Application of 4E-BP1 pathway inhibitor in preparation of intraocular pressure lowering drugs and trabecular meshwork injury and fibrosis repairing drugs

Abstract

According to the application of the 4E-BP1 pathway inhibitor in preparation of intraocular pressure lowering drugs and trabecular mesh injury and fibrosis repairing drugs, based on the fibrosis research progress of other tissues and organs of an organism, the key regulation point of a trabecular mesh fibrosis pathway network is focused, and it is found that 4E-BP1 is a core intersection point of trabecular mesh injury caused by various stress stimuli, so that the application of the 4E-BP1 pathway inhibitor in preparation of intraocular pressure lowering drugs and trabecular mesh injury and fibrosis repairing drugs is achieved. An ideal intervention target is provided for damage and repair of local ocular trabecular meshwork tissue, the invention provides a local ocular 4E-BP1 inhibitor and a strategy for preparing an intraocular pressure reducing drug, and aims to protect an intraocular pressure reducing method for down-regulating trabecular meshwork 4E-BP1 expression and other strategies through gene intervention and other methods, and in-vitro experiments also show that in a mouse hormonal intraocular hypertension model, the intraocular pressure reducing drug can be used for reducing intraocular pressure. The 4E-BP1 inhibitor represented by torin 1 and AZD8055 shows a good intraocular pressure reducing effect when being used for eye local dripping, the trabecular network fibrosis related indexes are remarkably reduced, and the effect of the 4E-BP1 inhibitor is superior to that of rapamycin insensitive to 4E-BP1.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to the application of a 4E-BP1 pathway inhibitor in the preparation of drugs for reducing intraocular pressure and drugs for repairing trabecular meshwork injury and fibrosis. Background Art

[0002] Glaucoma is the world's leading irreversible blinding eye disease and an important public health problem in China. Currently, there are approximately 22 million glaucoma patients in China, resulting in 6.3 million blind people and more than 10 million visually handicapped people. The absolute or relative increase in intraocular pressure (IOP) is considered to be the most important risk factor for glaucomatous optic nerve damage. In both ocular hypertensive and normal-tension glaucoma, reducing intraocular pressure is currently the only proven method to effectively prevent or delay the progression of the disease. Exploring the pathological mechanism of increased intraocular pressure in glaucoma and developing safer and more effective drugs for reducing intraocular pressure remains an important challenge and topic in the field of glaucoma.

[0003] The maintenance of IOP depends on the dynamic balance between aqueous humor production and outflow. The trabecular meshwork (TM)-Schlemm's canal-collecting duct-aqueous vein pathway is the most important channel for aqueous humor drainage under physiological conditions, accounting for 70%-95% of the total. Apoptosis of trabecular cells and fibrosis of the trabecular meshwork are common pathological features of glaucomatous trabecular meshwork tissue. In theory, ideal intraocular pressure reduction effects may be achieved based on the regulation of the physiological function of the trabecular meshwork, but its mechanism involves numerous signaling pathways and cytokines, and there are cross-interactions between the pathways. Interventions targeting a single pathway usually have unsatisfactory effects, and the treatment is complex and challenging. Currently, there are very limited intraocular pressure-lowering drugs targeting the regulation of trabecular meshwork cell function and increasing aqueous humor outflow through the trabecular meshwork pathway. Recently, newly introduced Rho kinase inhibitors are considered to be able to increase the formation of pores in Schlemm's canal endothelial cells, relax smooth muscle fibers in the trabecular meshwork, increase aqueous humor outflow through the trabecular meshwork pathway, and reduce the outflow resistance. However, such drugs have not been officially clinically applied in China, and their intraocular pressure-lowering effect is not superior to prostaglandin drugs that increase the uveoscleral pathway, and they also have a high ocular surface irritation rate (50%-59%). This not only significantly affects the quality of life of patients and reduces medication compliance, but also the chronic inflammatory irritation reaction may reduce the success rate of future glaucoma surgeries. Exploring the key signaling pathways of extracellular matrix remodeling and fibrosis in trabecular meshwork cells and developing safe and effective drugs for regulating trabecular meshwork function and inhibiting fibrosis are hotspots, difficulties, and important topics in the field of glaucoma.

[0004] The mTORC1 pathway is considered to play an important role in the process of fibrosis. Its inhibitor, rapamycin and its derivatives, have shown certain anti-fibrotic and anti-cell proliferation effects in various tissues and organs. However, it should be noted that as an incomplete inhibitor of the mTORC1 pathway, rapamycin only shows moderate anti-tumor and anti-fibrotic effects in clinical trials. This may be related to the limited inhibitory effect of rapamycin on collagen synthesis. Recently, the rapamycin-insensitive mTORC1 / 4E-BP1 pathway has been proven to be a key signal pathway intersection in the collagen synthesis of physiological and pathological fibrosis in various tissues and organs. At present, the role of 4E-BP1 in trabecular meshwork fibrosis and its intraocular pressure-lowering effect have not been reported. Summary of the Invention

[0005] To solve the technical defects existing in the prior art, the present invention first confirms that 4E-BP1 participates in trabecular meshwork injury as a common pathway for various currently recognized stress stimuli, and at the same time provides the application of a 4E-BP1 pathway inhibitor in the preparation of intraocular pressure-lowering drugs and drugs for the repair of trabecular meshwork injury and fibrosis. In the TGF-β1-induced trabecular meshwork cell injury model, both 4E-BP1 inhibitors torin 1 and AZD8055 can significantly inhibit the expression of trabecular meshwork cell fibrosis-related proteins, especially the inhibitory effect on collagen 1 expression is significantly better than that of rapamycin insensitive to 4E-BP1. In vitro experiments also show that in the mouse model of steroid-induced ocular hypertension, local eye drops of 4E-BP1 inhibitors torin 1 and AZD8055 show good intraocular pressure-lowering effects, and the trabecular meshwork fibrosis-related indicators are significantly reduced, and the effect is better than that of rapamycin.

[0006] The technical solution adopted by the present invention is: 1. The application of a 4E-BP1 pathway inhibitor in the preparation of intraocular pressure-lowering drugs.

[0007] Further, the 4E-BP1 pathway inhibitor is 4E-BP1 inhibitor torin 1 or AZD8055 and their mimetics.

[0008] Further, the concentration of the 4E-BP1 inhibitor torin 1 is 1 mg / ml.

[0009] Further, the concentration of the AZD8055 is 1 mg / ml.

[0010] Further, the intraocular pressure-lowering drug is a drug for treating pathological intraocular pressure elevation caused by trabecular meshwork injury and fibrosis.

[0011] The application of a 4E-BP1 pathway inhibitor in the preparation of drugs for the repair of trabecular meshwork injury and fibrosis.

[0012] Furthermore, the 4E-BP1 pathway inhibitor is 4E-BP1 inhibitor torin 1 or AZD8055 and its mimetics.

[0013] Furthermore, the concentration of the 4E-BP1 inhibitor torin 1 is 1 μM.

[0014] Furthermore, the concentration of AZD8055 is 10 μM.

[0015] The beneficial effects of the present invention are as follows: The present invention provides an application of a 4E-BP1 pathway inhibitor in the preparation of intraocular pressure-lowering drugs and drugs for trabecular meshwork injury and fibrosis repair. Based on the research progress of fibrosis in other tissues and organs of the body, focusing on the key regulatory points of the trabecular meshwork fibrosis pathway network, it is found that 4E-BP1 is the key intersection point of trabecular meshwork injury caused by various stress stimuli, providing an ideal intervention target for local trabecular meshwork tissue injury and repair in the eye. The present invention provides local 4E-BP1 inhibitors and strategies for preparing intraocular pressure-lowering drugs, and aims to protect the intraocular pressure-lowering methods such as down-regulating the expression of 4E-BP1 in the trabecular meshwork by gene intervention and other methods. In vitro experiments also show that in a mouse model of steroid-induced ocular hypertension, local eye drops of 4E-BP1 inhibitors torin 1 and AZD8055 exhibit good intraocular pressure-lowering effects, and the trabecular meshwork fibrosis-related indicators are significantly reduced, and the effect is better than that of rapamycin insensitive to 4E-BP1. Description of the Drawings

[0016] Figure 1 To confirm by western blot method that in the three currently most important trabecular meshwork injury stress stimuli, namely hormone (DEX-induced model), inflammation (TGF-β1-induced model), and oxidative stress (tBHP-induced model), the 4E-BP1 pathway is consistently activated, but the changes in other mTOR-related pathways are different. Among them Figure 1 A shows the protein expression of 4E-BP1 and other mTOR pathways in trabecular meshwork cells after treatment with the hormone dexamethasone; Figure 1 B shows the protein expression of 4E-BP1 and other mTOR pathways in trabecular meshwork cells after treatment with TGF-β; Figure 1 C shows the protein expression of the 4E-BP1 pathway in trabecular meshwork cells after treatment with different oxidative stress inducers tBHP and rotenone.

[0017] Figure 2 To confirm by immunofluorescence and western blot methods that the 4E-BP1 pathway inhibitors torin 1 and AZD8055 can effectively inhibit TGF-β1-induced fibrosis of trabecular meshwork cells, and the effect is better than that of rapamycin insensitive to 4E-BP1; among them Figure 2A is the immunofluorescence staining result of the morphological changes of trabecular meshwork cells and the expression of fibrosis markers after TGF-β1 treatment; Figure 2 B is the western blot detection and semi-quantitative analysis of the expression of fibrosis markers in trabecular meshwork cells after TGF-β1 treatment; Figure 2 C shows the effects of 4E-BP1 pathway inhibitors torin1 and AZD8055, as well as rapamycin insensitive to the 4E-BP1 pathway, on the expression of fibrosis markers in TGF-β1-induced trabecular meshwork cells.

[0018] Figure 3 To explore the anti-fibrotic mechanism of 4E-BP1 pathway inhibitors on trabecular meshwork using western blot method, it was found that 4E-BP1 pathway inhibitors torin 1 and AZD8055 could significantly inhibit the expression of 4E-BP1 and its phosphorylation in TGF-β1-induced trabecular meshwork cells. Western blot detection (left) and semi-quantitative analysis (right) of the effects of 4E-BP1 pathway inhibitors torin1 and AZD8055, as well as rapamycin insensitive to the 4E-BP1 pathway, on the expression of 4E-BP1 and other mTOR pathway proteins in TGF-B1-induced trabecular meshwork cells.

[0019] Figure 4 In vivo animal experiments confirmed that topical eye drops of 4E-BP1 pathway inhibitors torin 1 and AZD8055 could effectively reduce intraocular pressure in mice with hormone-induced ocular hypertension model, and the intraocular pressure reduction was significantly greater than that of rapamycin insensitive to 4E-BP1.

[0020] Figure 5 It was confirmed by immunofluorescence staining that 4E-BP1 pathway inhibitors torin 1 and AZD8055 could effectively reduce the expression of α-SMA, a fibrosis marker in trabecular meshwork of mice with hormone-induced ocular hypertension model. Detailed implementation mode

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0022] Example 1 This experiment used in vitro cell experiments to explore and confirm that 4E-BP1 participates in trabecular meshwork fibrosis as a common pathway for various trabecular meshwork injury stimuli. Human trabecular meshwork cells (hTM) were used in the study, seeded in 10-cm culture dishes, and cultured in DEMD medium containing 10% fetal bovine serum. When the trabecular cells reached 80% confluence, a drug intervention experiment was conducted.

[0023] Experimental grouping: TGF-β1 treatment group: Cultured in 10% FBS-DMEM medium containing 3 mg / ml TGF-β1.

[0024] Dexamethasone (DEX) treatment group: Cultured in 10% FBS-DMEM medium containing 500 nM DEX.

[0025] tBHP treatment group: Cultured in 10% FBS-DMEM medium containing 3 μM or 5 μM tBHP.

[0026] Rotenone treatment group: Cultured in 10% FBS-DMEM medium containing 500 nM rotenone.

[0027] After 24 hours of drug intervention, the medium was aspirated, and the cells were washed 3 times with PBS, then digested and collected with 0.25% trypsin. Western Blot was used to detect the expression levels of 4E-BP1 and its phosphorylated proteins in cells of each group. At the same time, the expression of other proteins in the mTOR pathway was detected. The results showed that in human trabecular cells treated with the hormone dexamethasone, the inflammatory factor TGF-β1, the oxidative stress drug tBHP, and rotenone, the activation of the mTOR pathway was different, but there was a significant increase in the expression of 4E-BP1 and its phosphorylated proteins in all cases, suggesting that 4E-BP1 participates in cell injury and fibrosis as a common key pathway for various trabecular meshwork injury stress stimuli, as Figure 1 shown.

[0028] Example 2 The role of the 4E-BP1 signaling pathway in human trabecular cell fibrosis and its potential therapeutic effect.

[0029] This example aims to explore the effect of the activation of the 4E-BP1 signaling pathway on human trabecular cell fibrosis and the inhibitory effect of the 4E-BP1 signaling pathway inhibitor on human trabecular cell fibrosis. According to the results of Example 1 above, we have demonstrated that the 4E-BP1 signaling pathway in human trabecular cells is activated after TGF-β1 treatment. In this example, we directly observed the morphology of human trabecular cells 24 hours after TGF-β1 treatment through phase contrast microscopy, and at the same time through immunofluorescence and The expression of fibrosis-related index proteins was detected by Western Blot assay, and the anti-fibrotic effect of 4E-BP1 inhibitor was explored.

[0030] Experimental grouping: Normal control group: Human trabecular cells cultured in 10% FBS-DMEM medium.

[0031] Experimental group: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, the medium was replaced with 10% FBS-DMEM medium and cultured for 24 hours. Rapamycin intervention group: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, the medium was replaced with 10% FBS-DMEM medium containing 10 μM Rapamycin and cultured for 24 hours. 4E-BP1 inhibitor intervention group 1: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, the medium was replaced with 10% FBS-DMEM medium containing 1 μM Torin1 and cultured for 24 hours. 4E-BP1 inhibitor intervention group 2: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, the medium was replaced with 10% FBS-DMEM medium containing 10 μM AZD8055 and cultured for 24 hours. Experimental results: The morphological changes of human trabecular cells after TGF-β1 treatment were observed by phase contrast microscopy. Figure 2 A The results showed that the number and density of trabecular cells were significantly increased compared with the control group after TGF-β1 treatment. Immunofluorescence results showed that the fluorescence intensities of fibrosis marker proteins α-SMA, fibronectin and collagen I in trabecular cells were significantly increased after TGF-β1 treatment. Figure 2 B The Western Blot results further proved this by quantitative analysis. The 4E-BP1 inhibitors Torin 1 (1 μM) and AZD8055 (10 μM) acting for 24 hours could effectively inhibit the expression of the above fibrosis markers induced by TGF-β1, and the effect was better than that of rapamycin insensitive to 4E-BP1, as Figure 2 shown in C.

[0032] Example 3 This example aims to compare the different effects and functions of 4E-BP1 pathway inhibitors and rapamycin on mTOR and downstream related signaling pathways by Western Blot assay.

[0033] Experimental grouping: Normal control group: Human trabecular cells cultured in 10% FBS-DMEM medium.

[0034] Experimental group: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, it was changed to 10% FBS-DMEM medium and cultured for 24 hours. Rapamycin intervention group: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, it was changed to 10% FBS-DMEM medium containing 10 μM Rapamycin and cultured for 24 hours. 4E-BP1 inhibitor intervention group 1: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, it was changed to 10% FBS-DMEM medium containing 1 μM Torin1 and cultured for 24 hours. 4E-BP1 inhibitor intervention group 2: After pretreatment with 3 mg / ml TGF-β1 for 24 hours, it was changed to 10% FBS-DMEM medium containing 10 μM AZD8055 and cultured for 24 hours. Experimental results: The results showed that in the TGF-β-induced fibrosis model of human trabecular cells, the application of 4E-BP1 inhibitors Torin 1 (1 μM) and AZD8055 (10 μM) could effectively inhibit 4E-BP1 and its phosphorylation, and at the same time down-regulate mTOR and downstream related signaling pathways. However, Rapamycin, which is insensitive to 4E-BP1, could significantly inhibit the mTOR signaling pathway and its downstream Akt and P70S6K1 signals, but only showed a mild inhibitory effect on 4E-BP1 and its phosphorylation, as Figure 3 shown. Combining with the experimental results of Example 2, the inhibition of the 4E-BP1 signaling pathway could obtain a more significant and better anti-trabecular cell fibrosis effect than the mTOR pathway inhibitor Rapamycin, which is insensitive to 4E-BP1.

[0035] Example 4 4E-BP1 inhibitors can effectively reduce the intraocular pressure of mice in a steroid-induced ocular hypertension model. This example aims to explore the in vivo effect of 4E-BP1 inhibitors on reducing intraocular pressure. 100 mg of dexamethasone powder was dissolved in 1 ml of physiological saline to prepare a 10% DEX stock solution. Another physiological saline was used to dilute the vitamin A palmitate ophthalmic gel in a 1:1 ratio, and then the DEX stock solution was diluted with the diluted gel to a concentration of 0.1%. 6-8-week-old adult male C57BL / 6J mice were used to make a high intraocular pressure model by instilling 0.1% DEX gel into the eyes twice a day (8-9 AM and 13-14 PM). Subsequently, the intraocular pressure of the mice was measured and recorded every 2 days using an icare tonometer until the intraocular pressure of the mice increased and stabilized. 10 mg of Rapamycin, 5 mg of Torin1, and 5 mg of AZD8055 were weighed and placed in an EP tube, and 50 μl of DMSO, 400 μl of PEG300, 50 μl of Tween-80, and 500 μl of physiological saline were added in sequence to prepare the corresponding stock solutions. Then, the respective stock solutions were diluted to a concentration of 1 mg / ml with the diluted gel. While continuing to instill DEX to maintain high intraocular pressure, the above-mentioned therapeutic drugs were instilled twice a day, and the changes in intraocular pressure of each group of mice were monitored and recorded.

[0036] Experimental grouping: Control group: Diluted gel without any drug.

[0037] Experimental group: 0.1% DEX gel.

[0038] Rapamycin treatment group: 0.1% DEX gel + 1 mg / ml Rapamycin gel.

[0039] 4E-BP1 inhibitor treatment group 1: 0.1% DEX gel + 1 mg / ml Torin1 gel.

[0040] 4E-BP1 inhibitor treatment group 2: 0.1% DEX gel + 1 mg / ml AZD8055 gel.

[0041] Experimental results: The intraocular pressure of the mice began to gradually increase on the 4th day after instilling 0.1% DEX gel, and reached a peak of 17-18 mmHg around the 20th day. After local instillation of the 4E-BP1 inhibitors Torin 1 (1 mg / ml), AZD8055 (1 mg / ml), and the mTOR inhibitor Rapamycin (1 mg / ml), the intraocular pressure of the mice decreased. However, both Torin 1 and AZD8055 showed a better intraocular pressure-lowering effect than Rapamycin, which is insensitive to 4E-BP1, manifested as a shorter time to reach the maximum intraocular pressure-lowering effect and a greater amplitude of intraocular pressure reduction. As Figure 4 shown.

[0042] Example 5 This example aims to prove that the 4E-BP1 inhibitor can inhibit hormone-induced fibrosis of mouse trabecular cells in vivo. The expression of α-SMA, an index related to trabecular cell fibrosis in mice, was detected by immunofluorescence technology to analyze the anti-fibrotic effect of the 4E-BP1 inhibitor on trabecular cells in vivo.

[0043] Experimental grouping: Control group: Gel diluted with blank.

[0044] Experimental group: 0.1% DEX gel.

[0045] 4E-BP1 inhibitor treatment group 1: 0.1% DEX gel + 1 mg / ml Torin1 gel.

[0046] 4E-BP1 inhibitor treatment group 2: 0.1% DEX gel + 1 mg / ml AZD8055 gel.

[0047] After completing the in vivo intraocular pressure monitoring experiment on mice, the eyeballs of the mice were dissected, fixed with 4% PFA at 4 °C for 2 hours, and then subjected to gradient dehydration and embedding. The expression of α-SMA, an index related to trabecular cell fibrosis in mice, was detected by immunofluorescence staining.

[0048] Experimental results: Local eye drops of 4E-BP1 inhibitors torin 1 (1 mg / ml) and AZD8055 (1 mg / ml) could effectively inhibit the expression of the fibrosis marker α-SMA (red fluorescence signal) in the trabecular meshwork and the inner wall of Schlemm's canal in the mouse model of steroid-induced ocular hypertension. As Figure 5 shown.

[0049] Conclusion In the TGF-β1-induced trabecular meshwork cell injury model, both 4E-BP1 inhibitors torin 1 and AZD8055 could significantly inhibit the expression of proteins related to trabecular meshwork cell fibrosis, especially the inhibitory effect on collagen 1 expression was significantly better than that of rapamycin. In vitro experiments also showed that in the mouse model of steroid-induced ocular hypertension, local eye drops of 4E-BP1 inhibitors torin 1 and AZD8055 showed good intraocular pressure-lowering effects, and the indexes related to trabecular meshwork fibrosis were significantly reduced, and the effect was better than that of rapamycin.

[0050] Innovations of the present invention 1. Target innovation: Trabecular meshwork damage and fibrosis are important etiologies for the pathological elevation of intraocular pressure leading to glaucoma and related optic nerve damage, but its pathogenesis is complex, and it is difficult to achieve ideal therapeutic effects based on the intervention of a single pathway. Based on the research progress of fibrosis in other tissues and organs of the body, this invention focuses on the trabecular meshwork fibrosis pathway The key regulatory points of the network were found, and it was discovered that 4E-BP1 is the intersection point of trabecular meshwork damage caused by various stress stimuli.

[0051] It provides an ideal intervention target for local trabecular meshwork tissue damage and repair in the eye.

[0052] 2. The present invention aims to provide a local 4E-BP1 inhibitor in the eye and a strategy for preparing an intraocular pressure-lowering drug.

[0053] The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should be regarded as within the protection scope of the present invention.

Claims

1. Use of 4E-BP1 pathway inhibitor in the preparation of an intraocular pressure-lowering drug.

2. The application according to claim 1, wherein The 4E-BP1 pathway inhibitor is 4E-BP1 inhibitor torin 1 or AZD8055 and its mimetics.

3. The application according to claim 2, characterized in that, The concentration of the 4E-BP1 inhibitor torin 1 is 1 mg / ml.

4. The application according to claim 2, wherein The concentration of the AZD8055 is 1 mg / ml.

5. The application according to claim 4, wherein The intraocular pressure-lowering drug is a drug for treating pathological intraocular pressure elevation caused by trabecular meshwork damage and fibrosis.

6. Use of 4E-BP1 pathway inhibitor in the preparation of a drug for repairing trabecular meshwork damage and fibrosis.

7. The application according to claim 6, wherein The 4E-BP1 pathway inhibitor is 4E-BP1 inhibitor torin 1 or AZD8055 and its mimetics.

8. The application according to claim 7, characterized in that, The concentration of the 4E-BP1 inhibitor torin 1 is 1 μM.

9. The application according to claim 7, wherein The concentration of the AZD8055 is 10 μM.

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