JP2009067738A - Prophylactic or therapeutic agent for ischemia-related eye disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent useful for prophylaxis or therapy of ischemia-related eye diseases. <P>SOLUTION: The prophylactic or the therapeutic agent for ischemia-related eye diseases comprises a P2X<SB>7</SB>receptor antagonist as an active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a drug useful for the prevention or treatment of eye diseases associated with ischemia, for example, diseases such as diabetic retinopathy and glaucoma.

  For eye diseases related to ischemia such as diabetic retinopathy and glaucoma, drugs for improving ischemia such as Ca antagonists and peripheral circulation improving agents have been used so far. However, these drugs may cause side effects such as a decrease in blood pressure and flushing of the skin, and may not be used depending on cases.

On the other hand, P2X ₇ receptors in retinal ganglion cells, increased intracellular Ca ^2+, in which (see Non-Patent Document 1) which is reported to be involved in cell death. In addition, it has been reported that expression of P2X ₇ receptor is enhanced in an ischemic model of cultured brain cells (see Non-Patent Document 2).

In addition, the present inventors have found that the P2X ₇ receptor is related to cell death of retinal microvascular cells (see Non-Patent Document 3), and that the P2X ₇ receptor is involved in retinal blood flow reduction in diabetes. (See Non-Patent Document 4).

However, there have been no reports that relate retinal neuronal cell death due to ischemia to the P2X ₇ receptor.
Zhang X et al, IOVS, 2005,46 (6): 2183-91 Wirkner K et al, J Neurochem, 2005,95,1421-1437 Sugiyama T et al, IOVS, 2004,45 (3): 1026-32 Sugiyama T et al, Arch Ophthalmol, 2006,124 (8): 1143-9

  The main object of the present invention is to provide a drug useful for the prevention or treatment of ophthalmic diseases related to ischemia.

As a result of intensive studies on the relationship between retinal neurons and P2X ₇ receptors, the present inventor has found that P2X ₇ receptors are involved in retinal neuronal cell death caused by ischemia. The invention has been completed.

That is, the present invention relates to an agent for the prevention or treatment of ischemia-related eye diseases comprising a P2X ₇ receptor antagonist as an active ingredient.

  Hereinafter, the present invention will be described in more detail.

1. P2X ₇ receptor antagonist In the present invention, the P2X ₇ receptor antagonist means a compound that can completely or partially suppress the activity of the P2X ₇ receptor. A P2X ₇ receptor antagonist can also be referred to as a P2X ₇ receptor antagonist.

Confirmation of the action of the compound on the P2X ₇ receptor can be performed according to a known method. For example, the presence of ethidium bromide P2X ₇ receptor (fluorescent DNA probe), when activated by P2X ₇ receptor agonist, increase in fluorescence of intracellular DNA- binding ethidium bromide is observed. Thus, by quantifying the change in fluorescence can confirm the effect of the substance on P2X ₇ receptor.

Specifically, P2X ₇ receptor antagonists include KN-62 (1- [N, O-dis- (5-iso
-quinolinesulfonyl) -N-methyl-L-tyrosyl] -4-phenylpiperazine), HMA (hexamethylene amiloride), Oxidized-ATP (periodate-oxidized adenosine triphosphate), or BBG (Brilliant blue G).

Moreover, the following compounds can be illustrated:
2-chloro-5-[[2- (2-hydroxy-ethylamino) -ethylamino] -methyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide Dihydrochloride;
2-chloro-5- [3-[(3-hydroxypropyl) amino] propyl] -N- (tricyclo [3.3.1.1] dec-1-ylmethyl) -benzamide hydrochloride;
(R) -2-Chloro-5- [3-[(2-hydroxy-1-methylethyl) amino] propyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl ) -Benzamide hydrochloride;
2-Chloro-5-[[2-[(2-hydroxyethyl) amino] ethoxy] methyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide acetic acid (1: 1) salt, 2-chloro-5- [3- [3- (methylamino) propoxy] propyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) Benzamide hydrochloride;
2-chloro-5- [3- (3-hydroxy-propylamino) -propoxy] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide hydrochloride;
2-Chloro-5- [2- (3-hydroxypropylamino) ethylamino] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide acetic acid (1: 1 )salt;
2-chloro-5- [2- (3-hydroxypropylsulfonyl) ethoxy] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide;
2-Chloro-5- [2- [2-[(2-hydroxyethyl) amino] ethoxy] ethoxy] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide Hydrochloride salt;
2-chloro-5-[[2-[[2- (1-methyl-1H-imidazol-4-yl) ethyl] amino] ethyl] amino] -N- (tricyclo [3.3.1.1.3 ^{, 7} ] Dec-1-ylmethyl) -benzamide;
2-chloro-5-piperazin-1-ylmethyl-N- (tricyclo [3.3.1.1] dec-1-ylmethyl) -benzamide dihydrochloride;
2-chloro-5- (4-piperidinyloxy) -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -benzamide hydrochloride;
2-chloro-5- (2,5-diazabicyclo [2.2.1] hept-2-ylmethyl) -N- (tricyclo [3.3.1.1] dec-1-ylmethyl) -benzamide hydrochloride;
2-chloro-5- (piperidin-4-ylsulfinyl) -N- (tricyclo [3.3.1.1 ^{3, 7]} dec-1-ylmethyl) - benzamide;
5-chloro-2- [3 - [(3-hydroxypropyl) amino] propyl] -N- (tricyclo [3.3.1.1 ^{3, 7]} dec-1-ylmethyl) -4-pyridinecarboxamide;
2-chloro-5- [3-[[(1R) -2-hydroxy-1-methylethyl] amino] propyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl ) -3-pyridinecarboxamide;
5-chloro-2- [3- (ethylamino) propyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -4-pyridinecarboxamide hydrochloride;
5-Chloro-2- [3-[(2-hydroxyethyl) amino] propyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -4-pyridinecarboxamide hydrochloride ;
5-chloro-2- [3-[[(2S) -2-hydroxypropyl] amino] propyl] -N- (tricyclo [3.3.1.1 ^3,7 ] dec-1-ylmethyl) -4- Pyridinecarboxamide dihydrochloride; and N- [2-methyl-5- (9-oxa-3,7-diazabicyclo [3.3.1] non-3-ylcarbonyl) phenyl] -tricyclo [3.3. 1.1 ^3.7 ] Decan-1-acetamide hydrochloride.

Of these, in the present invention, high antagonist selective to the P2X ₇ receptors, for example, Oxidized-ATP is preferably used.

2. Eye disease preventive or therapeutic agent for ischemia-related eye disease preventive or therapeutic agent present invention, by containing, as an active ingredient, P2X ₇ receptor antagonist, according to process for the preparation of known pharmaceutical preparations can be prepared.

The said formulations, in addition to P2X ₇ receptor antagonist, may include a suitable additive or pharmaceutically acceptable carrier. It can also contain other pharmaceutically active ingredients. For example, a medicinal component having a different mechanism of action such as a Ca antagonist and a peripheral circulation improving drug can be contained.

  The form of the preparation is not particularly limited, and can be selected according to the purpose of prevention or treatment. For example, it can be prepared in the form of tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections, aerosols, syrups, eye drops, eye ointments and the like. .

  For example, preparations such as eye drops and eye ointments are produced according to a conventional method using a normal ophthalmic preparation carrier. For example, to produce eye drops, sterilized distilled water is used as a base, and a solubilizing agent, buffer, antioxidant, preservative, isotonic agent, pH adjuster, etc. are blended as necessary. Can be prepared. For example, in order to produce an eye ointment, it can be prepared using a known emulsion base, water-soluble base, suspending base and the like. Representative examples of the base include white petrolatum, purified lanolin, liquid paraffin and the like.

The amount of P2X ₇ receptor antagonist in the prevention or treatment agent for ischemia-related eye diseases can be set according to the degree and type of the disease, formulation, patient age, gender, and other conditions. However, for example, in the case of Oxidized-ATP, it is usually formulated at an appropriate dose to produce an effective tissue concentration of about 10 to 1000 μM, particularly about 30 to 100 μM. In the case of BBG, it is usually formulated at a dose suitable for producing an effective tissue concentration of about 5 to 500 μM, particularly about 10 to 50 μM.

  Also, the administration method is not particularly limited, and administration is performed by a method according to various preparation forms, patient age, sex and other conditions, the degree of disease, and the like. For example, oral administration, intravenous administration, intradermal or subcutaneous administration can be used. In addition, for example, in the case of eye drops, administration can be performed by dropping 1 to 2 drops into the eye from an infusion container. In the case of an eye ointment, it can be administered by application to the eye.

  The dose can also be appropriately set depending on the administration method, patient age, sex and other conditions, and the degree of disease.

  The agent for preventing or treating eye diseases of the present invention is useful for the prevention or treatment of ischemia-related eye diseases. An ischemia-related eye disease is an eye disease that is possibly associated with ischemia, and means an eye disease caused by ischemia.

  Specifically, the ischemia-related eye diseases include diabetic retinopathy, ischemic optic neuropathy, glaucoma, retinal artery occlusion, retinal vein occlusion, retinopathy of prematurity, pulseless disease, and the like.

  In particular, the eye disease preventive or therapeutic agent of the present invention is useful for the prevention or treatment of eye diseases in which ischemia is associated with retinal nerve cell death, in other words, eye diseases associated with retinal nerve cell death due to ischemia.

  In addition, the present invention can be used for suppressing retinal nerve cell death due to ischemia and protecting retinal neurons against ischemia or hypoxia.

  The present invention is useful for prevention or treatment of ischemia-related ocular diseases such as diabetic retinopathy, ischemic optic neuropathy, glaucoma, retinal artery occlusion, retinal vein occlusion, retinopathy of prematurity, and pulseless disease. . The mechanism of action differs from Ca antagonists, peripheral circulation improving agents, and the like that have been applied to these diseases, and can be a new therapeutic method.

  Hereinafter, in order to describe the present invention more specifically, examples and experimental examples will be described. However, the present invention is not limited to these examples.

  In the following, “%” represents “% by weight” unless otherwise specified.

1. Preparation of Retinal Nerve Cells Retinas were collected from embryonic day 18-19 fetal Wistar rats, and their cell suspension (1.0 × 10 ⁶ cells / ml) was seeded on a coverslip and subjected to culture. The culture medium is Earle's Minimal Essential Medium (MEM) containing ₂ mM glutamine, penicillin-streptomycin (100 units / ml-50μg / ml), 25 mM HEPES, 10% fetal bovine serum, and contains 5% CO ₂ Cultured under air. In order to remove non-neuronal cells, 10 μM cytosine arabinoside (ara-C) was added to the culture solution on the fifth day of culture. For the experiment, retinal neurons on the 10th day of culture were used.

  In addition, when the cells were stained with Syntaxin-1, which is a specific marker for amacrine cells, it was found that about 80% were stained, and in particular, amacrine cells accounted for the majority.

2. Confirmation of P2X ₇ receptor by immunohistochemical staining Whether or not the cells prepared in 1 above had P2X ₇ receptor was examined by immunohistochemical staining (fluorescence method).

(Immunohistochemical staining)
Retinal neurons were fixed with 100% ethanol and then placed in PBS containing 5% donkey serum and 1% fetal calf serum for 1 hour at room temperature. After washing with PBS for 5 minutes, soaked in the primary antibody rabbit anti-P2X ₇ antibody solution (1: 1000, Sigma) for 1 hour, and then added to the secondary antibody FITC-conjugated donkey anti-rabbit IgG antibody (1: 300). Soaked for 45 minutes. All were kept at room temperature. Finally, nuclear staining was performed with DAPI, and observation and imaging were performed with an inverted fluorescence microscope system (VZ-8000, Keyence).

FIG. 1 shows the results of immunohistochemical staining. As a result, most neurons were stained, and the presence of P2X ₇ receptor in the cells could be confirmed.

3. Examination of retinal neuronal cell death due to hypoxic load (3-1) Using oxygen / carbon dioxide controller (PROOX Model 110, Physiotech), maintaining oxygen at 5% as usual, maintaining oxygen normally Retinal nerve cells were cultured in a state where the oxygen concentration was decreased from about 20% to 1%, 3%, and 5%. The culture time was 24 hours, or 12 or 6 hours. Cell death was confirmed by the following trypan blue excretion ability.

(Trypan blue discharge capacity)
The cultured retinal neurons are soaked in 1.5% trypan blue solution for 10 minutes at room temperature, fixed with isotonic formalin solution (pH 7.0, 2-4 ° C), washed with physiological saline, and Hoffman modulation microscope (Olympus, Tokyo). Observed at a magnification of 200 times. Count more than 200 cells per coverslip, with cells stained in trypan blue as dead cells and unstained cells as live cells, cell death rate (%), ie, the number of dead cells relative to the total number of cells measured The rate (%) was calculated. In addition, the solidified cells were not counted, and the measurement contents were made unknown to the measurer.

  FIG. 2 shows an example of cell death of retinal nerve cells subjected to hypoxia for 24 hours.

  As a result, it was shown that most cells were stained with trypan blue at an oxygen concentration of 1%, causing cell death.

  (3-2) The results of the above experiments were examined statistically. Using the technique described in (3-1) above, cover slips of n = 6 to 10 were performed using 8 animals, and expressed as mean ± SE. Statistical significance was tested using Bonferroni-test, and less than 5% was considered significant.

  FIG. 3 is a graph showing the oxygen concentration dependence of cell death due to low oxygen load. As a result, it was found that cell death increased significantly as the oxygen concentration decreased.

  (3-3) Furthermore, the time-dependent change of the cell death by hypoxic load was investigated. The experiment was performed on a cover slip of n = 5 to 10 using five animals in the same manner as in the above (3-1), and expressed as mean ± SE. Statistical significance was tested using Bonferroni-test, and less than 5% was considered significant.

  FIG. 4 is a graph showing changes over time in cell death when cultured at an oxygen concentration of 1%.

  As a result, almost no significant cell death occurred until 6 hours after hypoxia, but the cell death increased significantly as 12 hours and 24 hours passed.

4). Effects of P2X ₇ receptor antagonist
(4-1) was examined influence of P2X ₇ receptor antagonists for retinal neuronal cell death due to hypoxia. Retina with low oxygen load as in (3-1) above, except that Oxidized ATP (Sigma Aldrich Japan Co., Ltd.) is used as a P2X ₇ receptor antagonist and Oxidized ATP is dissolved in the culture medium and exposed for 24 hours. Neuronal cell death was examined. Hereinafter, Oxidized ATP is also referred to as “Ox-ATP”.

FIG. 5 shows an example of the results of analyzing retinal neuronal cell death caused by hypoxic load and inhibition by P2X ₇ receptor antagonist.

As a result, it was found that when Oxidized ATP, a P2X ₇ receptor antagonist, was exposed, cell death under hypoxic load was suppressed.

  (4-2) Furthermore, examination was performed by changing the concentration of Oxidized ATP added. The experiment was performed on cover slips with n = 9 to 10 using 8 animals in the same manner as in (4-1) above, and expressed as mean ± SE. Statistical significance was tested using Bonferroni-test, and less than 5% was considered significant.

  FIG. 6 is a graph showing the results of examining the inhibitory effect of Ox-ATP on retinal neuronal cell death caused by low oxygen load at different concentrations.

  As a result, it was found that cell death was suppressed depending on the concentration of Oxidized ATP.

(4-3) Furthermore, the same examination was performed using BBG (Sigma Aldrich Japan Co., Ltd.) as a P2X ₇ receptor antagonist. The experiment was performed in the same manner as in the above (4-1) except that BBG was used as a P2X ₇ receptor antagonist. Using 8 animals, n = 5-10 cover slips were performed and expressed as mean ± SE. Statistical significance was tested using Bonferroni-test, and less than 5% was considered significant.

  FIG. 7 shows the results of analyzing the inhibitory effect of BBG on retinal neuronal cell death caused by low oxygen load at different concentrations.

As a result, as in the case of Ox-ATP described above, it was found that cell death was suppressed depending on the BBG concentration. However, its action was weak compared to Oxidized ATP, which is more selective for the P2X ₇ receptor.

  (4-4) Further, the retinal neuronal cell death in (4-1) above was examined by the following TUNEL staining (fluorescence method). It is said that TUNEL staining can mainly confirm apoptosis.

(TUNEL staining)
Prior to TdT-mediated dUTP nick-end labeling (TUNEL) assay, the cells were fixed by soaking in PBS containing 4% paraformaldehyde for 25 minutes at room temperature. It was immersed in PBS containing 0.2% Triton X-100 for 5 minutes. After that, DeadEnd Fluometric TUNEL System (Promega) was used according to the protocol of the attachment. Finally, DAPI was used as a nuclear stain, and observed and photographed with an inverted fluorescence microscope system (VZ-8000, Keyence). More than 200 cells were counted for each coverslip and distinguished into TUNEL positive cells and negative cells.

  FIG. 8 shows an example of the result of analyzing the apoptosis of retinal neurons by hypoxic load by TUNEL staining.

As a result, it was confirmed that apoptosis increased due to low oxygen load. It was also confirmed that apoptosis was suppressed by the presence of the P2X ₇ receptor antagonist.

  (4-5) Furthermore, the ratio of apoptosis under low oxygen load was examined. The experiment was performed on 164 to 186 retinal neurons on a coverslip using one animal in the same manner as in (4-4) above, and expressed as a percentage (%) of each positive / negative cell. did. Statistical significance was tested using Fisher's exact test, and less than 5% was considered significant.

It shows the result of analyzing the influence of the proportion and the P2X ₇ receptor antagonists of apoptosis (TUNEL-positive cells) by hypoxia in FIG.

As a result, it was confirmed that apoptosis was significantly suppressed by the P2X ₇ receptor antagonist.

5). Cell death due to ATP and effects of P2X ₇ receptor antagonist Furthermore, cell death due to ATP and the effect of P2X ₇ receptor antagonist were examined.

Retinal nerve cell death was examined in the same manner as in (3-1) above, except that ATP was dissolved in the culture and exposed to a normal oxygen concentration of 20% for 24 hours. Furthermore, in addition to ATP, a P2X ₇ receptor antagonist was dissolved, and the effect of the P2X ₇ receptor antagonist on cell death by ATP was examined. Using 8 animals, n = 4-10 cover slips were performed and expressed as mean ± SE. Statistical significance was tested using Bonferroni-test, and less than 5% was considered significant.

FIG. 10 shows the results of analyzing retinal neuronal cell death by ATP and inhibition by P2X ₇ receptor antagonist.

As a result, it was found that cell death increased in a dose-dependent manner with ATP. It was also found that cell death due to ATP was suppressed by the presence of the P2X ₇ receptor antagonist.

6). Cell death by P2X ₇ receptor stimulant (agonist) and influence of P2X ₇ receptor antagonist (6-1) Cell death by P2X ₇ receptor agonist and influence of P2X ₇ receptor antagonist were examined.

The culture solution, dissolving the P2X ₇ receptor agonist, in addition to 24-hour exposure normal oxygen concentration of about 20%, in the same manner as in the above (3-1), were investigated retinal neuronal cell death. Furthermore, in addition to the P2X ₇ receptor agonist, a P2X ₇ receptor antagonist was dissolved, and the influence of the P2X ₇ receptor antagonist on cell death by ATP was examined. Using 8 animals, n = 7-13 cover slips were performed and expressed as mean ± SE. Statistical significance was tested using Bonferroni-test, and less than 5% was considered significant.

BzATP (benzoylbenzoyl-adenosine triphosphate, Sigma Aldrich Japan Co., Ltd.) was used as a P2X ₇ receptor agonist. In addition, Ox-ATP was used as a P2X ₇ receptor antagonist.

FIG. 11 shows the results of analyzing the effects of retinal neuronal cell death by P2X ₇ receptor agonists and P2X ₇ receptor antagonists.

As a result, it was found that cell death increased depending on the concentration of the P2X ₇ receptor agonist. It was also found that it was suppressed by P2X ₇ receptor antagonists.

(6-2) Further, the cell death due to P2X ₇ receptor agonists in the above (6-1) (agonist) were examined by TUNEL staining according to (4-4). FIG. 12 shows an example of the result of analyzing retinal neuronal cell death by P2X ₇ receptor agonist by TUNEL staining.

As a result, it was also confirmed that apoptosis was increased with the P2X ₇ receptor agonist. It was also confirmed that it was suppressed by the P2X ₇ receptor antagonist.

(6-3) Further, the ratio of apoptosis by a P2X ₇ receptor stimulant (agonist) and the effect of the P2X ₇ receptor antagonist on it were examined. The experiment was performed in the same manner as in (6-2) above, and was performed on 158 to 172 retinal neurons on a coverslip using one animal, and expressed as a percentage (%) of each positive / negative cell. did. Statistical significance was tested using Fisher's exact test, and less than 5% was considered significant.

FIG. 13 shows the results of analyzing the proportion of apoptosis (TUNEL positive cells) by the P2X ₇ receptor agonist and the inhibitory effect of the P2X ₇ receptor antagonist.

As a result, it was confirmed that apoptosis was significantly suppressed by the P2X ₇ receptor antagonist.

7). Changes in intracellular Ca concentration by ATP and effects of P2X ₇ receptor antagonist
We examined changes in intracellular Ca concentration by ATP and the effects of P2X ₇ receptor antagonists on it. Changes in intracellular Ca concentration were examined as follows.

(Intracellular Ca measurement)
The retinal neurons attached on the coverslip were treated in a solution containing 2 μM fura2-AM (an ester of intracellular calcium-indicating fluorescent dye) at 37 ° C. for 30 minutes in the dark, and fura-2 was loaded into the cells. . The cells loaded with fura-2 were washed and used for the experiment. The cover slip with the microvessel specimen attached was set in a perfusion chamber on an inverted microscope (IX70 OLYMPUS, Tokyo) connected to an image analysis system (ARGUS / HISCA, Hamamatsu Photonics). The experiment was performed at 37 ° C. The volume of the perfusion chamber was about 80 μl. Fura2 was excited at two wavelengths of 340/380 nm, and the fluorescence intensity at 510 nm was measured. Changes in intracellular calcium concentration were expressed as a fluorescence intensity ratio (F340 / F380). One experiment was performed on 4-7 cover slips obtained from 3 animals and expressed as mean ± SD.

FIG. 14 shows the results of analysis of changes in intracellular Ca concentration by ATP and inhibition by P2X ₇ receptor antagonist. As a result, it was found that the intracellular Ca concentration increased with the addition of ATP, but it was suppressed by the P2X ₇ receptor antagonist.

8). From the above, it was suggested that P2X ₇ receptor is involved in hypoxia-induced retinal neuronal cell death (including apoptosis). Furthermore, it was suggested that retinal neuronal cell death (including apoptosis) due to ischemia can be suppressed by suppressing the activity of the P2X ₇ receptor. Moreover, it was suggested that the increase of intracellular Ca concentration and its suppression may be involved in the mechanism of this cell death (including apoptosis) and its inhibitory effect.

The result of immunohistochemical staining of retinal neurons is shown. The left of FIG. 1 shows the result of nuclear staining with DAPI. The right side of FIG. 1 shows the result of examining the immune activity of the receptor by FITC. The example of the result of having investigated the retinal nerve cell death by hypoxic load by the trypan blue discharge | emission ability is shown. The left side of FIG. 2 shows the result of culturing at a normal oxygen concentration of about 20% for control. The center of FIG. 2 is the result of culturing at an oxygen concentration of 5%. The right side of FIG. 2 shows the result of culturing at an oxygen concentration of 1%. It is the figure which graphed the oxygen concentration dependence of the retinal nerve cell death by a hypoxic load. The vertical axis | shaft of FIG. 3 shows the value of cell death (%). The horizontal axis in FIG. 3 indicates the oxygen concentration (%). C in FIG. 3 is a control, and shows a value when cultured at an oxygen concentration of about 20%. It is a graph which shows a time-dependent change of the retinal nerve cell death when it culture | cultivates by 1% of oxygen concentration. The vertical axis | shaft of FIG. 4 shows cell death (%). The horizontal axis in FIG. 4 shows the time after changing to this oxygen concentration, and Control shows the value when culturing at an oxygen concentration of about 20%. And retinal neuronal cell death due to hypoxia, which is a view showing an example of a result of analyzing the inhibition by P2X ₇ receptor antagonists. The left side of FIG. 5 shows the result of culturing at a normal oxygen concentration of about 20% for control. The center of FIG. 5 is the result of culturing at an oxygen concentration of 5%. The right side of FIG. 5 shows the examination results when Oxidized ATP was added to the culture solution at a concentration of 100 μM at an oxygen concentration of 5%. It is drawing which shows the result analyzed by changing the density | concentration about the inhibitory effect of Ox-ATP to the retinal nerve cell death by hypoxic load. The vertical axis in FIG. 6 represents cell death (%). The horizontal axis in FIG. 6 shows the added concentration of Oxidized ATP when cultured under the condition of an oxygen concentration of 5%. C shows the value when cultured at an oxygen concentration of about 20%. It is drawing which shows the result of having changed the density | concentration about the inhibitory effect of BBG on the retinal nerve cell death by a hypoxic load. The vertical axis in FIG. 7 represents cell death (%). The horizontal axis in FIG. 7 shows the added concentration of BBG when cultured under the condition of an oxygen concentration of 5%. C shows the value when cultured at an oxygen concentration of about 20%. It is drawing which shows the example of the result of having analyzed the apoptosis of the retinal nerve cell by a hypoxic load by TUNEL staining. The left side of FIG. 8 shows the result of culturing at a normal oxygen concentration of about 20% for control. The center of FIG. 8 shows the results of culturing at an oxygen concentration of 5%. The right side of FIG. 8 shows the results when Oxidized ATP was added at a concentration of 100 μM at an oxygen concentration of 5%. It is drawing which shows the result of having analyzed the ratio of the apoptosis of the retinal nerve cell by a hypoxic load. The vertical axis in FIG. 9 indicates the number of cells. Control in FIG. 9 shows the result of culturing at a normal oxygen concentration of about 20%. The center of FIG. 9 shows the results of culturing at an oxygen concentration of 5%. The right side of FIG. 9 shows the results when Oxidized ATP was added to the culture solution at a concentration of 100 μM at an oxygen concentration of 5%. It is a view showing a result of analyzing the suppression by retinal neuronal cell death and P2X ₇ receptor antagonist according to ATP. The vertical axis | shaft of FIG. 10 shows cell death (%). The numerical value on the horizontal axis in FIG. 10 indicates the dissolved concentration of ATP. On the other hand, the rightmost data shows the results when 100 μM Ox-ATP is added in addition to 300 μM ATP. C shows the value when cultured at an oxygen concentration of about 20%. P2X ₇ is a view showing a result of analyzing the suppression by retinal neuronal cell death and P2X ₇ receptor antagonist by receptor agonists. The vertical axis | shaft of FIG. 11 shows cell death (%). The numerical values on the horizontal axis in FIG. 11 indicate the dissolved concentration of BzATP. On the other hand, the rightmost data shows the result when 100 μM Ox-ATP is added in addition to 100 μM BzATP. C shows a value when cultured at an oxygen concentration of about 20% without adding BzATP. Apoptosis of retinal neurons by P2X ₇ receptor agonist is a graph showing the results of analysis by TUNEL staining (fluorescence method). The left side of FIG. 12 shows the results of culturing at an oxygen concentration of about 20% without adding BzATP as a control. The center of FIG. 12 shows the results when 100 μM BzATP was added. The right side of FIG. 12 shows the results when 100 μM Ox-ATP was dissolved in addition to 100 μM BzATP. P2X ₇ is a view showing a result of analyzing the inhibition by apoptosis and P2X ₇ receptor antagonist of retinal neuronal cells by receptor agonists. The vertical axis | shaft of FIG. 13 shows a cell number (%). Control of FIG. 13 shows the result of culturing without adding BzATP. The center of FIG. 13 shows the results when 100 μM BzATP was added. The right side of FIG. 13 shows the results when 100 μM Ox-ATP was added in addition to 100 μM BzATP. It is a view showing a result of analyzing the inhibition due to a change and P2X ₇ receptor antagonist of retinal neuronal intracellular Ca concentration by ATP. The left side of FIG. 14 shows the case where 300 μM ATP was added. The right side of FIG. 14 shows the case where 100 μM Ox-ATP was added before 300 μM ATP was added.

Claims (1)

An agent for the prevention or treatment of ischemia-related eye diseases comprising a P2X ₇ receptor antagonist as an active ingredient.