Use of a compound EMT-1 in the treatment of high myopia complicated with ocular hypertension

Abstract

The present invention provides an application of an inhibitor in the treatment of ocular hypertension, and the inhibitor is EMT-1, and its chemical formula is as follows: In the process of studying the YAP / TGF-β signaling pathway, it is found that the inhibitor EMT-1 can affect the expression levels of genes related to trabecular meshwork cells, thereby changing the morphology and microenvironment of the trabecular meshwork and regulating intraocular pressure. This discovery provides new technologies and ideas for screening small molecule drugs for targeted intervention in patients with high myopia complicated with POAG and for targeted intervention.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to application of a compound EMT-1 in treating high myopia combined with ocular hypertension. Background Art

[0002] Glaucoma is a group of diseases with common characteristics of optic disc atrophy and depression, visual field damage and decreased visual field. Pathological increase in intraocular pressure is the main risk factor for glaucoma. There are various types of glaucoma, including primary and secondary glaucoma, among which primary open-angle glaucoma (POAG) is the main type of glaucoma. Epidemiology believes that high myopia and related genetic factors are high-risk groups for POAG. The risk of glaucoma increases by 20% for every 1D increase in myopia. The incidence of POAG in people with high myopia is 7.3 times that of normal people. The increase in intraocular pressure in glaucoma patients is mostly caused by an imbalance between the outflow rate of aqueous humor and the production rate of aqueous humor.

[0003] The trabecular meshwork (TM) is the main functional part of the aqueous humor outflow channel. It is a three-dimensional mesh structure composed of trabecular meshwork cells in multiple layers of extracellular matrix (ECM). The change in its structure leads to an increase in the resistance to aqueous humor outflow, which is the main reason for the occurrence and development of POAG. Studies have found that the basement membrane of the TM tissue of POAG patients is thickened, the extracellular matrix is ​​remodeled, and the elastic fibers are increased and arranged in a disordered manner. It can be seen that increased intraocular pressure is closely related to the structural changes of TM. Therefore, understanding the morphological and structural changes of TM tissue at different time periods of high intraocular pressure can provide a preliminary reference for related research on controlling intraocular pressure and treating glaucoma. As the main channel for aqueous humor outflow, TM is a tissue in the eye that is highly sensitive to mechanical stimulation. It regulates intraocular pressure by adjusting cell morphology. Because intraocular pressure changes with fluid movement and ciliary muscle pressure gradient, and because of the unique structural characteristics of TM and the particularity of its location, it will be affected by pressure, tension and shear force. TM cells are biomechanically sensitive cells. Changes in pressure may lead to changes in gene expression, which in turn affects physiological functions such as cell proliferation, differentiation, migration, and apoptosis, and even causes changes in the structural morphology of tissues. A series of changes such as reduced gaps and increased tissue hardness were found in TM of open-angle glaucoma, resulting in changes in the structure and function of the trabecular meshwork.

[0004] Currently, most studies believe that the fibrous ECM deposition in the juxtacanalicular region of the trabecular meshwork is the most obvious change in the trabecular meshwork area of POAG. Under the action of high intraocular pressure or mechanical stress, the expression of related factors regulating extracellular matrix remodeling in the TM increases significantly, resulting in abnormal deposition of the extracellular matrix, thereby affecting the structure of the TM. Recent studies have shown that TM cells respond to mechanical strain by remodeling the ECM and cytoskeleton, altering gene expression, releasing cytokines, and regulating signal transduction.

[0005] Commonly used intraocular pressure-lowering drugs clinically include prostaglandin derivatives, cholinergic drugs, β-adrenergic receptor blockers, α-adrenergic receptor agonists, etc. There are also surgeries to reduce intraocular pressure, which helps prevent or slow down vision loss, but it cannot solve the problem of the degradation and loss of TM cell function.

[0006] In the process of studying the changes in the morphology and microenvironment of the trabecular meshwork, the present invention affects the expression levels of genes related to trabecular meshwork cells, thereby changing the morphology and microenvironment of the trabecular meshwork. This discovery is of great significance for the regulation of intraocular pressure, thus completing the present invention. Summary of the Invention

[0007] In a first aspect, the present invention provides the use of compound EMT-1 in the preparation of a drug for treating diseases caused by insufficient secretion of the extracellular matrix of trabecular meshwork cells. The inhibitor can regulate the expression of genes in trabecular meshwork cells, remodel the extracellular matrix, and slow down apoptosis to achieve the effect. The compound EMT-1 has the following chemical formula:

[0008]

[0009] Furthermore, the gene is selected from one or more of ECM (COL1, FN), AQP1, MLC, α-SMA.

[0010] Furthermore, it is found that in patients with high myopia complicated with high intraocular pressure, the gene expression levels of ECM (COL1, FN) and AQP1 are decreased, while the gene expression levels of MLC and α-SMA are increased. The compound EMT-1 of the present invention can up-regulate the gene expression levels of ECM (COL1, FN) and AQP1, and down-regulate the gene expression levels of MLC and α-SMA. Brief Description of the Drawings

[0011] Figure 1 Changes in IOP values of the control group, microsphere group, high myopia group, and high myopia with microsphere injection group;

[0012] Figure 2 Changes in the levels of genes related to the extracellular matrix and function of the TM in high myopia complicated with high intraocular pressure detected by PCR and fluorescence staining before drug treatment;

[0013] Figure 3Changes in the levels of apoptosis-related genes detected by PCR before drug treatment;

[0014] Figure 4 Expression of YAP1, Smad2, and Smad3 genes before drug treatment;

[0015] Figure 5 Changes in IOP values in the control group, high myopia with high intraocular pressure group, and high myopia with high intraocular pressure drug administration group after subconjunctival injection of the inhibitor EMT-1;

[0016] Figure 6 Changes in the levels of TM extracellular matrix and function-related genes in high myopia complicated with high intraocular pressure detected by PCR and fluorescence staining after drug treatment;

[0017] Figure 7 Changes in the levels of apoptosis-related genes detected by PCR after drug treatment;

[0018] Figure 8 Expression of YAP1, Smad2, and Smad3 genes after drug treatment; Specific implementation manners

[0019] The following specifically refers to the embodiments to illustrate the present invention. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0020] Example 1 Construction of an animal model of high myopia complicated with high intraocular pressure

[0021] Construction of a high myopia model

[0022] Experimental animals: Clean-grade three-color guinea pigs were purchased from Changyi Experimental Animal Breeding Co., Ltd., Danyang City, with 30 males and 30 females, 2 weeks old, and a body weight of about 100 g. They were raised in the experimental animal center of the ophthalmic hospital. Before the experiment, all guinea pigs were in good condition, with a binocular anisometropia value less than 2.50 D and all being hyperopic, and no other eye diseases. The breeding environment conformed to the management regulations of the experimental animal center, with a constant room temperature of about 22 ± 2 °C, a 12-hour light-dark cycle, and free access to food and water.

[0023] First, a high myopia model was established. Using 2-week-old three-color guinea pigs, after detecting and recording the AL length, refractive power, and IOP, the right single eye was covered with a rubber headgear, and a high myopia animal model was constructed by the method of visual deprivation. From the 8th week, the axial length and refractive power were detected weekly. After the refractive power exceeded -6.00 D, the high intraocular pressure model was established. The IOP was detected before the operation, and then 3 μl of latex microspheres were injected into the anterior chamber with a 32G needle, and pressed for a moment to ensure no leakage. The IOP was detected starting from the second day after the operation, 3 times each in the morning, afternoon, and evening.

[0024] 1. Detection of AL length, refractive power, and IOP in the high myopia model

[0025] 1.1 Axial length measurement:

[0026] Calibrate the instrument regularly to ensure the accuracy of the measurement data. Before the examination, instill proparacaine hydrochloride eye drops for surface anesthesia. Disinfect the probe, usually with 75% ethanol. Note that the measurement should be carried out after the alcohol on the probe surface has completely evaporated to avoid corneal burns caused by residual alcohol. During the measurement, the probe is perpendicular to the center of the cornea without pressing on the cornea to avoid scratching the cornea. Measure more than 5 groups of data, delete the values with large differences, and finally take the average of 5 groups of similar measurement results.

[0027] 1.2 Retinoscopy:

[0028] Prepare a retinoscope and a trial lens case. Place the plus lenses on the right side of the trial lens case and the minus lenses on the left side. Place the spherical lenses on the outside and the cylindrical lenses in the middle. Prepare a flexible ruler and fix the guinea pig device at a distance of 50 cm from the examiner. The height of the device needs to be parallel to the examiner's visual axis. Prepare a semi-dark room so that it is easy to see the light band and also the refractive power on the lenses. Dilate the pupils with atropine sulfate eye drops before the examination. First, observe and judge whether it is with-the-rule or against-the-rule movement, that is, whether the far point is in front of or behind the examiner. If with-the-rule movement is seen, add plus lenses. When with-the-rule movement is seen, the far point is located somewhere behind the examiner, and plus lenses (converging the outgoing light) need to be added to move the far point to the retinoscope. At this time, the neutralization process can be seen. If against-the-rule movement is seen, add minus lenses. When the examiner sees against-the-rule movement, the far point is located somewhere in front of the examiner, and minus lenses (diverging the outgoing light) need to be added to move the far point to the retinoscope. At this time, the examiner can see the neutralization phenomenon. Retinoscopy is performed at 0 weeks, 4 weeks, 8 weeks, and 11 weeks of visual deprivation in guinea pigs. Retinoscopy is performed by the same optometrist using the single-blind method, and each eye is measured three times repeatedly and the average value is finally taken.

[0029] 1.3 Intraocular pressure measurement:

[0030] Instill proparacaine hydrochloride eye drops for ocular surface anesthesia before the examination. Soothe the guinea pig's emotions. During the measurement, the probe of the rebound tonometer is perpendicular to the cornea of the guinea pig to measure the IOP of the guinea pig. Take the average of 6 measurement results as the intraocular pressure value. IOP measurements are performed before and after visual deprivation (0 weeks, 11 weeks) and before and after anterior chamber injection (0 days, and then once every other day for 5 days). All measurements are completed between 9 am and 12 pm.

[0031] Experimental results: Before visual deprivation, there were no significant differences in axial length (AL), refractive power, and intraocular pressure (IOP) among the control group, natural control group, and visual deprivation group. At 5 weeks in the visual deprivation group, the AL was approximately 8.19 mm and the refractive power was approximately -5.00 D, showing significant differences in AL and refractive power compared with those of the control group and natural control group (P < 0.001). By 8 weeks, the AL (8.57 ± 0.16 mm) and refractive power (-6.54 ± 0.56 D) of the visual deprivation group were statistically significant compared with those of the control group (AL: 8.09 ± 0.09 mm; refractive power: -0.91 ± 1.16 D) and natural control group (AL: 8.16 ± 0.15 mm, refractive power: -0.11 ± 1.38 D) (P < 0.001), but there was no statistical difference in IOP (control group: 17.57 ± 3.00 mmHg; natural control group: 18.62 ± 2.93 mmHg; visual deprivation group: 19.26 ± 4.95 mmHg) (see Table 1).

[0032] Table 1. Changes in refractive error of guinea pigs at different time points

[0033]

[0034]

[0035] Construction of a model of high myopia combined with high intraocular pressure by injecting latex microspheres into the anterior chamber in guinea pigs with high myopia:

[0036] Guinea pigs were anesthetized with 0.6% sodium pentobarbital (20 g / 0.125 ml) at a dose of per kilogram. After topical anesthesia with proparacaine hydrochloride eye drops, the eyelids were opened with an eyelid retractor. One hand held forceps to fix the eyeball, and the other hand held a 32G needle Hamilton microsyringe. The cornea was punctured and inserted into the anterior chamber at a 45° angle, and 3 μl of latex microspheres were injected. The needle was slowly withdrawn, and a little eye ointment was applied (see Figure 1 ).

[0037] Results: After injecting an equal volume of microspheres into the anterior chamber of the guinea pig high myopia model, compared with the other three groups (normal group: 19.4 ± 4.219 mmHg; normal microsphere injection group: 20 ± 2.121 mmHg; high myopia group: 17.6 ± 3.209 mmHg), the IOP of the high myopia microsphere injection group (33.2 ± 5.119 mmHg) was significantly increased (***P < 0.001). (An IOP higher than 25 mmHg for more than 2 weeks is a model of high myopia combined with high intraocular pressure)

[0038] Example 2 Subconjunctival drug treatment for a model of high myopia combined with high intraocular pressure

[0039] After topical anesthesia with proparacaine hydrochloride eye drops, guinea pigs with high myopia complicated with high intraocular pressure were anesthetized by inhalation of ether and isoflurane. The eyelids were retracted with an eyelid retractor. One hand held forceps to fix the eyeball, and the other hand held a syringe. The needle was at a 15° angle to the eyeball, and the bulbar conjunctiva near the temporal fornix (5 mm from the corneal limbus) was punctured. The bulbar conjunctiva was gently lifted and the needle was inserted about 3-4 mm. EMT-1 (50 ng) was slowly injected, and the bulbar conjunctiva at this site bulged like a fish bubble. IOP was measured three times a day, morning, noon, and evening, for 7 consecutive days before and after the injection.

[0040] Example 3 Changes in the gene expression levels of TM cells before and after drug treatment in a model of high myopia complicated with high intraocular pressure

[0041] Experimental method:

[0042] 1. Detection of TM gene expression level by qPCR

[0043] 1 ml of Trizol was added to the collected TM tissue, and vortexed for 5 minutes to fully lyse it. 200 μl of chloroform was added, vortexed again for 30 s, and then left standing for 3 minutes. The centrifuge was pre-cooled to 4 °C, and the samples were centrifuged at 12,000 rpm for 15 minutes. After centrifugation, the samples showed three layers of stratification. Carefully aspirate the upper colorless aqueous phase and transfer it to a 1.5 ml EP tube. An equal volume of absolute ethanol was added, inverted and mixed well, and then transferred to a centrifugal column. Centrifuge at 10,000 rpm for 30 s, and discard the filtrate. Add 500 μl of Clean Buffer, centrifuge at 10,000 rpm for 30 s, and discard the filtrate. Add 500 μl of Clean Buffer, centrifuge at 10,000 rpm for 30 s, and discard the filtrate. Add 500 μl of WashBuffer, centrifuge at 10,000 rpm for 30 s, and discard the filtrate. Add 500 μl of Wash Buffer, centrifuge at 10,000 rpm for 30 s, and discard the filtrate. Centrifuge again at 10,000 rpm for 2 minutes to remove residual ethanol. The centrifugal column was placed in a new EP tube, and 20 μl of Nuclease-free Water was added, and centrifuged at 12,000 rpm for 2 minutes to obtain mRNA, which was stored in a -80 °C refrigerator. The RNA was incubated at 65 °C for 5 minutes and then immediately cooled on ice. This step helps RNA with a higher-order structure to provide reverse transcription efficiency.

[0044] Reverse transcription was carried out at 37 °C for 15 minutes. The total volume of the reaction solution was configured to 10 μl.

[0045]

[0046] Enzyme inactivation reaction was carried out at 98 °C for 5 minutes. After the reaction, it was stored at -20 °C. The qPCR reaction system was configured on ice, and the reaction system was a 10 μL system.

[0047]

[0048] Set the program as pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for 40 cycles. Obtain fluorescence signals during the cycling reaction, and finally add the melting curve. After the PCR experiment, export the corresponding data analysis results.

[0049] 2. Detect the expression level of TM gene by tissue immunofluorescence

[0050] Fix the guinea pig eyeballs in 4% paraformaldehyde solution for 2 hours. Add embedding gel into the embedding cassette, then put the guinea pig eyeballs in, adjust the position and direction, and place it in a -80°C refrigerator. Take it out after the embedding gel solidifies. Turn on the microtome in advance, and lower the blade temperature and the operating room temperature to about -20°C. Set the section thickness to 12 μm. Wash the embedding gel on the glass slide with PBS and put it in a wet box for subsequent operations. Fix it with 4% paraformaldehyde at room temperature for 30 minutes. Wash it three times with PBS, 5 minutes each time. Treat it with 0.3% Tritonx-100 for 10 minutes. Wash it three times with PBS, 5 minutes each time. Incubate it with the blocking solution (5% BSA) at room temperature for 1 hour. Incubate it with a certain proportion of primary antibody overnight in a 4°C refrigerator. Wash it 3 times with PBS, 5 minutes each time. Incubate it with a certain proportion of secondary antibody in the dark at room temperature for 1 hour. Wash it three times with PBS, 5 minutes each time. Incubate it with DAPI in the dark at room temperature for 20 minutes. Wash it three times with PBS, 5 minutes each time. Drop anti-fluorescence quencher and then seal the coverslip. Take pictures under a confocal microscope. Experimental results:

[0051] Detection results before drug treatment (see Figure 2— Figure 4 )

[0052] As shown in Figures 2(a) and 2(b), detect the changes in the levels of TM extracellular matrix and function-related genes in high myopia with high intraocular pressure by PCR and fluorescence staining. Compared with the control group, the gene expression levels of cell ECM (COL1, FN), AQP1 in the high myopia with high intraocular pressure group were down-regulated, and the expression levels of MLC and α-SMA were up-regulated (*P<0.05, ***P<0.001).

[0053] As Figure 3 shown, detect the change levels of apoptosis-related genes by PCR. Compared with the control group, the gene expression of anti-apoptotic protein Bcl2 was down-regulated, and the gene expressions of pro-apoptotic proteins Caspase3 and BAX were up-regulated in the high myopia with high intraocular pressure group (*P<0.05, ***P<0.001).

[0054] As Figure 4As shown in the figure, PCR detection found that compared with the control group, the gene expression levels of YAP1, Smad2, and Smad3 in the YAP / TGF-β pathway in the high myopia combined with high intraocular pressure group were significantly up-regulated (***P<0.001).

[0055] The detection results after drug treatment (see Figure 5 — Figure 8 ):

[0056] As Figure 5 shown, after subconjunctival injection of the inhibitor EMT-1, the IOP in the high myopia combined with high intraocular pressure group decreased from 33.0±6.33 mmHg to 18.4±3.36 mmHg after 7 days, and could be restored to the normal intraocular pressure level (16.6±3.05 mmHg) (*P<0.05, **P<0.01).

[0057] As shown in Figures 6(a) and 6(b), compared with the high myopia combined with high intraocular pressure group, the gene expression levels of ECM (COL1, FN) and AQP1 in the cells of the EMT-1 administration group were up-regulated, and the expression levels of MLC and α-SMA were down-regulated (**P<0.01, ***P<0.001).

[0058] As Figure 7 shown, PCR was used to detect the change levels of apoptosis-related genes. Compared with the high myopia combined with high intraocular pressure group, the gene expressions of the pro-apoptotic proteins Caspase3 and BAX in the EMT-1 administration group were down-regulated, and the gene expression of the anti-apoptotic protein Bcl2 was up-regulated (*P<0.05, **P<0.01, ***P<0.001).

[0059] As Figure 8 shown, PCR was used to detect the change levels of related genes. Compared with the high myopia combined with high intraocular pressure group, the gene expressions of YAP1, Smad2, and Smad3 in the EMT-1 administration group were significantly down-regulated (***P<0.001).

[0060] Thus, it can be seen that EMT-1 in the present invention significantly reduces IOP to the normal intraocular pressure by up-regulating the gene expressions of ECM (COL1, FN) and AQP1 and down-regulating the gene expressions of MLC and α-SMA.

Claims

1. Use of an inhibitor in the preparation of a drug for treating ocular hypertension diseases, wherein the ocular hypertension diseases are concurrently complicated with high myopia; the inhibitor can regulate the expression of genes of trabecular meshwork cells, and the inhibitor is EMT-1, and the chemical formula is as follows: 。

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