMDA-induced nerve injury than the base dose in the case of intravitreal administration (based on × p <0.001, Tukey-kramer multiple comparison test). In addition, all-trans forms also showed significantly superior neuroprotective effects compared to 0.1% (Qianshou pharmaceutical) of AIPHAGAN eye drops purportedly having neuroprotective effects (based on p <0.05, Tukey-kramer multiple comparison test).

Fig. 8 shows a photomicrograph of the tissue section of test example 2.

The results of test example 3 are shown in FIG. 9. As can be seen from fig. 9, in the case of eye drop administration, all-trans bodies showed a more significant neuroprotective effect on nerve damage caused by NMDA than the base (based on the x p <0.05, t test).

(7) Test for suppressing cloudiness during Low-temperature storage

Preparation of eye drops

Eye drops containing commercially available teprenone, GGA at each weight ratio (all-trans: 5Z mono-cis: 7:3, 8:2, 9:1, etc.) and all-trans purified by the above method were prepared as follows.

Specifically, GGA or all-trans form was added to a surfactant (polysorbate 80) heated to 65 ℃, dissolved with stirring in a hot water bath at 65 ℃ for 2 minutes, added with water at 65 ℃, and then mixed with each buffer solution to prepare a homogeneous solution, and the pH and osmotic pressure were adjusted with hydrochloric acid and/or sodium hydroxide. The solution was filtered through a membrane filter (Saimeishik Co., Ltd., bottle top filter) having a pore diameter of 0.2. mu.m, to prepare a clear sterile eye drop.

The compositions of the respective eye drops are shown in tables 3 to 8, which are disclosed later.

In each operation, after confirming that the content of GGA is not reduced by adsorption to a device or the like by HPLC described later, sterile eye drops are prepared.

Method for confirming GGA concentration

The GGA concentration in each eye drop was measured from the area value (Ac) of the 5Z single cis form and the area value (At) of the all-trans form under the following HPLC measurement conditions under the measurement conditions of the dissolution test described in "teprenone 100mg/g fine" No. 0412007 of the japanese pharmacopoeia "teprenone standard (the all-trans form: 5Z single cis form: about 6:4 by weight ratio, manufactured by general dentistry and pharmacosome レギュラトリーサイエンス )" or teprenone (and wako pure chemical industries) as a standard. In addition, the total amount of all-trans and 5Z mono-cis in teprenone (all-trans: 5Z mono-cis in a weight ratio of 3:2) was calculated as GGA content.

< HPLC measurement conditions >

A detector: ultraviolet absorption photometer (measuring wavelength: 210nm)

A chromatographic column: YMC-Pack ODS-A (inner diameter 4.6mm, length 15cm, particle diameter 3 μm)

Column temperature: 30 deg.C

Mobile phase: 90% acetonitrile solution

Flow rate: 1.2 to 1.3 mL/min (5Z dissolution in the order of monocistronic form and all-trans form)

Injection amount: 5. mu.L of a 0.05g/100mL sample was injected

Preservation at Low temperature

The prepared eye drop was filled in a container (daily electrochemical nitro product) made of transparent glass and having a capacity of 10mL in full volume (without any space), and after being tightly closed, it was stored at 4 ℃. The samples immediately after the preparation and stored at 4 ℃ for 3 days were dispensed onto a 96-well plate (flat bottom, polystyrene) by a glass scale pipette, and the absorbance at 660nm (20-25 ℃ C. in the apparatus) was measured by an enzyme-labeling instrument (VersaMax, manufactured by molecular instruments). With reference to JIS K0101 (measurement of transmitted light turbidity by Industrial Water test method), the absorbance of each sample at 660nm was used as an index of white turbidity (turbidity).

The test was conducted after the progress of the test was made quickly and it was confirmed that the GGA content did not decrease during storage at 4 ℃ and absorbance measurement.

The turbidity is shown in tables 3 to 6 below.

[ Table 3]

[ Table 4]

[ Table 5]

[ Table 6]

As is clear from tables 3 to 6, when the all-trans isomer ratio is 80% by weight or more, the white turbidity during storage at 4 ℃ can be significantly suppressed.

The prepared eye drop was filled in a container (made of daily electrical glass) made of transparent glass having a capacity of 10mL in a full amount (without any void), and after being tightly closed, it was stored at 4 ℃. The absorbance at 660nm of the product stored at 4 ℃ for 6 days or 14 days was measured by the above-described method and the measured value was defined as turbidity. Each sample was measured for n-4 or n-5, and compared with a control (water) by the Dunnett method.

The turbidity and the results of the test are shown in table 7 below. Further, photographs of the preserved products stored at 4 ℃ for 14 days are shown in FIG. 10 (left: comparative example 10, right: example 13).

[ Table 7]

As is clear from table 7, when the ratio of all-trans forms is 80% by weight or more, white turbidity during storage at 4 ℃ can be significantly suppressed.

The results of examples 7 to 12 are summarized in Table 8 from Table 5 and Table 6.

[ Table 8]

When the buffer solution contains a phosphoric acid buffer, the cloudiness during low-temperature storage can be more remarkably suppressed than when the buffer solution contains a boric acid buffer.

(8) Test for suppressing cloudiness at ambient temperature storage

Preparation of eye drops and confirmation of GGA concentration were carried out in the same manner as in "(7) test for suppressing white turbidity during low-temperature storage". However, since GGA concentration is high, filtration is not performed during the preparation of eye drops. The composition of the eye drops is shown in table 9.

The prepared eye drop was filled in a container (daily electrochemical nitro product) made of a transparent glass having a capacity of 10mL in full volume (without any space), and after being tightly closed, it was stored at room temperature (about 25 ℃). The product stored for 3 days was dispensed onto a 96-well plate (flat bottom, polystyrene) by a glass scale pipette in an amount of 0.2mL, and the absorbance at 660nm (20 to 25 ℃ C. in the apparatus) was measured by a microplate reader (VersaMax, molecular instruments). With reference to JIS K0101 (measurement of transmitted light turbidity by Industrial Water test method), the absorbance of each sample at 660nm was used as an index of white turbidity (turbidity).

Each sample was measured for n-4, and comparative example 11 and example 13, and comparative example 12 and example 14 were performed by the t-test method. The results are shown in Table 9.

[ Table 9]

As is clear from table 9, when the concentration of all-trans isomer was high, the white turbidity was more significantly suppressed than that of teprenone (weight ratio of all-trans isomer: 5Z mono-cis isomer: 6: 4).

(9) Functional test

Adding teprenone or all-trans form into surfactant (polysorbate 80, POE castor oil) heated to 65 deg.C, dissolving in hot water bath at 65 deg.C under stirring for 2 min, adding water at 65 deg.C, mixing, stirring to obtain uniform solution, and adjusting pH and osmotic pressure with hydrochloric acid and/or sodium hydroxide. The solution was filtered through a membrane filter (Saimeishik Co., Ltd., bottle top filter) having a pore diameter of 0.2. mu.m, to prepare a clear sterile eye drop. The composition of each eye drop is shown in table 10 disclosed later. These eye drops were aseptically filled in a container (8mL) made of polyethylene terephthalate.

About 30. mu.L of these eye drops were applied to 9 healthy volunteers (age 33.8. + -. 6.6 years: 8 men and 1 woman) sensitive to irritation without wearing a contact lens, and the degree of "stinging" felt immediately after the application of the eye drops and 3 minutes after the application of the eye drops was evaluated by the VAS method (visual analog Scale): visual evaluation Scale) (double blind test).

The results are shown in Table 10.

[ Table 10]

As is clear from table 10, the all-trans form suppressed "stinging" more significantly than teprenone in both cases immediately after eye drop and 3 minutes after eye drop.

(10) Heat stability test

Adding all-trans-isomer into surfactant (polysorbate 80, POE castor oil) heated to 65 deg.C, dissolving in hot water bath at 65 deg.C under stirring for 2 min, adding water at 65 deg.C, mixing, stirring to obtain uniform solution, and adjusting pH and osmotic pressure with hydrochloric acid and/or sodium hydroxide. The solution was filtered through a membrane filter (Saimeishik Co., Ltd., bottle top filter) having a pore diameter of 0.2. mu.m, to prepare a clear sterile eye drop. The composition of each eye drop is shown in table 11 disclosed later. These eye drops were aseptically filled in a container made of polyethylene terephthalate (container for Rohto dryoid EX, london pharmaceutical) in a full amount of 8 mL.

These eye drops were allowed to stand at 40 ℃, 50 ℃ or 60 ℃ for 10 days or 20 days, whereby a stability test was performed. The content of all-trans forms in each sample was quantified and the residual ratio (%) was calculated using the japanese pharmacopoeia "teprenone standard (all-trans form: single cis form: weight ratio of about 6:4, manufactured by general financial and human medical instrument レギュラトリーサイエンス ") as a standard. In addition, the total amount of all-trans and single-cis in the standard was calculated as GGA.

Residual ratio (%) < 100 × [ all-trans body content after leaving for a predetermined time (g/100 mL)/all-trans body content immediately after production (g/100mL) ]

The results are shown in Table 11.

[ Table 11]

When the buffer contains a phosphoric acid buffer, the residual rate of all-trans forms is significantly higher than that when the buffer contains a boric acid buffer, and the thermal stability is excellent. In addition, when the pH is within the range of 5.7 to 7.5, the residual rate of all-trans forms is higher and the thermal stability is more excellent at a higher pH.

(11) Light stability test

Adding all-trans-isomer into surfactant (polysorbate 80, POE castor oil) heated to 65 deg.C, dissolving in hot water bath at 65 deg.C under stirring for 2 min, adding water at 65 deg.C, mixing, stirring to obtain uniform solution, and adjusting pH and osmotic pressure with hydrochloric acid and/or sodium hydroxide. The solution was filtered through a membrane filter (Saimeishik Co., Ltd., bottle top filter) having a pore diameter of 0.2. mu.m, to prepare a clear sterile eye drop. The composition of each eye drop is shown in table 12 disclosed later. These eye drops were aseptically filled in a container made of polyethylene terephthalate (container for Rohto dryoid EX, london pharmaceutical) in a full amount of 8 mL.

Each eye drop was irradiated with light under the following conditions, and the content of all-trans forms in the sample immediately after production and after irradiation was quantified, and the residual ratio (%) was calculated.

An irradiation device: LTL-200A-15WCD (manufactured by Nagano Science)

Light source: d-65 lamp

Total exposure and temperature and humidity: 130 kalx. hr (4000 lx. times.325 hr), 25 ℃ 60% RH light irradiation direction: irradiating from above while standing the container on a rotating disk in the container

The residual ratio (%) < 100 × [ all-trans body content after light irradiation (g/100 mL)/all-trans body content immediately after production (g/100mL) ]

The results are shown in Table 12.

[ Table 12]

g/100mL	Example 22	Example 23	Example 24	Example 25
					All-trans form	0.005	0.05	0.005	0.05
Sodium dihydrogen phosphate dihydrate	0.30	0.30	―	―
					Disodium hydrogen phosphate dodecahydrate	3.20	3.20	―	―
Boric acid	―	―	1.30	1.30
					Borax	―	―	0.40	0.40
POE Castor oil	0.002	0.02	0.002	0.02
					Polysorbate 80	0.050	0.50	0.050	0.50
Hydrochloric acid	Proper amount of	Proper amount of	Proper amount of	Proper amount of
					Sodium hydroxide	Proper amount of	Proper amount of	Proper amount of	Proper amount of
Purified water	Proper amount of	Proper amount of	Proper amount of	Proper amount of
					pH	7.5	7.5	7.5	7.5
Osmolarity mOsm	260	260	240	240
					Residual ratio (%)	90.5	92.8	86.1	89.0

When the buffer contains a phosphate buffer, the residual rate of all-trans forms is significantly higher than that when the buffer contains a borate buffer, and the photostability is excellent.

(12) Test for inhibiting adsorption to corneal contact lens

All-trans form or a mixture of all-trans form and 5Z monocistronic form (8: 2 by weight) is added to a surfactant (polysorbate 80) heated to 65 ℃, dissolved in a hot water bath at 65 ℃ for 2 minutes under stirring, water at 65 ℃ is added, then the buffers are mixed and stirred to prepare a homogeneous solution, and the pH and osmotic pressure are adjusted with hydrochloric acid and/or sodium hydroxide. The solution was filtered through a membrane filter (Saimeishik Co., Ltd., bottle top filter) having a pore diameter of 0.2. mu.m, to prepare a clear sterile eye drop. The composition of each eye drop is shown in table 13 disclosed later. These eye drops were filled in a 4mL transparent glass container (Nicotiana daily).

1 piece of soft contact lens (hereinafter referred to as SCL: ACUVUE OASIS (manufactured by Johnson & Johnson, authentication No. 21800BZY10252000, base arc 8.4mm, diameter 14.0mm, degree-3.00D)) was immersed in each 4mL of eye drop, and the resulting solution was stored in an upright state at 25 ℃ for 14 hours (immersion liquid). In addition, soft corneal contact lenses were used, which were initialized by immersing 1 SCL in 10mL of physiological saline (an Otsuka direct injection) overnight after the SCL was taken out from the packaging solution.

4mL of eye drops (blank solution) without the SCL impregnated were treated under the same conditions as those of eye drops (impregnated solution) after the SCL impregnation. The content of each all-trans isomer or a mixture of all-trans isomer and 5Z monocsyn isomer in the blank solution and the immersion solution was determined by HPLC, and the amount adsorbed to SCL (μ g/tablet) was calculated from the difference in the content (each n is 2).

The adsorption amount (μ g/sheet) ═ content (g/100mL) of all-trans form or a mixture of all-trans form and 5Z single-cis form in the blank liquid-content (g/100mL) — content (g/100mL) of all-trans form or a mixture of all-trans form and 5Z single-cis form in the immersion liquid (weight ratio 8: 2))/100 × 4 × 1000 × (g/sheet) }

The results are shown in Table 13.

[ Table 13]

When the phosphate buffer is contained, GGA adsorption to the corneal contact lens is more significantly inhibited than when the borate buffer is contained.

Industrial applicability

The agent of the present invention is excellent in the effect of preventing, ameliorating or treating retinal diseases, and can suppress low-temperature white turbidity, and is also excellent as a pharmaceutical preparation.

Claims

1. Use of geranylgeranylacetone for the manufacture of a prophylactic, ameliorating or therapeutic agent for inflammation of a retinal disease,

the geranylgeranyl acetone is used as a raw material,

(b) consisting of 5E,9E,13E geranylgeranylacetone only; or

(c) Consisting of only 5Z,9E,13E geranylgeranylacetone.

2. Use according to claim 1, which is an ophthalmic preparation.

3. Use according to claim 2, wherein geranylgeranylacetone is contained in an amount of 0.00001 to 10% by weight relative to the total amount of the formulation.

4. The use according to claim 1, which is an oral formulation.

5. Use according to claim 4, wherein geranylgeranylacetone is present in an amount of 0.001 to 80% by weight relative to the total amount of the preparation.

6. The use according to claim 1, wherein the retinal disease is a disease selected from the group consisting of glaucoma, retinitis pigmentosa, age-related macular degeneration and diabetic retinopathy.

7. Use according to claim 1, wherein the pH is 6 to 8.

8. The use of claim 1, further comprising a phosphate buffer.

9. Use of geranylgeranylacetone for the manufacture of a formulation for protecting retinal cells from inflammatory damage,

the geranylgeranyl acetone is used as a raw material,

(b) consisting of 5E,9E,13E geranylgeranylacetone only; or

(c) Consisting of only 5Z,9E,13E geranylgeranylacetone.

10. Use according to claim 9, which is an ophthalmic preparation.

11. Use according to claim 10, wherein geranylgeranylacetone is contained in an amount of 0.00001 to 10% by weight relative to the total amount of the formulation.

12. The use according to claim 9, which is an oral formulation.

13. Use according to claim 12, wherein geranylgeranylacetone is present in an amount of 0.001 to 80% by weight relative to the total amount of the preparation.

14. Use according to claim 9, wherein the retinal cell is a retinal ganglion cell or a retinal pigment epithelial cell.

15. Use according to claim 9, wherein the pH is 6 to 8.

16. The use of claim 9, further comprising a phosphate buffer.

17. Use of geranylgeranylacetone in the manufacture of an inhibitor of degeneration, damage or death caused by inflammation of retinal cells,

the geranylgeranyl acetone is used as a raw material,

(b) consisting of 5E,9E,13E geranylgeranylacetone only; or

(c) Consisting of only 5Z,9E,13E geranylgeranylacetone.

18. Use according to claim 17, in an ophthalmic formulation.

19. Use according to claim 18, wherein geranylgeranylacetone is contained in an amount of 0.00001 to 10% by weight relative to the total amount of the formulation.

20. The use according to claim 17, which is an oral formulation.

21. Use according to claim 20, wherein geranylgeranylacetone is present in an amount of 0.001 to 80% by weight relative to the total amount of the formulation.

22. The use of claim 17, wherein the retinal cell is a retinal ganglion cell or a retinal pigment epithelial cell.

23. The use according to claim 17, wherein the pH is 6 to 8.

24. The use of claim 17, further comprising a phosphate buffer.