KR102629963B1 - Composition for treating eye disease comprising mTOR activator and use thereof - Google Patents

Abstract

The present invention relates to compositions comprising mTOR activators and uses thereof. Specifically, the present invention provides a pharmaceutical composition for preventing or treating eye diseases containing an mTOR activator and a method for preventing or treating eye diseases using the same. The mTOR activator provided in the present invention has the effect of preventing retinal nerve damage and thereby preventing or treating eye diseases by inhibiting apoptosis or autophagy of retinal ganglion cells.

Description

Composition for treating eye disease comprising mTOR activator and use thereof}

The present invention relates to a composition for treating eye diseases containing an mTOR activator and its use. Specifically, it relates to an mTOR activator that can treat eye diseases including diabetic retinopathy. The mTOR activator of the present invention can be used to treat eye diseases including diabetic retinopathy by regulating the expression of proteins related to neuronal death and autophagy.

Diabetic retinopathy is a lesion caused by microvascular disorders that occurs in the retina of diabetic patients, and the mechanism of its development has not yet been fully revealed. It is known that hemodynamic mechanisms, biochemical mechanisms, hormonal influences, and genetic factors are involved in a complex manner, causing specific changes in retinal capillary occlusion, capillary aneurysm, retinal hemorrhage, exudate, and new blood vessels.

Among diabetic patients worldwide, 21.7-34.6% suffer from diabetic retinopathy, which is estimated to be 450 million adults as of 2017. Additionally, despite appropriate screening tests, it is known that approximately 2.6 million patients suffer from blindness due to diabetic retinopathy.

Currently, treatments for proliferative diabetic retinopathy are limited to anti-VEGF agents targeting the HIF-1α/VEGF pathway. Anti-VEGF agents are effective in suppressing the progression or worsening of proliferative diabetic retinopathy, but there are some cases in which they do not respond to treatment, and treatment using them is not a fundamental treatment method for vascular proliferative diabetic retinopathy. In addition, as described in Korean Patent Publication No. 10-2018-0109278, conventional treatments for diabetic retinopathy have been studied focusing on mTOR inhibitors to inhibit VEGF activity, and no treatment for diabetic retinopathy using mTOR activators has been studied. .

The present inventors, for the first time, discovered the effectiveness of mTOR activators in treating diabetic retinopathy, breaking away from the existing treatments for diabetic retinopathy that had been studied mainly on mTOR inhibitors. Based on these therapeutic effects, the present inventors developed mTOR activators for the treatment of diabetic retinopathy. We would like to provide

1. Republic of Korea Patent Publication 10-2018-0109278.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating eye diseases containing an mTOR activator.

The present invention also aims to provide a method for preventing or treating eye disease, which includes administering a pharmaceutical composition containing an mTOR activator to an individual in need thereof.

The present invention provides a pharmaceutical composition for preventing or treating eye diseases containing an mTOR activator.

In one embodiment, the mTOR activator is 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine, Raptor expression enhancer, TSC1 or TSC2 expression Provided is a pharmaceutical composition for preventing or treating eye diseases, which is one or more substances selected from the group consisting of inhibitors, RHEB (Ras homolog enriched in brain) protein or nucleic acid encoding it, and PRAS expression inhibitors.

In one embodiment, the eye diseases include diabetic retinopathy, glaucoma, retinal detachments & breaks, retinal vein occlusion, retinal hemorrhage, and cataracts. ), Floaters, Dry eye syndrome, Vitreous hemorrhage, Central Serous ChorioRetinopathy, Iritis, one or more diseases selected from the group consisting of A pharmaceutical composition for preventing or treating diseases is provided.

In one embodiment, the mTOR activator provides a pharmaceutical composition for preventing or treating eye diseases that reduces apoptosis or autophagy of retinal ganglion cells.

The present invention provides a method for preventing or treating eye disease, comprising administering a pharmaceutical composition containing an mTOR activator to an individual in need thereof.

In one embodiment, the mTOR activator is 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine, Raptor expression enhancer, TSC1 or TSC2 expression Provided is a method for preventing or treating eye disease, which is one or more substances selected from the group consisting of inhibitors, RHEB (Ras homolog enriched in brain) protein or nucleic acid encoding it, and PRAS expression inhibitors.

In one embodiment, the eye diseases include diabetic retinopathy, glaucoma, retinal detachments & breaks, retinal vein occlusion, retinal hemorrhage, and cataracts. ), Floaters, Dry eye syndrome, Vitreous hemorrhage, Central Serous ChorioRetinopathy, Iritis, one or more diseases selected from the group consisting of Provides methods for preventing or treating diseases.

In one embodiment, the administration provides a method of preventing or treating eye disease, which is oral administration.

In one embodiment, the administration provides a method of preventing or treating eye disease, which is topical administration using eye drops.

In one embodiment, the administration provides a method of preventing or treating eye disease, which is administration using intraocular injection.

In one embodiment, the administration provides a method of preventing or treating eye disease, wherein the administration is administered using intravenous injection.

The present invention provides a pharmaceutical composition for preventing or treating eye diseases containing an mTOR activator, which has the effect of preventing or treating eye diseases, specifically diabetic retinopathy. In addition, the mTOR activator provided by the present invention has the effect of inhibiting retinal ganglion cell damage more directly by inhibiting apoptosis and autophagy of retinal ganglion cells.

Figure 1 shows the mouse experimental design. Bid stands for twice a day, and qd stands for once a day.
Figure 2 shows changes in molecular levels induced by hyperglycemia in STZ mouse retina.
Figure 3 shows cells expressing pmTOR S2448, pS6 and GLUT1.
Figure 4 shows the results of observing changes in expression of pS6 and GLUT1 in STZ-induced DM mice for 6 months.
Figure 5 shows autophagy and ganglion activity results in the retina of STZ-induced DM.
Figure 6 shows the effects of short-term hyperglycemia in STZ mouse retina.
Figures 7 and 8 show that long-term hyperglycemia causes autophagy dysregulation and nerve damage.
Figure 9 is a schematic diagram showing the mechanism of mTOR activator to inhibit nerve damage.

Term Definition

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

eye disease

As an example, the term “eye disease” used herein includes diabetic retinopathy and diseases related thereto. Specific examples of diseases related to diabetic retinopathy include Glaucoma, Retinal detachments & breaks, Retinal vein occlusion, Retinal hemorrhage, Cataract, Floaters. ), dry eye syndrome, vitreous hemorrhage, central serous chorioretinopathy, and iritis. However, eye diseases are not limited to the above examples and include all eye diseases known to be associated with damage to retinal ganglion cells.

mTOR activator

In the term "mTOR activator" used herein, mTOR is an abbreviation for mammalian target of rapamycin, and is a type of kinase also called FRAP1 (FK506 binding protein 12-rapamycin-associated protein 1). mTOR is known to bind to other proteins and act as a key component of the protein complexes mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2), thereby regulating cellular processes related to cell growth, proliferation, and survival. In the present specification, the mTOR activator serves to activate mTOR, and in one embodiment, 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazine-2-amine , Raptor expression enhancer, TSC1 or TSC2 expression inhibitor, RHEB (Ras homolog enriched in brain) protein or nucleic acid encoding it, PRAS expression inhibitor, etc.

At this time, the expression increasing agent includes a substance that increases the expression of a specific gene or protein, and the expression inhibitor includes a substance that reduces the expression of a specific gene or protein.

At this time, the gene expression enhancer or inhibitor includes deleting or inserting a target gene in the form of a gene therapy, and as an example, the gene therapy includes editing the target gene using CRISPR-Cas.

At this time, Rapor plays a role in constituting the mTORC1 complex, so mTOR can be activated by increasing Raptor expression. Because TSC1 and TSC2 play a role in regulating mTORC1 expression upstream of mTORC1, mTOR can be activated by suppressing the expression of TSC1 and TSC2. RHEB (Ras homolog enriched in brain) plays a role in activating mTOR, so mTOR can be activated through RHEB. Since PRAS is involved in the PI3K/Akt-mTOR pathway, mTOR can be activated by suppressing the expression of PRAS.

However, the mTOR activator is not limited to this and includes all substances that have the function of activating mTOR.

Ⅰ. Uses of mTOR activators

One. Pharmaceutical composition for preventing or treating eye diseases

The present invention provides a pharmaceutical composition for treating or preventing eye diseases containing an mTOR activator.

As used herein, the term "prevention" refers to all actions that suppress or delay the onset of a disease by administering the composition of the present invention, and "treatment" refers to the improvement of disease symptoms by administering the composition of the present invention. It means any action that becomes or changes beneficially.

The pharmaceutical composition provided by the present invention may further include a carrier, and pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, such as lactose, dextrose, sucrose, Sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate. Includes, but is not limited to, nitrite, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

When formulating the pharmaceutical composition according to the present invention, it can be prepared by mixing one or more diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in the field.

In one embodiment, solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc. These solid preparations include the mTOR activator of the present invention and at least one excipient, such as starch. It is prepared by mixing calcium carbonate, sucrose, lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. In another embodiment, liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents and sweeteners are used. , fragrances, preservatives, etc. may be included.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

In one embodiment, formulations for intraocular injection include both liquid and solid formulations, and when formulating pharmaceutical compositions, fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. commonly used in the field are used. It can be manufactured by mixing one or more diluents or excipients.

At this time, the intraocular injection preparation includes a preparation for intravitreal injection, a preparation for subretinal injection, and a preparation for subscleral or suprascleral injection.

In addition, when developed as a gene therapy, viral vectors for gene therapy such as different serotypes of recombinant Adeno-Associated Virus (rAAV), lentivirus vectors, LNP (lipid nanoparticle), CPP (cell -Penetrating peptides), etc. are included.

However, the formulation of the pharmaceutical composition is not limited to the above examples.

At this time, in the pharmaceutical composition, the disease includes eye disease and, as an example, may mean diabetic retinopathy and diseases related thereto. Specific examples of diseases related to diabetic retinopathy include glaucoma, retinal detachments & breaks, retinal vein occlusion, retinal hemorrhage, cataract, and rhinitis ( Floaters, dry eye syndrome, vitreous hemorrhage, central serous chorioretinopathy, and iritis.

Referring to an example of the present invention, it can be confirmed that mTOR activator inhibits apoptosis and increased autophagy in retinal ganglion cells induced by long-term hyperglycemia in a diabetic mouse model. That is, as an example, the mTOR activator of the present invention can have the effect of treating eye diseases, including diabetic retinopathy, by reducing apoptosis or autophagy of retinal ganglion cells.

2. Food composition for improving or preventing eye disease

The present invention provides a food composition for improving or preventing eye disease containing an mTOR activator. At this time, the eye disease includes all information related to eye disease described in 1. Pharmaceutical composition for treating eye disease.

The food composition includes a health functional food composition, and the health functional food is not particularly limited as long as it contains an mTOR activator.

The types of health functional foods are not particularly limited as long as they are commonly manufactured and/or sold. For example, meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. It can be used in the form of pills, powders, granules, precipitates, tablets, capsules or beverages, and includes all health functional foods in the conventional sense.

3. How to prevent or treat eye disease

The present invention provides a method for preventing or treating eye disease comprising administering an mTOR activator to an individual in need thereof.

The eye disease includes all information related to eye disease described in 1. Pharmaceutical composition for treating eye disease.

Administering the pharmaceutical composition to an individual in need thereof can be performed through various routes. For example, oral (e.g., drenches, tablets, capsules (including sprinkle capsules and gelatin capsules), lumps, powders, granules, pastes for application to the tongue, such as aqueous or non-aqueous solutions or suspensions) ; Absorption through oral mucosa (e.g. sublingual); Anus, rectum or vagina (e.g. pessary, cream or foam); Parenterally (intramuscularly, intravenously, subcutaneously or intrathecally, for example as a sterile solution or suspension); nasal cavity; intraperitoneal; subcutaneous; Transdermal (e.g., a patch applied to the skin); and topical (e.g., creams, ointments or sprays applied to the skin, or eye drops). In one embodiment, the pharmaceutical composition can be administered orally. In another example, the pharmaceutical composition may be administered topically using eye drops. In another example, the pharmaceutical composition may be administered through intravenous injection. In another example, the pharmaceutical composition may be administered using intraocular injection. At this time, the intraocular injection includes intraocular injection through an intravitreal injection route, a subretinal injection route, and a subsclera or suprascleral injection route.

However, the administration method is not limited to the above examples.

The subject may be a mammal such as a human, or may be a non-human mammal. The actual dosage of the pharmaceutical composition may vary for a particular patient, composition, and method of administration to obtain an amount of active ingredient effective to achieve the desired therapeutic response without toxicity to the patient. The selected dosage depends on the combination of compositions or their activity, route of administration, time of administration, rate of excretion of the composition, duration of treatment, other drugs, compounds and/or substances used in conjunction with the composition, age, gender, weight, condition, general health. and medical history of the subject being treated, and other factors well known in the medical field.

A physician or veterinarian skilled in the art can easily determine and prescribe the required therapeutically effective amount of a pharmaceutical composition. For example, a physician or veterinarian may start the dosage of the pharmaceutical composition at a lower level than necessary to achieve the desired therapeutic effect and slowly increase the dosage until the desired effect is achieved. “Therapeutically effective amount” means a concentration of a compound sufficient to produce the desired therapeutic effect. It is generally understood that the effective amount may vary depending on the individual's weight, gender, and medical history. Other factors that affect the effective amount may be the severity of the subject's condition, the stability of the composition, etc. The total dose may be delivered in multiple doses of the agent. Methods for determining potency and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

In certain instances, the compositions of the invention may be administered alone or in combination with other types of therapeutic agents. As used herein, the term “conjoint administration” refers to any administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body. (e.g., two compounds may be effective on a subject simultaneously, in which case the two compounds may have a synergistic effect). For example, different therapeutic compounds may be administered concomitantly or sequentially, in the same formulation or in separate formulations. In certain embodiments, different therapeutic compounds may be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 1 week of each other. Accordingly, individuals receiving such treatment may benefit from the combined effects of different therapeutic compounds.

In certain embodiments, co-administration of a composition of the invention and one or more additional therapeutic agents (e.g., one or more additional chemotherapy agents) results in improved efficacy compared to separate administration of the composition of the invention or one or more additional therapeutic agents. can be provided. Hereinafter, the present invention will be described in detail through examples.

At this time, the examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

Ⅱ. Example

One. Experiment method

- Animal testing

All animal experiments were designed and performed in accordance with the Guide for the Care and Use of Laboratory Animals at the Association for Vision Research, Ophthalmic Statement on the Use of Animals in Ophthalmic and Vision Research, and were approved by the Animal Care and Use Committee of Soonchunhyang University Bucheon Hospital. The mice used in this study were strain C57/BL6 (8 weeks old, male, 22-24 g) and were purchased from Orient Bio Inc. (Seongnam, Korea). All mice were housed in a room with a 12/12 hour light/dark cycle with free access to food and water, and humidity and temperature were maintained at 50% and 23-26 °C, respectively.

- Diabetes induction

Diabetes was induced by a single intraperitoneal injection of STZ ((150 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). STZ was prepared in 100 mM citrate buffer (pH 4.5). After injection of STZ into mice. To prevent sudden hypoglycemia, 10% sucrose was infused overnight. Two days and two weeks after the infusion, blood sugar was monitored using an Accu-Check active blood glucose monitor. As a result of blood sugar measurement, only mice with non-fasting blood sugar of 300 mg/dL or more and polyuria or gluconuria were classified as STZ-induced diabetic mice and used in the experiment. Non-diabetic mice of the same age were used as controls. The general parameters obtained on the day the mice were sacrificed are shown in Table 1.

Table 1 Weight and blood sugar levels

- Grouping and treatment regimen

A total of 140 diabetic animal models were used in this study. To evaluate the natural course of diabetic retinopathy, 60 mice were observed over 6 months. The animals were divided into six groups according to the observation period: (1) normal control, (2) 1 m DM, (3) 2 m DM, (4) 3 m DM, (5) 4 m DM and ( 6) 6 m DM (Figure 1a). The effect of short-term hyperglycemia on the retina was observed in 40 mice, divided into four groups: (1) normal control (2) 1m DM (3) 1m DM/PHL (4) 1m DM/Rapa ( Figure 1b).

In the 1m DM/PHL group, mice were injected with phlorizin (PHL) (Cayman Chemi cal, Ann Arbor, MI) dissolved in 60% propylene glycol in phosphate-buffered saline at a dose of 200 mg/kg, daily for the last 7 days. Injected subcutaneously twice each, on the morning of the eighth day and 3 hours before sacrifice. PHL, phloretin glucoside, restores blood sugar levels to normal by increasing glucose excretion from the kidneys. The dose and method of administration of PHL were selected based on previous studies [Fort PE, Losiewicz MK, Reiter CEN, et al. Differential roles of hyperglycemia and hypoinsulinemia in diabetes induced retinal cell death: evidence for retinal insulin resistance. PLoS ONE. 2011; Sδllstrϕm J, Eriksson T, Fredholm BB, Persson AEG, Palm F. Inhibition of sodium-linked glucose reabsorption normalizes diabetes-induced glomerular hyperfiltration in conscious adenosine A1-receptor deficient mice. Acta Physiol. 2014;210(2):440-5.]. Mice in the 1 m DM/Rapa group received rapamycin (Sigma-Aldrich, St. Louis, MO) diluted in 4% ethanol and 5% Tween-20 in distilled water at a dose of 3 mg/kg for the same period as the 1 m DM/PHL group. The dose was administered intraperitoneally daily. Rapamycin, a macrolide, forms a gain-of-function complex with FKBP12 and then inhibits mTORC1.

The effects of long-term hyperglycemia on the retina of diabetic mice were evaluated in 40 mice divided into four groups: (1) normal control, (2) 3m DM, (3) 3m DM/PHL, and (4) 3m DM. /MHY (Figure 1c).

In the 3m DM/PHL group, mice were injected subcutaneously with PHL twice daily for the last 10 days, and on the morning of day 11 and 3 hours before sacrifice. Mice in the 3m DM/MHY group were treated with MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-tris daily at a dose of 10 mg/kg for the same period as the 3m DM/PHL group. Azin-2-amine (MedChem Express, Monmouth Junction, NJ) was injected intraperitoneally.

- Tissue collection and preparation

After completion of the experiment, mice were used for Western blot and histological analysis. For histological analysis, mice were deeply inoculated by intraperitoneal injection of 40 mg/kg zolazepam/tiletamine mixture (Zoletil 50, Virbac, Carros Cedex, France) and 5 mg/kg xylazine (Rompun, Bayer Healthcare, Leverkusen, Germany). They were anesthetized, and after intracardiac perfusion with 0.1 M phosphate buffer (PB) containing 1,000 U/ml heparin, 4% paraformaldehyde (PFA) in 0.1 M PB was injected. The eye-cup was made by removing the front part of the enucleated mouse eye. They were then fixed in 4% paraformaldehyde (Biosesang, Seongnam, South Korea) for 1 hour, dehydrated in 30% sucrose overnight, and embedded in frozen section compound (Leica Biosystems Richmond, IL). To collect samples for Western blot experiments, neural retinas were isolated from enucleated mouse eyes and stored at -80°C until used in experiments.

- Western blot

Neuroretinal tissue was disrupted in RIPA lysis buffer (Gendepot, Barker, Tx) containing Xpert phosphatase inhibitor cocktail (Gendepot, Barker, Tx) and Xpert protease inhibitor cocktail (Gendepot, Barker, Tx) for one hour on ice. Insoluble material was removed by centrifugation (13,000 rpm, 15 min, 4°C), and the supernatant was obtained. Protein lysates were quantified using the Pierce BCA protein assay kit (Termo scientifc, Middlesex, MA) according to the manufacturer's instructions. Lysates were denatured in 4x Laemmli sample buffer (Gendepot, Barker, Tx) and heated at 95 °C for 10 min. Aliquots of each sample containing equal amounts of protein were separated using SDS-acrylamide gels in the range of 6 to 10% depending on the molecular weight of the target antibody and transferred to polyvinylidene fluoride membranes (ATTO, Amherst, NY). I ordered it. For detection of total proteins, cells were incubated with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20, and for detection of phosphorylated proteins, cells were incubated with 5% BSA in PBS containing 0.1% Tween-20 for 1 hour at room temperature. . At this time, anti-mTOR, anti-phospho-S6 ribosomal (pS6) protein (Cell signaling technology, Danvers, MA), anti-phospho-mTOR (S2448), anti-GLUT1 (Abcam, Cambridge, UK), anti-β actin (Santa Cruz Biotechnology, Dallas, TX) and incubated overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and goat anti-mouse IgG (Genedepot, Barker, Tx) for 2 hours at room temperature. Immunoreactive signals were developed using a Western blot detection kit (EzWestLumiplus, ATO Corporation, Tokyo, Japan) and read with Azure BiosystemsTM c280 (Azure Biosystems, Dublin, CA, USA). Bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

- Immunofluorescence

Eye cup cryosections (10 μm thick) were prepared using a frozen tissue sectioner (Termo Fisher Scientifc Shandon Cryotome, Middlesex, MA). Slides were washed with 1xPBS containing 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and blocked with 5% donkey serum in PBST for 1 hour. Blocked slides were then incubated with anti-phospho-mTOR (S2448) and anti-GLUT1 from Abcam (Cambridge, UK) and anti-phospho-S6 ribosomal protein (S240/244) from Cell signaling technology (Danvers, MA). and anti-glial fibrillary acidic protein (GFAP) (CST), anti-glutamine synthetase (GS) from Millipore (Billerica, MA), anti-brain-specific homeobox/POU domain protein 3A (Brn3a), and anti-neuronal nuclear protein. (NeuN), anti-calbindin (Calb) from Sigma-Aldrich (St. Louis, MO), anti-cleaved caspase-3 from Cell signaling technology (Danvers, MA), anti-cleaved caspase-3 from Novus Biologicals (Littleton, CO). They were incubated with Beclin1 antibody for 2 hours and detected with secondary antibodies (Alexa Fluor 488 or 568 or 647, dilution 1:1000; Invitrogen Corp., Carlsbad, CA). Nuclear counterstaining was performed with Hoechst 33,342 (Invitrogen Corp, Carlsbad, CA). After washing, they were mounted with fluorescent mounting medium (Dako, Santa Clara, CA).

- Image analysis and cell counting

All slides were imaged using a Leica SP8 (Leica Microsystems, Wetzlar, Germany) confocal microscope. Cleaved caspase-3, NeuN and Hoechst positive cells (n=6/group) were counted using Image J software.

- Statistical analysis

Data were expressed as mean ± standard deviation. Differences between groups were analyzed using the Mann-whitney U test. Differences were considered statistically significant when p≤0.05.

2. Determination of the effects of hyperglycemia on mTOR-related proteins and GLUT1

After 1 month of STZ injection, the levels of pmTOR (S2448) and pS6 (S240/244) increased and decreased significantly after 2, 3, and 4 months. The 6-month sample showed the lowest levels of pmTOR and pS6 (Fig. 2a, b). GLUT1 also increased one month after diabetes induction, but gradually decreased until six months of observation (Figure 2a, b).

3. Confirmation of cell death results in diabetic retina

To evaluate neuronal cell death in the diabetic retina, cleaved caspase-3 and NeuN were immunostained using STZ retinal frozen sections at 1, 3, and 6 months, and normal mouse retina was used as a control ( Figure 2c). Cleaved caspase-3 was detected in the GCL (ganglion cell layer) of the diabetic retina, showing weak signals at 1 month and strong signals at 3 and 6 months. On the other hand, no signal was detected in the normal retina. The number of cleaved caspase-3 positive cells indicated that the number of cell deaths gradually increased during 6 months of diabetes induction (Figure 2d). Additionally, it was confirmed that the NeuN immune response in ganglion cells gradually decreased at 3 and 6 months (Figure 2e).

4. Observation of pmTOR S2448, pS6 and GLUT1 expressing cells in normal mouse retina

pmTOR S2448 was confirmed to be expressed in GCL and INL (inner nuclear layer), and GLUT1 was confirmed to be expressed in GCL, INL, and ONL (outer nuclear layer) (Figure 3a). Co-staining results of pmTOR S2448 and NeuN confirm the expression of activated mTORC1 in ganglion cells (Figure 3b). S2448 phosphorylated mTOR is expressed in calbindin-positive cells (Figure 3c). Furthermore, pmTOR S2448 is expressed in the endfeet and cell body of Müller cells stained with glutaime systhetase (GS) (Figure 3d) and co-localizes with GLUT1 (Figure 3e).

pS6, a downstream effector of the mTORC1 complex, is highly expressed in the outermost part of the GCL and INL, and is less expressed in the cell body of the inner half of the INL. The signal of pS6 in the GCL arises from NeuN-positive ganglion cells (Figure 3f), and the outermost signal in the INL arises from the cell bodies of horizontal cells stained with calbindin (Figure 3g). The weak pS6 signal co-localized with NeuN-positive cells in the INL may be amacrine cells or ectopic ganglion cells ( Fig. 3F ).

5. Confirmation of hyperglycemia-induced changes in pS6 and GLUT1 expression in STZ mouse retina

After 1 month of hyperglycemia, pS6 expression increased in the inner retina. After 3 months of hyperglycemia, pS6 expression was decreased. After 6 months of diabetes, it was observed that there were almost no pS6 positive ganglion cells and horizontal cells (Figure 4). GLUT1 expression also increased after 1 month of diabetes, and gradually decreased after 3 and 6 months (Figure 4).

6. Hyperglycemia-induced ganglion activity and autophagy dysregulation in STZ mouse retina.

Cryosections of normal mouse retina and 3m STZ mouse retina were stained with an antibody against GFAP to observe ganglion reactivity. In normal mouse retina, GFAP was expressed limited to the nerve fiber layer (NFL), Müller cell endpits of the GCL, and astrocytes (Figure 5). On the other hand, in the 3m diabetic mouse retina, GFAP expression increased and extended to the ganglia of the NFL, GCL, and IPL (Figure 5). Moreover, the expression of pmTOR was decreased, unlike normal mouse retina (Figure 5).

To assess autophagy, normal and 3m STZ mouse retinas were stained with Beclin1. In normal mouse retina, Beclin1 expression was observed in the GCL, INL, and OPL. In the 3m STZ mouse retina, Beclin1 was observed to extend to the IPL, ONL, and photoreceptor cell layers and increased expression (Figure 5).

7. Effects of short-term hyperglycemia on STZ mouse retina

pmTOR S2448 expression was increased at 1 month of diabetes, and treatment with phlorizin (PHL) brought it to a level similar to that of normal mouse retina (Figure 6). The hyperglycemia-induced increase in pS6 and GLUT1 was reduced by glycemic control due to PHL treatment. Treatment with rapamycin reduced pmTOR S2448 and pS6 levels, as expected with mTOR inhibition, but no reduction in GLUT1 was observed (Figure 6).

In normal mouse retina, GFAP expression was observed in NFL and GCL, but in 1m diabetic retina, it was also observed in astrocytes and some Müller cells located in IPL. Treatment with PHL and rapamycin caused GFAP expression levels to return to normal (Figure 6c).

8. Nerve damage confirmed after prolonged high blood sugar levels

pmTOR S2448 and pS6 expression decreased at 3 months of diabetes, and PHL treatment restored the activity of mTOR (Figures 7a, 8d). GLUT1 expression in the retina of 3m STZ mice was increased by PHL treatment ( Fig. 7A ). MHY1485 treatment activated mTOR in the retina of 3m STZ-mice (Figures 7A, 8D).

Beclin1 immunoreactivity in the retina of the 3m DM/PHL group showed a similar expression pattern to that of the normal control group. That is, signals were observed in GCL, INL, and OPL. However, with MHY1485 treatment, the signal of Beclin1 was observed only in GCL, showing strong inhibition of autophagy (Figure 7b). Calbindin staining showed no differences in the OPL of the retinas of all groups, but did show differences in the GCL.

To determine how autophagy affects neurons in GCL, retinal tissue was stained with cleaved caspase 3 and NeuN, and retinal tissue from a normal mouse was used as a control. Cleaved caspase 3 of GCL was increased in 3m STZ mice, indicating increased apoptotic activity after prolonged hyperglycemia (Figure 8A). PHL treatment reduced the immune response and number of cleaved caspase 3-positive cells (Figure 8 a,b). However, mTOR activator blocked autophagy, significantly reducing the immune response and number of cleaved Caspase 3 positive cells compared to the 3m DM and 3m DM/PHL groups (Figure 8 a,b). Quantification of neurons in the GCL of tissue from the treatment group indicated that neurons were protected from apoptotic activity induced by high-period hyperglycemia (Figure 8C). The total cell number of GCL in the retinal tissue of the 3mDM group was reduced compared with the normal group and 3mDM/PHL. However, the number of NeuN-positive cells increased in tissues of the 3mDM/PHL group compared to the 3mDM group. In the tissues of the 3mDM/MHY group, the total cell number and NeuN-positive cells were highest among the STZ-injected groups (Figure 8c).

To confirm nerve recovery according to autophagy regulation, the response of ganglia was confirmed by GFAP labeling. GFAP-positive astrocytes and Müller ganglion cells were observed in the NFL, GCL, and IPL of 3m STZ-mice. In the MHY1485-treated mouse retina, GFAP signals were observed only in the NFL and GCL, as in the normal mouse retina (Figure 8D).

Claims (12)

A pharmaceutical composition for preventing or treating eye disease containing an mTOR activator,
The mTOR activator is 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazine-2-amine,
The eye disease is diabetic retinopathy,
Pharmaceutical composition for preventing or treating eye diseases. delete delete According to paragraph 1,
The mTOR activator is a pharmaceutical composition for preventing or treating eye disease, which reduces apoptosis or autophagy of retinal ganglion cells. A food composition for improving or preventing eye disease containing an mTOR activator,
The mTOR activator is 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazine-2-amine,
The eye disease is diabetic retinopathy,
Food composition for improving or preventing eye disease. A method for preventing or treating eye disease, comprising administering a pharmaceutical composition containing an mTOR activator to an entity other than a human in need thereof, comprising:
The mTOR activator is 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazine-2-amine,
The eye disease is diabetic retinopathy,
How to prevent or treat eye disease. delete delete According to clause 6,
The method of preventing or treating eye disease includes oral administration, local administration using eye drops, administration using intraocular injection, administration using intravenous injection, or administration using intraperitoneal injection. delete delete delete