Mouse myopia induction model and endoplasmic reticulum stress inhibitor for myopia prevention / suppression

Abstract

To obtain a therapeutic agent for suppressing myopia by analyzing the process of myopia progression using a mouse myopia induction model and searching for myopia treatment drugs based on the obtained knowledge.SOLUTION: By attaching a minus lens that can adjust the angle and width according to the growth of the mouse, and a protector, to a young mouse, a model that closely resembles human high myopia can be made. Additionally, it has been shown that from this model analysis, endoplasmic reticulum stress is generated in the sclera by myopia induction and that ER stress induces myopia. Further, it has been revealed that ER stress inhibitors, especially phenylbutyric acid and tauroursodeoxycholic acid, act as myopia prevention / suppression agents.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a method for producing a mouse myopia induction model to elucidate the mechanism by which myopia occurs, and to a drug that suppresses myopia. [Background technology]

[0002] It is said that East Asians have a higher rate of myopia than Westerners, and at least one-third of the Japanese population, or about 40 million people, are myopic. Despite this, the molecular mechanisms involved in the onset and progression of myopia have not been elucidated at all, and although correction is possible with glasses and contact lenses, there is no fundamental treatment.

[0003] Myopia is a condition in which the light is focused in front of the retina, making it difficult to see clearly. There are two main types of myopia: refractive myopia, which occurs when the refractive index of the cornea or crystalline lens is too strong, and axial myopia, which occurs when the axial length, or the length of the eyeball in the front-to-back direction, is too long. Refractive myopia occurs when the thickness of the crystalline lens, which acts as a lens, cannot be adjusted properly, so light focuses in front of the retina. Axial myopia occurs when the axial length is too long, so light focuses in front of the retina (see Figure 1, Axial Myopia). The majority of myopic patients have axial myopia.

[0004] When axial myopia becomes severe, that is, when it reaches a state known as severe myopia, the degree of elongation of the eye axis increases. As a result, the retina and choroid are stretched backwards, increasing the load on them and causing various abnormalities in the fundus. A condition in which abnormalities occur in the fundus is called pathological myopia, which is one of the leading causes of blindness in developed countries. According to a report by the Ministry of Health, Labor and Welfare, pathological myopia is the fourth leading cause of blindness in Japan (2005 Report of the Ministry of Health, Labor and Welfare Retina and Choriooptic Atrophy Research Group). Although pathological myopia can lead to blindness, there is currently no effective treatment, and the establishment of a treatment is desired.

[0005] Traditionally, myopia research has been conducted by inducing myopia in animals such as chicks, tree shrews, and guinea pigs (Non-Patent Document 1). Among them, chicks are often used as model animals for myopia research because they are diurnal, have relatively large eyes, and are easy to handle. Even now, more than 35 years after the introduction of a myopia model using chicks, they are still used as the main myopia model. In addition to chicks, various animals such as tree shrews, marmosets, guinea pigs, and rhesus monkeys are used in myopia research. However, none of these animals are easy to genetically manipulate, and are not suitable for studying the genetic factors of myopia. Since the incidence of myopia varies between races as mentioned above, it is thought that genetic factors play a large role as well as environmental factors. However, one of the reasons why a treatment method to suppress myopia has not been established is that a myopia-induced model animal that can study genetic factors has not been obtained.

[0006] In recent years, myopia models in mice have been reported, and progress in genetic research into myopia is expected. Initially, there was skepticism as to whether myopia could be induced using minus lenses in the same way as in chicks, because mice are nocturnal, but a myopia-inducing model using minus lenses has been created (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2015 / 064768 [Patent Document 2] Special Publication No. 2013-534902 [Non-patent literature]

[0008] [Non-Patent Document 1] Schaeffel, F. & Feldkaemper, M., 2015, Clin. Exp.Optom.,Vol.98,p.507-517. [Non-Patent Document 2] Tkatchenko, T.V. et al., 2010, Invest. Ophthalmol. Vis. Sci., Vol.51, p.1297-1303. [Non-Patent Document 3] Kolb, P.S. et al., 2015, Int. J. Biochem. Cell Biol., Vol.61, p.45-52. [Non-Patent Document 4] Chen, Y. et al., 2016, Scientific Reports, 6:27486, DOI:10.1038 / srep27486. [Non-Patent Document 5] Hatz, C. et al., 2013, Nature Rev. Drug Discov. Vol.12, p.703-719. [Non-Patent Document 6] Hetz, C., 2012, Nature Rev. Mol. Cell Biol., Vol.13, p.89-102. [Non-Patent Document 7] Guillen, C., 2016, Ann. Transl. Med. Suppl.1, S45 [Non-Patent Document 8] Gu, Y., et al., 2012, Acta Pharmacol. Sinica, Vol.33, p.941-952. [Non-Patent Document 9] Liu, H. et al., 2016, Evidence-Based Comple. Alter. Med., Vol.2016, Article ID 7831282 [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] However, the currently reported mouse models do not meet all the symptoms known as symptoms of high myopia in humans. In other words, they do not meet the symptoms such as the degree of axial length elongation, refractive index, and sclera being thinner than normal, making them incomplete as myopia models. The present invention aims to create a mouse model with myopia that closely resembles the symptoms of high myopia in humans. In the conventional mouse model, as described in the model in Non-Patent Document 2, the lens is sutured to the skin and further reinforced and fixed with an adhesive, so it cannot be easily removed. Therefore, it is difficult to observe the changes over time associated with the progression of myopia. The present invention aims to analyze the process of myopia progression using a mouse myopia induction model, search for a treatment drug for myopia based on the knowledge obtained, and obtain a treatment drug that suppresses myopia. [Means for solving the problem]

[0010] The present invention relates to the following preventive / suppressive agent for myopia, a method for producing a mouse myopia-induced model, a mouse myopia-induced model produced by said production method, and a method for screening a drug using the mouse myopia-induced model. (1) An agent for preventing and suppressing myopia, comprising an endoplasmic reticulum stress inhibitor as an active ingredient. (2) The agent for preventing and suppressing myopia according to (1), characterized in that the endoplasmic reticulum stress inhibitor is phenylbutyric acid, tauroursodeoxycholic acid, salubrinal, guanabenz, GSK2606414, GSK2656157, ISRIB, azoramide, arctigenin, or a pharmacologically acceptable salt thereof. (3) The agent for preventing and suppressing myopia according to (1) or (2), wherein the endoplasmic reticulum stress inhibitor is phenylbutyric acid, tauroursodeoxycholic acid, or a pharmacologically acceptable salt thereof. (4) The agent for preventing and suppressing myopia according to any one of (1) to (3), wherein the myopia is axial myopia. (5) The agent for preventing and suppressing myopia according to any one of (1) to (4), wherein the myopia is pathological myopia. (6) The agent for preventing and suppressing myopia according to any one of (1) to (5), which is in the form of eye drops. (7) A method for producing a mouse myopia induction model, comprising attaching a protector and a minus lens to the eyes of a young mouse and adjusting the angle and width using an adjustment mechanism according to the mouse's growth. (8) A method for screening drugs to prevent and suppress myopia, comprising administering a candidate substance to a mouse model in which protectors and minus lenses are placed in front of the eyes of a young mouse and the angle and width are adjusted according to the growth of the mouse to induce myopia. (9) A mouse myopia induction model created by placing protectors and minus lenses in front of the eyes of young mice and adjusting the angle and width using an adjustment mechanism according to the mouse's growth. Effect of the Invention

[0011] We created a mouse model of induced myopia that exhibits symptoms similar to those seen in severe myopia in humans. Compared to other animals, mice have more advanced techniques for genetic manipulation, making them a good tool for studying the genetic causes of myopia. Analysis of this model mouse revealed that endoplasmic reticulum stress occurs in the sclera as myopia is induced. Furthermore, myopia is induced by endoplasmic reticulum stress, indicating that myopia is induced by endoplasmic reticulum stress. Experiments using the mouse model of induced myopia also revealed that an endoplasmic reticulum stress inhibitor is effective in suppressing myopia. Therefore, it is now possible to provide a treatment for myopia, for which there was no effective treatment until now. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an outline of a method for producing a mouse myopia induction model. [Diagram 2] Graph showing changes in refractive index, axial length, and sclera in a mouse myopia induction model. [Diagram 3] Electron microscopy images showing changes in the sclera of a mouse myopia-induced model. [Figure 4] Figure 4 shows the effect of sodium phenylbutyrate (4-PBA), an endoplasmic reticulum stress inhibitor, on myopia induction. Figure 4 (A) shows the refractive index, Figure 4 (B) shows the axial length, and Figure 4 (C) shows the change in axial length after one week and three weeks of lens wear. [Diagram 5] FIG. 1 shows the effect of tauroursodeoxycholic acid, an endoplasmic reticulum stress inhibitor, on myopia induction. [Figure 6] FIG. 1 shows the effect of instilling sodium phenylbutyrate on myopia induction. [Figure 7] FIG. 1 shows the results of an analysis of myopia induction by endoplasmic reticulum stress-inducing agents. [Figure 8] Figure 8 shows the effect of compounds inhibiting different endoplasmic reticulum stress sensor signal pathways. Figure 8(A) shows the results of refractive index analysis, and Figure 8(B) shows the results of axial length analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present inventors have demonstrated for the first time that induction of myopia causes endoplasmic reticulum stress in the sclera. It has been known that endoplasmic reticulum stress affects corneal endothelial cells and causes a number of diseases (Patent Document 1). However, there have been no reports to date that myopia is correlated with endoplasmic reticulum stress, or that endoplasmic reticulum stress induces myopia. Furthermore, the present inventors were the first to discover that the progression of myopia can be inhibited by an endoplasmic reticulum stress inhibitor.

[0014] In addition, myopia induction models have been created in various animals, and myopia induction models in mice have also been reported (Non-Patent Documents 1 and 2). However, no model has been reported that shows symptoms equivalent to those of human myopia, i.e., changes in refractive index, axial length, and sclera. For the first time, a mouse model exhibiting symptoms similar to those of humans has been created by the inventor's method. This makes it possible to study the genetic factors of myopia, which have not been clarified until now, and to screen medicines that provide fundamental treatment for myopia. In fact, the inventors have found that endoplasmic reticulum stress inhibitors suppress the induction of myopia in mouse models.

[0015] When screening for a myopia prevention / suppression agent, the administration of the candidate compound may be performed at any timing. That is, the administration of the candidate compound may be started immediately after the start of myopia induction, or after the symptoms of axial myopia are observed to some extent after the start of myopia induction. The administration period and timing may also be appropriately determined depending on the candidate compound.

[0016] The administration of the myopia suppressant is performed here by intraperitoneal administration and eye drops, but any administration form may be used. Specifically, it may be administered by injection, application as eye drops or eye ointment, or oral administration. Therefore, the dosage form may be an injection, eye drops, eye ointment, or a dosage form suitable for oral administration such as tablets or capsules. In particular, eye drops and eye ointment are preferable because they can be applied directly to the eyes.

[0017] In addition, although 4-phenylbutyric acid (4-PBA) sodium and tauroursodeoxycholic acid (TUDCA) are used as endoplasmic reticulum stress inhibitors here, other pharmacologically acceptable salts may also be used in addition to these compounds. Examples of pharmacologically acceptable salts include alkali metal salts, alkaline earth metal salts, and addition salts of amines or basic amino acids.

[0018] Furthermore, any drug that can suppress endoplasmic reticulum stress may be used. Chemical chaperones that contribute to the formation and stabilization of the higher-order structure of proteins, such as sodium phenylbutyrate, tauroursodeoxycholic acid, and trehalose, are said to reduce endoplasmic reticulum stress. In addition, endoplasmic reticulum stress signals can also be suppressed by inhibiting downstream signals of endoplasmic reticulum stress sensors (Non-Patent Documents 3-5). However, even if the mechanism of action is different, compounds that reduce endoplasmic reticulum stress or suppress signals generated by endoplasmic reticulum stress sensors may act as myopia prevention and suppression agents.

[0019] ER stress is sensed by three stress sensors, which transmit signals downstream to prevent excessive accumulation of misfolded proteins. Three pathways are known to be ER stress sensors: the PERK (PKR-like endoplasmic reticulum kinase) pathway, the IRE1 (Inositol requiring 1) pathway, and the ATF6 (Activating transcription factor 6) pathway (Non-patent Document 6). Therefore, drugs that reduce ER stress by inhibiting the signal transduction of any of these pathways may be used.

[0020] Examples of such drugs include Salubrinal, Guanabenz, GSK2606414, GSK2656157, ISRIB, STF-083010, MKC-3946, Toyocamycin, Nelfinavir, Sunitinib, and 4μ8C (7-Hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-carboxaldehyde) (Non-Patent Document 5). Among these, as shown in the Examples, it has been shown that drugs of the PERK pathway and the ATF6 pathway are effective. Therefore, the PERK pathway inhibitors Salubrinal, Guanabenz, GSK2606414, GSK2656157, and ISRIB can function as effective myopia suppressants. Furthermore, Patent Document 2 discloses PERK inhibitors including GSK2606414 and GSK2656157. It goes without saying that the inhibitors described in Patent Document 2 can also be used.

[0021] In addition, Azoramide and Arctigenin are thought to inhibit endoplasmic reticulum stress at a more upstream stage (Non-Patent Documents 7 and 8). Therefore, these drugs are also thought to act as myopia suppressants.

[0022] In addition, natural compounds contained in plants, such as astragaloside IV, baicalein, berberine, crosin, elatoside C, ginsenoside Rb1, honokiol, icariin, mangiferin, notoginsenoside R1, and pterostilbene, are listed as compounds that suppress endoplasmic reticulum stress (Non-Patent Document 9).

[0023] [Example 1] Preparation of a mouse myopia induction model First, the method for producing the mouse model of the present invention will be described. The mechanism by which axial myopia is induced by wearing a minus lens is shown in FIG. 1. Embophthalmos refers to a state in which an image is clearly seen because parallel light rays entering the eye form an image on the retina. On the other hand, axial myopia refers to a state in which an image is not clearly seen because parallel light rays entering the eye form an image in front of the retina due to the elongated axial length. The eyes of animals, including humans, grow larger as they grow. When a minus lens is worn on a young mouse, the eye axis elongates to the position where an image is formed when wearing a minus lens, that is, to the state where an image is clearly seen when wearing a minus lens. As a result, the eye axis elongates, and an eye state similar to axial myopia can be created.

[0024] Specifically, a mouse myopia induction model is prepared as follows. Since it is easier to induce myopia in young mice, it is desirable to fit minus lenses as soon as possible after weaning. Three-week-old C57BL6J mice are used here. The mice are anesthetized with a triple anesthetic mixture of Domitor (Nihon Zenyaku Kogyo Co., Ltd.), Betolfar (Meiji Seika Pharma Co., Ltd.), and Midazolam (Sandoz Corporation), and the skull is exposed with scissors. A support 1 is erected on the skull and fixed in place with dental cement (Super-Bond, Sun Medical Co., Ltd.). The support has a screw thread so that the adjustment tool described below can be fixed with a nut.

[0025] To induce myopia, a minus lens of -30 diopter (Rainbow Contact, Rainbow Optical Laboratory) 2 is attached to one side, and a lens of 0D as a control or only the frame 3 is attached to the other side. A protector 4 protruding laterally is attached to the frame at the bottom of the lens to prevent the mouse from scratching the lens with its front legs when the lens is attached to the mouse. The protector 4 prevents the mouse from touching the lens, and the lens is not scratched. The protector 4 used here is attached to the frame and integrated, but it does not have to be integrated with the lens as long as the lens is not scratched by the mouse's behavior. For example, it may be shaped like an Elizabethan collar worn by animals that have sustained trauma.

[0026] An adjustment tool 5 is attached to the frame above the lens to adjust the width and angle of the attached lens as the mouse grows. The adjustment tool 5 is bent in an "L" shape, with the lens attached to one end and a long hole 6 provided on the other end so that it can be attached to a support 1 erected on the head. By passing the long hole 6 through the support 1 and screwing it in place with a nut 7, the lens can be attached tightly to the skin and fixed in place without compressing the edges of both eyes of the mouse.

[0027] The adjustment mechanism, consisting of three parts: the support 1, nut 7, and adjustment tool 5, allows the width and angle to be adjusted according to the growth of the mouse, and the lens to be adjusted so that it is positioned at the mouse's eye. In addition, since the lens is removable, it is possible to measure changes in axial length and refractive value over time. As mentioned above, with conventional models it was not possible to observe changes in myopia progression over time, but with this myopia induction model the lens can be easily removed, making it possible to analyze myopia progression in more detail.

[0028] The left eye was given a frame only as a control, and the right eye was given a -30D lens for three weeks. The refractive index, axial length, and scleral thickness were measured, and the difference before and after wearing was calculated. The refractive index was measured using a refractometer (Infrared photorefractor for mice, made by Professor Schaeffel, University of Tubingen) and SD-OCT (Spectral-domain OCT, Envisu R4310, bioptigen Inc.), and the scleral thickness was measured using the imaging software cellSens after taking optical images of HE-stained paraffin sections using an optical microscope (BX53, Olympus Corporation). The results were analyzed using ANOVA and Turkish HSD.

[0029] As shown in Figure 2, the refractive index, axial length, and scleral thickness of eyes in which myopia was induced using -30D lenses were significantly different from the control (in the figure, * indicates p<0.05, ** indicates p<0.01. The same applies to the following figures). Regarding scleral thickness, it was observed that the scleral thickness was thinner in myopic eyes than in normal eyes, regardless of the distance from the optic disc.

[0030] None of the myopia induction models reported so far have been reported to meet all the symptoms of severe myopia, such as refraction, axial length, and scleral changes reported in humans. In contrast, the myopia induction model created in this embodiment has all the characteristics of human axial myopia, indicating that it can be an excellent model. This is thought to be because this myopia induction model allows the lens position to be fine-tuned as the child grows, and furthermore, a protector is provided to protect the lens, so the lens is not scratched, and axial myopia can be induced more significantly.

[0031] [Example 2] Screening of therapeutic drugs using a mouse myopia induction model To investigate the pathology of the myopia induction model in detail, analysis was performed using a transmission electron microscope (TEM). Eyeballs in which axial myopia was induced by wearing minus lenses for 3 weeks and eyeballs in which only a frame was worn as a control were removed from the mice and fixed in 2.5% glutaraldehyde / saline for 1 hour at 4°C. The corneas were removed and post-fixed overnight in 2.5% glutaraldehyde / saline, embedded in Epok812 (Oken Shoji Co., Ltd.), sliced, and observed by TEM (JEM-1400plus, JEOL Ltd.). The upper panel of Figure 3 shows the sclera of a sample obtained from a mouse in which myopia was induced by wearing a control lens, and the lower panel shows the sclera of a sample obtained from a mouse in which myopia was induced by wearing a -30D lens. The scales are 1.0 μm, 500 nm, and 500 nm from the left.

[0032] As shown in the control image in the upper row, the mouse sclera is mostly composed of collagen fibers and fibroblasts. The control fibroblasts are rich in mitochondria and rough endoplasmic reticulum (indicated by the upper arrow). On the other hand, in the sclera of the mouse in which myopia was induced using a minus lens, numerous dilated vacuolated ERs were observed (indicated by the lower arrow), suggesting the occurrence of endoplasmic reticulum stress.

[0033] (1) Effects of endoplasmic reticulum stress inhibitor, sodium phenylbutyrate Observation using an electron microscope suggested that endoplasmic reticulum stress occurs with myopia induction. Therefore, an endoplasmic reticulum stress inhibitor was administered to analyze whether myopia induction could be suppressed. As an endoplasmic reticulum stress inhibitor, sodium phenylbutyrate (Cayman Corporation) was administered intraperitoneally at a dose of 200 mg / kg / day every day from the second day to the 21st day after lens wearing, and refraction and axial length were measured on the 21st day. The control group was administered only PBS.

[0034] Figure 4(A) shows the change in refractive value. In the control group administered PBS, a significant change in refractive value was observed when the -30D lens was worn, but in the sodium phenylbutyrate (4-PBA) administration group, there was no change in refractive value between the eyes wearing the -30D lens and the eyes wearing only the frame, indicating that sodium phenylbutyrate has a myopia suppression effect.

[0035] The change in axial length is shown in Figure 4(B). In the sodium phenylbutyrate group, no difference in axial length was observed when comparing the axial length of eyes wearing minus lenses with eyes wearing only the frame (control). On the other hand, in the PBS group, the axial length of eyes wearing minus lenses was significantly longer than that of the sodium phenylbutyrate group wearing minus lenses.

[0036] The axial length increases with growth, but sodium phenylbutyrate does not inhibit this growth. Figure 4C shows the axial length after 1 week and 3 weeks of lens wear. The axial length of the minus lens-wearing eyes in the PBS-administered group was significantly longer than that of the non-lens-wearing control eyes and the lens-wearing and non-lens-wearing eyes in the sodium phenylbutyrate-administered group 1 week after lens wear. The axial length of the PBS-administered group was longer than that of the frame-wearing eyes 1 week after lens wear in both groups. The axial length of the PBS-administered group was longer than that of the frame-wearing eyes 1 week after lens wear in the sodium phenylbutyrate-administered group, indicating that sodium phenylbutyrate does not affect the normal axial length increase that occurs with growth.

[0037] Sodium phenylbutyrate is a drug that has already been approved for use in the treatment of urea cycle disorders, and its safety in humans has been confirmed. As shown above, it is also clear that it does not interfere with normal axial length growth, making it a very promising drug for inhibiting the progression of severe myopia.

[0038] (2) Effects of tauroursodeoxycholic acid, an endoplasmic reticulum stress inhibitor Next, the effect of tauroursodeoxycholic acid, which is also known as an endoplasmic reticulum stress inhibitor, was analyzed. As in Example 1, the analysis was performed using 3-week-old male C57BL6J mice. The mice were fitted with a -30D lens in the right eye and a frame only in the left eye. From the day of lens fitting, 100 mg / kg tauroursodeoxycholic acid (SIGMA-Aldrich Co., Ltd.) was administered intraperitoneally once a day (n=4), and an equal amount of PBS was administered intraperitoneally to the control group (n=4). Axial length and refraction were measured before and one week after lens fitting, and the amount of change was calculated. The left side of FIG. 5 shows the refraction value, and the right side shows the change in axial length.

[0039] In the control group administered PBS, a significant change in refractive value was observed in the eyes wearing -30D lenses and the eyes wearing only the frame, but in the group administered tauroursodeoxycholic acid, no difference was observed between the two, demonstrating the effect of tauroursodeoxycholic acid in suppressing myopia.In addition, when comparing the change in axial length of the eyes that underwent myopia induction between the group administered tauroursodeoxycholic acid and the group administered PBS, a significant difference was observed, demonstrating the effect of tauroursodeoxycholic acid in suppressing axial length.

[0040] (3) Effect of instilling endoplasmic reticulum stress inhibitors As a drug for suppressing myopia, a dosage form that can be administered directly to the eye, such as eye drops or eye ointment, is desirable because it is highly effective and can be administered by the patient himself. Therefore, a mouse myopia induction model was created in the same manner as in Example 1, and the effect of instilling sodium phenylbutyrate was analyzed.

[0041] Three-week-old male C57BL6J mice were fitted with lenses, and from the day of lens fitting, sodium phenylbutyrate was dissolved in PBS at 0.2% (n=4) or 2% (n=4) and administered as eye drops once a day to both eyes. PBS was administered as eye drops to the control group (n=4). Refraction (Figure 6, left) and axial length (Figure 6, right) were measured before and 3 weeks after lens fitting, and the amount of change was calculated.

[0042] When comparing the refractive index and axial length of eyes in which myopia was induced by wearing lenses, a significant difference was observed in the 2% sodium phenylbutyrate group compared to the PBS group. Therefore, it is recognized that sodium phenylbutyrate is effective in suppressing myopia even when administered by eye drops. In addition, although there was no significant difference in the 0.2% sodium phenylbutyrate group compared to the PBS group, there was a tendency to suppress changes in the refractive index and axial length.

[0043] [Example 3] Effect of endoplasmic reticulum stress induction on myopia As shown above, since inhibitors of endoplasmic reticulum stress have an inhibitory effect on myopia induction, it is believed that endoplasmic reticulum stress is directly involved in myopia induction. Therefore, we analyzed whether myopia can be induced by administering drugs that induce endoplasmic reticulum stress. The subjects were 3-week-old male C57BL6J mice (n = 12). The mice were administered 50 μg / ml tunicamycin (Tm) (Sigma Aldrich Co., Ltd.) or 10 μM thapsigargin (TG) (Wako Pure Chemical Industries, Ltd.) in the right eye, and PBS (Veh) in the left eye once. The refractive index and axial length were measured before and one week after administration of tunicamycin or thapsigargin, and the changes were calculated (Figure 7).

[0044] Administration of tunicamycin and thapsigargin, both of which are known to induce endoplasmic reticulum stress, induced significant differences in both refractive index and axial length compared to PBS-administered eyes, inducing myopia. In other words, it was shown that endoplasmic reticulum stress directly induces myopia.

[0045] [Example 4] Effect of endoplasmic reticulum stress pathway inhibitors on myopia induction As mentioned above, it is known that there are three pathways downstream of endoplasmic reticulum stress: the IRE1 pathway, the PERK pathway, and the ATF6 pathway. We analyzed whether inhibitors of the three endoplasmic reticulum stress pathways have an effect of suppressing myopia induction.

[0046] We used STF-083010 (STF) as an inhibitor of the IRE1 pathway, GSK265615 (GSK) as an inhibitor of the PERK pathway, and Nelfinavir (NFV) as an inhibitor of the ATF6 pathway. We administered these drugs to a mouse myopia induction model and analyzed whether myopia induction was suppressed.

[0047] As in Example 1, 3-week-old male C57BL6J mice were fitted with a -30D lens in the right eye and only a frame in the left eye. From the day of lens fitting, 60 μM STF-083010 (SIGMA Aldrich Co., Ltd.) (n=3), 50 μM GSK2656157 (Cayman Co., Ltd.) (n=3), or 50 μM Nelfinavir (Tokyo Chemical Industry Co., Ltd.) (n=3) dissolved in PBS were administered once a day to both eyes. 0.1% DMSO (SIGMA Aldrich Co., Ltd.) was dissolved in PBS and administered as eye drops to the control group (n=3). Refraction and axial length were measured before and one week after lens fitting, and the amount of change was calculated (FIG. 8). In Figure 8, the differences (amount of change) in refractive values ​​(Figure 8(A)) and axial length (Figure 8(B)) before and after lens wearing are shown on the left, and to compare the difference in the amount of change between the eyes wearing lenses and the control eyes, the difference in the amount of change was calculated and shown as the graph on the right.

[0048] Like DMSO, STF-083010 caused significant changes in both refractive index and axial length compared to control eyes that did not wear lenses, and did not suppress myopia induction. On the other hand, in the GSK2656157 and Nelfinavir eye drop groups, changes in refractive index and elongation of axial length were observed even in control eyes that did not wear lenses. However, since the difference in the amount of change compared to eyes that wore lenses and had myopia induced was significantly reduced (Figure 8 (A) and (B) right graph), it is thought that there is an effect of suppressing myopia induction.

[0049] These results indicate that myopia is induced by endoplasmic reticulum stress, and that myopia induction can be suppressed by inhibiting endoplasmic reticulum stress. In addition, myopia can be suppressed not only by using drugs such as phenylbutyric acid and tauroursodeoxycholic acid, which suppress endoplasmic reticulum stress itself, but also by inhibiting the downstream signal that transmits endoplasmic reticulum stress. In particular, myopia was effectively suppressed by inhibiting the signal generated by endoplasmic reticulum stress sensors PERK and ATF6. [Industrial Applicability]

[0050] It has become clear that endoplasmic reticulum stress inhibitors can suppress the progression of myopia, a condition for which there has been no effective treatment until now. Therefore, endoplasmic reticulum stress inhibitors may act as a treatment for myopia. In addition, using mice, which are easy to genetically analyze, we were able to create an induced myopia model that exhibits symptoms similar to those of human myopia. In the future, by using the induced myopia mouse model, it will be possible to elucidate the molecular mechanisms of myopia development and develop molecular targeted drugs. [Explanation of symbols]

[0051] 1 Support, 2 Minus lens, 3 Frame, 4 Protector, 5 Adjustment tool, 6 Slot, 7 Nut

Claims

[Claim 1] An agent for preventing, treating, or suppressing myopia, comprising as an active ingredient a drug that suppresses endoplasmic reticulum stress itself.

Images