JP2003313123A - Composition for preventing and/or treating ophthalmopathy (excluding ophthalmopathy caused by dry eye) caused by apoptosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a composition for preventing and/or treating ophthalmopathy (excluding ophthalmopathy caused by dry eye) caused by apoptosis. <P>SOLUTION: This composition for preventing and/or treating the ophthalmopathy (excluding ophthalmopathy caused by dry eye) caused by apoptosis contains 3-hydroxybutyric acid and/or its salt as an active ingredient. Further, the composition for preventing and/or treating the ophthalmopathy (excluding the ophthalmopathy caused by the dry eye) caused by the apoptosis, especially keratoconjunctivitis, contains the above active ingredient. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition containing 3-hydroxybutyric acid and / or a salt thereof as an active ingredient for the prevention and / or treatment of eye disorders due to apoptosis (excluding disorders due to dry eye). Regarding things. More specifically, it relates to a composition for preventing and / or treating an eye disorder due to apoptosis (excluding a disorder caused by dry eye), particularly a disorder of the corneal conjunctiva, containing the above-mentioned active ingredient.

[0002]

2. Description of the Related Art Apoptosis
Is cell death caused by activation of a cell self-destructing program controlled by a cell gene. The characteristics of this apoptosis include cell shrinkage, chromatin aggregation, chromatin DNA fragmentation, appearance of apoptotic bodies and cell fragmentation, and it is considered to be a form of active cell death that progresses in a short time. ing.

In recent years, much research has been done on this apoptosis, and its mechanism is gradually being elucidated. In particular, activation of caspase, which is a part of the mechanism, plays an important role in inducing apoptosis, and caspase 3 is considered to play a particularly important role. Therefore, suppressing caspase 3 activity is very important for suppressing apoptosis.

Apoptosis is an essential phenomenon in the developmental process of organisms and maintenance of homeostasis, but is induced by natural occurrence, deficiency of various factors such as nutritional factors and growth factors, and physical stress such as radiation. In addition, it is also known to be induced by removing serum from the medium in, for example, cell culture.

At present, there is a decrease in lymphocytes in AIDS, cancer cell death due to various anticancer agents, Parkinson's disease, dementia,
In diseases such as ischemic nerve cell death, it has been suggested to be associated with apoptosis.

Furthermore, it is considered that apoptosis is involved in various diseases other than the above-mentioned various diseases. Of the disorders suggested to be involved in apoptosis, examples of disorders related to the eye include:
Examples include retinal damage due to ischemia / reperfusion, glaucoma, optic nerve transection, retinal detachment, retinal degeneration, cataract and the like. Furthermore, it has recently been suggested that apoptosis is involved in disorders of the keratoconjunctiva. In particular, it has been suggested that apoptosis is involved in herpes virus, acanthamoeba, platelet activating factor, benzalkonium chloride, ultraviolet rays, and other disorders, keratoconus pathogenesis, and corneal damage after excimer laser irradiation.

Among the above-mentioned eye-related disorders, some drugs or methods for treating retinal disorders, glaucoma and cataract, which are said to be involved in apoptosis, have been disclosed. For example, a therapeutic agent for a retinal disease containing α-phenyl-t-butyl-nitrone and a derivative thereof (see, for example, Patent Document 1), a cell of a retinal nerve that can cause a disorder in which intraocular pressure increases (eg, glaucoma) Apoptotic cell death inhibitors that prevent retinal disorders associated with death or aging processes (eg, age-related macular degeneration) (see, eg, US Pat. No. 5,837,819), methods of interfering with cell regeneration after cataract surgery (eg, US Pat. 3) are disclosed respectively.

However, it is not known that 3-hydroxybutyric acid, which is an active ingredient of the present invention, is effective for the prevention and / or treatment of eye disorders due to apoptosis (excluding disorders due to dry eye).

[0009]

[Patent Document 1] Japanese Patent Laid-Open No. 9-278652

[Patent Document 2] Japanese Patent Publication No. 2001-513358

[Patent Document 3] Japanese Patent Publication No. 10-508575

[0010]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
It is intended to provide a composition containing hydroxybutyric acid and / or a salt thereof as an active ingredient, for preventing and / or treating an eye disorder due to apoptosis (excluding a disorder caused by dry eye). More specifically, it is intended to provide a composition containing the above-mentioned active ingredient for the prevention and / or treatment of eye disorders due to apoptosis (excluding disorders due to dry eye), particularly disorders of the corneal conjunctiva.

[0011]

According to the present invention, the above objects and advantages of the present invention are achieved by the following. (1) A composition for preventing and / or treating an eye disorder due to apoptosis (excluding a disorder due to dry eye) containing 3-hydroxybutyric acid and / or a salt thereof as an active ingredient. (2) The composition according to (1) above, wherein 3-hydroxybutyric acid is a D-form. (3) The salts of 3-hydroxybutyric acid are sodium salt, potassium salt, L-lysine salt, L-histidine salt and L-
The composition according to (1) or (2) above, which is at least one selected from the group consisting of arginine salts. (4) The above (1), wherein the concentration of 3-hydroxybutyric acid and / or its salt is 0.1 to 1000 mmol / L.
~ The composition according to any one of (3) above. (5) The composition according to any one of (1) to (4) above, wherein the concentration of 3-hydroxybutyric acid and / or its salt is 1 to 150 mmol / L. (6) The composition according to any one of (1) to (5) above, wherein the eye disorder due to apoptosis (excluding the disorder due to dry eye) is a disorder of the keratoconjunctiva.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, 3-hydroxybutyric acid used as an active ingredient is known as a biological component, is produced by the oxidation of fatty acids in the liver, and is used as an energy source in peripheral tissues. It is known that (Ikuo Yamashina's supervision, "The New Chemistry of Leninger (1)", 2nd Edition, Hirokawa Shoten, April 1993.
P.625-626, 15th of March). Regarding the configuration at the C3 position in the chemical structural formula of 3-hydroxybutyric acid, D-
Form, D, L-racemate and L-form are known. In the present invention, all of them can be used, but of these, the D-form is most preferable in terms of prevention and / or treatment of eye disorders due to apoptosis (excluding disorders due to dry eye). The salts of 3-hydroxybutyric acid are preferably sodium salt, potassium salt and L salt.
-At least one kind is appropriately selected from the group consisting of lysine salt, L-histidine salt and L-arginine salt. These 3-hydroxybutyric acid and / or salts thereof can be used alone or in combination of two or more. The concentration of 3-hydroxybutyric acid and / or its salts in the composition of the present invention is 0.1 to 1000 mmol / L, more preferably 0.5 to 500 mmol / L, depending on the age and symptoms of the patient and its use. , Furthermore, 1-150
The range of mmol / L is particularly preferable.

In the present invention, 3-hydroxybutyric acid, which is an active ingredient, is effective for the prevention and / or treatment of eye disorders due to apoptosis (excluding disorders due to dry eye). More specifically, the composition of the present invention containing the above-mentioned active ingredient is effective for the prevention and / or treatment of eye disorders due to apoptosis (excluding disorders due to dry eye), particularly disorders of the corneal conjunctiva. Examples of the ocular disorders due to apoptosis, to which the composition of the present invention is particularly effective, include herpes virus, acanthamoeba, platelet activating factor, benzalkonium chloride, ultraviolet rays and the like, keratoconus and corneal damage after excimer laser irradiation, etc. Is mentioned.

The composition of the present invention is appropriately used orally (tablets, granules, etc.) or parenterally (eye drops, drops, etc.). The form of the formulation comprising the composition of the present invention includes, for example, eye drops, drops, injections, eye ointments, tablets, granules, powders, capsules, etc., all of which are appropriately prepared by known methods. be able to. For these preparations, isotonic agents, buffers, stabilizers, preservatives, which are usually used,
Thickeners, pH adjusters, excipients, binders, dispersants, reabsorption promoters, surfactants, solubilizers, emulsifiers, humectants, colorants, fragrances, organic solvents (eg alcohols, oils) ),
It may contain silicones, polymers, solid fatty substances (such as waxes), etc., and can be appropriately selected and prepared according to the application.

Among the forms of the above-mentioned preparations, the composition of the present invention is preferably an eye drop or an eye ointment, especially for topical administration to the eye, and further, More preferably.

For example, when the composition of the present invention is used as an eye drop, an isotonic agent, a buffering agent, a stabilizing agent, a stabilizing agent, and a stabilizer may be added, if necessary, for the purpose of obtaining stability and comfortability of the eye drop. An agent, a thickening agent, a pH adjusting agent and the like can be contained. In addition, these components can be contained alone or in appropriate combination of two or more kinds, and in many cases, it is preferable.

The isotonicity agent that can be contained when the composition of the present invention is used as an eye drop is not particularly limited as long as it is generally used as an eye drop. When the composition of the present invention is used as an eye drop, a tonicity agent can be included for the purpose of adjusting the osmotic pressure of the eye drop.
Examples of such isotonicity agents include inorganic salts such as alkali or alkaline earth metal salts consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride and magnesium sulfate, and glucose, mannitol, sorbitol, xylitol and Examples include sugars such as dextran. These can be used alone or in combination of two or more. The concentration of these isotonicity agents is preferably 0.001 to 5 w / v%, more preferably 0.01 to 3 w / v%.

The buffering agent that can be contained in the composition of the present invention as an eye drop is not particularly limited as long as it is one generally used as an eye drop. When the composition of the present invention is used as an eye drop, a buffer may be contained for the purpose of stabilizing the pH of the eye drop. Such buffers include, for example, phosphate buffers such as disodium monohydrogen phosphate, sodium dihydrogen phosphate, dipotassium monohydrogen phosphate and potassium dihydrogen phosphate,
Examples thereof include boric acid buffers such as boric acid and sodium borate, and tris buffers such as trisaminomethane and dilute hydrochloric acid, and trismalate and dilute caustic soda solution. These can be used alone or in combination of two or more. The concentration of these buffers is 0.001
It is preferably 5 w / v%, and 0.01 to 1 w / v%
Is more preferable.

As the stabilizer that can be contained when the composition of the present invention is used as an eye drop, any stabilizer generally used as an eye drop may be used. When the composition of the present invention is used as an eye drop, the stabilizer may be contained for the purpose of stabilizing the active ingredient of the eye drop. Examples of such stabilizers include disodium ethylenediaminetetraacetate, citric acid and citrates. These can be used alone or in combination of two or more. As the concentration of these stabilizers,
It is preferably 0.001 to 5 w / v%, and 0.01
More preferably, it is ˜1 w / v%.

When the composition of the present invention is used as an eye drop, the thickening agent that can be contained is not particularly limited as long as it is a commonly used eye drop. When the composition of the present invention is used as an eye drop, the thickening agent can be contained for the purpose of adjusting the viscosity of the eye drop. Examples of such a thickening agent include polyols such as glycerin, ethylene glycol, propylene glycol, polyethylene glycol and polyvinyl alcohol, trehalose, sucrose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cyclodextrin and other sugars. And polycarboxylic acids / salts such as carboxyvinyl polymers, citrates and edetates, and may also contain hyaluronate, povidone and the like. These can be used alone or in combination of two or more. The concentration of these thickeners is 0.001 to 10w
/ V% is preferable, and 0.01 to 5 w / v% is more preferable.

The pH adjusting agent that can be contained in the composition of the present invention as an eye drop is not particularly limited as long as it is generally used as an eye drop.
When the composition of the present invention is used as an eye drop, the pH adjuster can be contained for the purpose of adjusting the pH of the eye drop. Examples of such a pH adjusting agent include hydrochloric acid, citric acid or its salt, boric acid or its salt, phosphoric acid or its salt, acetic acid or its salt, tartaric acid or its salt, sodium hydroxide, potassium hydroxide and the like. Can be mentioned. These can be used alone or in combination of two or more.
The composition of the present invention is adjusted to a desired pH by adding an appropriate amount of these pH adjusters.

On the other hand, when the composition of the present invention is used as an eye drop, it may contain an antiseptic agent for the purpose of imparting an antiseptic effect to the eye drop. In this case, the preservative that can be contained is not particularly limited as long as it is generally used as an eye drop, but since benzalkonium chloride may cause apoptosis, it may be used together with the composition of the present invention. It is not preferable to use it. Examples of preservatives that can be contained in the composition of the present invention include ethylparaben, butylparaben, benzethonium chloride, chlorhexidine gluconate, citric acid and boric acid. These can be used alone or in combination of two or more. The concentration of these preservatives is preferably 0.0001 to 0.1 w / v%, more preferably 0.001 to 0.05 w / v%.

However, in general, when an eye disorder is caused, the eye disorder may be further aggravated by instilling an eye drop containing a preservative,
It is more preferable not to include a preservative. In addition, even if the eye is not damaged, if frequent eye drops are required, the preservative may cause damage to the eye as well. preferable. In addition, when the preservative is not contained, the eye drop of the present invention
It is preferable to fill a so-called “disposable container” of a type that is disposable after one use.

When the composition of the present invention is used as an eye drop, other components include an antihistamine, a vasoconstrictor, an antiphlogistic / astringent, which are generally used as ophthalmic agents.
Lubricants, amino acids, antiallergic agents, vitamins, sulfa drugs, local anesthetics, miotics, etc. may also be contained. In addition, when these components are contained, it is necessary to have components and concentrations that do not affect the effect of 3-hydroxybutyric acid, which is an active ingredient, depending on the age and symptoms of the patient.

When the composition of the present invention is used as an eye drop, various components are combined to adjust the pH range to pH 5 to 8.
Is preferred. An acidic region of pH 5 or lower or an alkaline region of pH 8 or higher may cause eye irritation or eye damage, which is not preferable. Regarding the osmotic pressure, it should be 150 to 700 mOsm.
The range is preferably

When the composition of the present invention is used as an eye drop, the dose is not particularly limited as long as it is within the range provided ophthalmically, and usually 1 to 3 drops per dose is 1 drop.
It is preferable to administer several times a day.

[0027]

EXAMPLES The present invention will be specifically described below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 To investigate the effect of serum and D-3-hydroxybutyric acid (hereinafter abbreviated as HBA) on the cell activity of human conjunctival epithelial cells subjected to serum removal stimulation,
The experiment was conducted according to the following method. Human conjunctival epithelial cells (CCL2
0.2) was seeded at 5 × 10 ⁴ / well and cultured in a culture solution containing 10% serum (serum manufactured by Invitrogen) for 2 days. After completion of the culture, the culture solution was removed, washed twice with a phosphate buffer solution (hereinafter abbreviated as PBS), replaced with each culture solution containing various concentrations of serum or HBA, and cultured for 24 hours. The osmotic pressure of each culture solution was 470 mOsm. Was prepared. After completion of the culture, each culture solution was removed to give 10 cells per well.
A culture solution containing% Alamar Blue was added. The Alamar Blue-containing culture medium contained 10% serum. Fluorescent plate reader (Applied Bio S)
The fluorescence intensity (fluorescence intensity 1) of each well at an excitation wavelength of 530 nm and a fluorescence wavelength of 580 nm was measured by using Y. Systems. Then, the cells were cultured in a CO ₂ incubator for 30 minutes, and the fluorescence intensity (fluorescence intensity 2) was measured again. Fluorescence intensity 1 was subtracted from fluorescence intensity 2 to calculate the amount of increase in fluorescence intensity, and this value was defined as cell activity. Regarding the control, the cell activity after the first two days of culture was measured and the value was used as a control. Further, in all Examples, 199 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was used as the culture solution. Furthermore, in this example, 4 wells were used for each condition other than the control. The results obtained according to the above method are shown in FIG. In addition, various concentrations of serum or H
The cell activity of each culture medium containing BA was shown as a relative activity value with respect to the control cell activity.

From the results of FIG. 1, the activity of cells was significantly reduced by removing serum, which was about 5% of that of the control.
It fell to 0%. HBA suppressed this decrease in cell activity in a concentration-dependent manner, and at 80 mmol / L HBA, it showed about 70% of the cell activity of the control, which was equivalent to a value of 5% serum. From this result, it was found that HBA suppresses the decrease in cell activity due to the removal of serum, and has a suppressive effect almost equivalent to that of serum.

Example 2 Whether the inhibitory effect of HBA on the decrease in cell activity was suppressed by suppressing the decrease in cell activity itself or by suppressing the decrease in cell number was investigated. By the method shown in Example 1, using 10% serum-containing culture solution, 80 mmol / L HBA-containing culture solution and serum-free culture solution, the treatment time of each culture solution was 3, 6, 12, respectively.
And at 24 hours, the cell activity was measured, and at the same time, the cell number was also measured. 6.6 × 10 ⁵ cells were seeded in a 25 cm ² flask. The results for cell activity are shown in FIG. 2 and the results for cell number are shown in FIG.

From the results of FIG. 2 and FIG. 3, it was found that the decrease in cell activity was due to the decrease in cell number as the cell activity decreased as the cell number decreased. That is, it was found that cell death occurred. Also, 80
Since cell death was suppressed in the mmol / L HBA-containing culture medium as compared with the serum-free culture medium, it was found that HBA has an excellent cell death-suppressing effect on cell death due to serum removal. Further, it is known that cell death due to serum removal is due to apoptosis, and it was found that HBA has an effect of suppressing cell death due to apoptosis.

Example 3 The activity of caspases involved in the induction of apoptosis was investigated using serum and HBA. In addition, this example was performed by the following method. In a 75 cm ² flask, human conjunctival epithelial cells (CCL2
0.2) was seeded in 2 × 10 ⁶ cells and cultured for 2 days in a culture solution containing 10% serum (serum manufactured by Invitrogen). After completion of the culture, the culture solution was removed, washed twice with PBS, replaced with each culture solution containing serum or HBA, and cultured. The osmotic pressure of each culture solution was 470 mOsm. Adjusted to. After treatment for each culture treatment time of 3, 6, 12 and 24 hours, the culture medium was removed, washed twice with PBS, and cells in each flask were recovered by trypsin treatment,
Cells were pelleted by centrifugation. PBS cooled with ice was added to resuspend the cells, and then the cells were precipitated by centrifugation. The above procedure was repeated once. After the cells were precipitated by centrifugation and PBS was removed, lysis buffer (50 mmol / L HEPES,
1 mmol / L DTT, 0.1 mmol / LEDT
A, 0.1% CHAPS, 0.1 mmol / L PMS
F, pH 7.4). After centrifugation, 1 μL of each supernatant was collected, and the protein concentration in the collected supernatant was measured by the BCA micro method. 96 well plate, supernatant protein, assay buffer (50 mmol / L HEPES, 100 mmol
/ L NaCl, 10 mmol / L DTT, 1 mmo
1 / L EDTA, 10% glycerol, 0.1% CH
APS, 0.1 mmol / L PMSF, pH7.
4), 100 μmol / L caspase substrate [Ac-As
p-Glu-Val-Asp-AFC (Enzyme
Systems Products)]] with a total capacity of 1
It was prepared to be 00 μL. Here, the caspase acts on this substrate to release AFC from this substrate, and the released AFC fluoresces. Therefore, after incubating this solution at 37 ° C. for 1 hour, using a fluorescence plate reader, excitation wavelength 400 nm, fluorescence wavelength 508 nm
The fluorescence intensity of each well was measured. The fluorescence intensity was measured in the same manner only for the AFC standard which is a fluorescent substance and the substrate. The value obtained by subtracting the value of only the substrate from the value of each well was taken as the value of each sample, the AFC production amount of each sample was calculated from the AFC standard straight line, and the caspase activity per protein amount was calculated. The results obtained according to the above method are shown in FIG.

From the results shown in FIG. 4, 80 mmol /
The L HBA-containing culture solution showed an excellent caspase activity inhibitory effect after 3 hours, 6 hours, and 12 hours, as compared with the serum-free culture solution. In addition, an excellent caspase activity inhibitory effect was exhibited after 6 hours and 12 hours with respect to the serum-containing culture solution, and substantially the same effect was exhibited after 24 hours. From these, it was found that HBA suppresses the activity of caspase involved in the induction of apoptosis.

Example 4 In Example 3, when the cell morphology was also observed, cell loss due to apoptosis was already observed after 3 hours of culture. Therefore, for the purpose of investigating the inhibitory effect of HBA on caspase activity, the caspase activity before cell loss was measured according to the method described in Example 3. In addition,
Each processing time is 0 minutes, 15 minutes, 30 minutes, 45 minutes and 60 minutes.
I went as a minute. The result is shown in FIG.

From the results shown in FIG. 5, at the time of main culture treatment, the 80 mmol / L HBA-containing culture solution showed an excellent caspase activity suppressing effect as compared with both the serum-containing culture solution and the serum-free culture solution. Indicated. From these, it was found that HBA suppresses the activity of caspase involved in the induction of apoptosis in a short time.

Example 5 Chromatin aggregation involved in the induction of apoptosis was investigated using serum and HBA. In addition, this example was performed by the following method. Human conjunctival epithelial cells (CCL20.2) cultured by the method described in Example 1 were cultured in a culture medium containing 10% serum, 80 m
The culture solution was replaced with a culture solution containing mol / L HBA and a culture solution containing no serum, and each culture solution was treated for 1, 3 and 6 hours. The 80 mmol / L HBA-containing culture solution and the serum-free culture solution were adjusted to an osmotic pressure of 470 mOsm. Adjusted to. After the treatment, each culture medium was removed, and Hoechst3334 was removed.
2 (hereinafter abbreviated as Ho) dye is dissolved in PBS and 10 μg /
Add 100 μL of the solution in mL to each well,
Incubated for 30 minutes in a CO ₂ incubator. Collect the solution and use a fluorescence plate reader (Applie).
d Bio Systems), excitation wavelength 36
The fluorescence intensity of each well at 0 nm and a fluorescence wavelength of 450 nm was measured. After the measurement, the cultured cells were washed with PBS, and neutral red (hereinafter abbreviated as Nr) was dissolved in 199 medium to 0.00.
200 μL of a 5% solution was added to each well,
Incubated for 3 hours in a CO ₂ incubator. After removing the staining solution and washing the cultured cells twice with PBS, the cells were treated with a 1% acetic acid / 50% ethanol solution for 15 minutes at room temperature to extract Nr. Extract the extract with a fluorescent plate reader (Applied B)
io Systems), excitation wavelength 535n
m, the fluorescence intensity of each well at a fluorescence wavelength of 600 nm was measured. The fluorescence intensity of each solution obtained as a blank from the fluorescence intensity of each solution (Ho solution and Nr solution) (only Ho solution and N solution)
Ho value and N
The value was r. The Ho relative value and the Nr relative value of each individual in the treated group were calculated when the average value of the Ho value and the Nr value of the untreated group was 1.0. Divide the Ho relative value by the Nr relative value to obtain Ho / Nr of each individual.
The ratio was calculated. The results obtained according to the above method are shown in FIG. here,
The Ho / Nr value indicates the degree of chromatin aggregation per unit cell. The Ho / Nr value in the untreated group was 1.0,
A value larger than 1.0 indicates that apoptosis is occurring in proportion to the value. Furthermore, 1.0
A smaller value indicates that necrosis is occurring.

From the results shown in FIG. 6, 80 mmol /
The cells treated with the L HBA-containing culture medium and the 10% serum-containing culture medium had significantly less degree of chromatin aggregation per unit cell than the cells treated with the serum-free culture medium. It turns out that you are suppressing death. Further, since the value is close to 1.0, it was found that HBA has a very excellent inhibitory effect on apoptosis induced by serum withdrawal. Furthermore, it was found that the 80 mmol / L HBA-containing culture solution had a slightly superior inhibitory effect than the 10% serum-containing culture solution.

[0038]

EFFECTS OF THE INVENTION The present invention can prevent and / or treat ocular disorders (excluding disorders due to dry eye) due to apoptosis by containing 3-hydroxybutyric acid and / or its salts as an active ingredient. it can. Furthermore, by containing the above-mentioned active ingredient, it is possible to prevent and / or treat ocular disorders due to apoptosis (excluding disorders due to dry eye), particularly keratoconjunctival disorders.

[Brief description of drawings]

FIG. 1 Serum and D-3-hydroxybutyric acid (HB
It is a graph which showed the cell activity in each concentration condition of A).

FIG. 2 is a graph showing the inhibitory effect on the decrease in cell activity by serum and HBA.

FIG. 3 is a graph showing the inhibitory effect on cell number reduction by serum and HBA.

FIG. 4 is a graph showing the inhibitory effect on caspase activity at each treatment time by serum and HBA.

FIG. 5 is a graph showing the inhibitory effect on caspase activity at each treatment time by serum and HBA.

FIG. 6 is a graph showing the degree of chromatin aggregation per unit cell at each treatment time with serum and HBA.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kazuo Tsubota             5-26-7 Nishifune, Funabashi City, Chiba Prefecture (72) Inventor Akihiro Higuchi             1-15-5 Nishiyama, Kashiwa City, Chiba Prefecture F-term (reference) 4C206 AA02 DA07 KA17 MA01 MA04                       MA78 NA14 ZA33

Claims (6)

[Claims] 1. Prevention and / or prevention of eye damage due to apoptosis (excluding dry eye damage), which contains 3-hydroxybutyric acid and / or a salt thereof as an active ingredient.
Or a composition for treatment. 2. The composition according to claim 1, wherein 3-hydroxybutyric acid is D-form. 3. The salt of 3-hydroxybutyric acid is at least one selected from the group consisting of sodium salt, potassium salt, L-lysine salt, L-histidine salt and L-arginine salt. The composition according to 2. 4. The composition according to claim 1, wherein the concentration of 3-hydroxybutyric acid and / or its salt is 0.1 to 1000 mmol / L. 5. The concentration of 3-hydroxybutyric acid and / or its salts is 1 to 150 mmol / L.
~ The composition according to claim 4. 6. The eye disorder caused by apoptosis (excluding the disorder caused by dry eye) is a disorder of the keratoconjunctiva.
~ The composition according to claim 5.