Use of lifitegrast in preparation of drug for treating retinal artery occlusion injury

Abstract

The present invention provides use of lifitegrast in the preparation of a drug for treating retinal artery occlusion injury. Lifitegrast can effectively reduce retinal artery occlusion injury and apoptosis of retinal ganglion cells and has the effect of inhibiting inflammatory infiltration in the retina and activation of microglia. Moreover, lifitegrast has the effect of reducing retinal ganglion cell injury. Lifitegrast can be used for related diseases such as neuroinflammatory injury caused by retinal artery occlusion.

Description

Use of lifitegrast in preparation of a medicament for treating retinal artery occlusion injury TECHNICAL FIELD

[0001] The present application relates to the technical field of ophthalmic drugs, and particularly relates to use of lifitegrast in preparation of a medicament for treating retinal artery occlusion injury. BACKGROUND

[0002] Retinal artery occlusion (RAO) is characterized by sudden loss of unilateral vision without pain, and a large number of deaths of retinal ganglion cells (RGC) and strong inflammatory reactions, especially activation of microglia, due to sudden interruption of blood flow, resulting in loss of visual function. At present, the treatment measures for RAO mainly include thrombolytic therapy, hyperbaric oxygen therapy, intraocular pressure reduction and eye massage. In actual clinical work, most patients have missed the time window for thrombolytic therapy, and other treatment measures can only alleviate the symptoms of a part of patients, but it is difficult to obtain satisfactory improvement of visual acuity. The pathogenic factors of ROA are complex, and there is a lack of rapid and effective treatment drugs and programs in the clinic. Therefore, it is of great significance to develop a low-cost drug for treating retinal artery injury.

[0003] Lifitegrast is a new small molecule integrin antagonist, which is an artificially synthesized compound, and has a molecular formula of C 29 H 24 Cl2N2O7S, a CAS number of 1025967-78-5, and a structural formula as follows:

[0004] Studies have shown that lifitegrast is a new inhibitor of intercellular adhesion factor, which can act by blocking the binding between intercellular adhesion molecule-1 and integrin protein lymphocyte function-associated antigen-1. In July 2016, the U.S. Food and Drug Administration (FDA) officially approved the application of 5% lifitegrast eye drops (trade name XiidraTM), which is the first new drug approved by the FDA for improving and treating the symptoms of dry eye. The clinical trials of the drug mainly include one 12-week phase II clinical trial and three 12-week phase III clinical trials for patients with dry eye, and the research results fully prove the effectiveness and safety of the drug.

[0005] Retinal artery occlusion injury belongs to the category of fundus diseases, and there is currently no effective treatment drug. Lifitegrast has not been reported in the treatment of this disease, and therefore it is necessary to develop a new treatment drug for treating retinal artery occlusion injury.

[0006] The present application intends to develop a new use of litaoer in retinal artery obstruction injury, and provide a treatment method and idea for clinical research. SUMMARY

[0007] The present application intends to provide a new use of litaoer to solve the problem of lack of treatment methods for retinal artery obstruction. Through experiments, it is found that litaoer can effectively reduce the retinal artery obstruction injury and the apoptosis of retinal ganglion cells in mice. At the same time, litaoer also has the effect of inhibiting the inflammatory infiltration and activation of microglia cells in the retina. At the same time, litaoer also has the effect of reducing the damage of retinal ganglion cells, and can be used for treating diseases related to retinal artery obstruction and neuroinflammatory injury, and has a wide application prospect.

[0008] The purpose of the present application is achieved by the following technical solutions:

[0009] On the one hand, the purpose of the present application is to provide a use of litaoer in the preparation of a drug for treating retinal artery obstruction injury.

[0010] On the second aspect, the purpose of the present application is to provide a use of a composition containing litaoer in the preparation of a drug for treating retinal artery obstruction injury.

[0011] On the third aspect, the purpose of the present application is to provide a use of litaoer in the preparation of a drug for inhibiting the gene expression of at least one of Inos, Tnf-alpha, Il-1beta and IL-6.

[0012] On the fourth aspect, the purpose of the present application is to provide a use of a composition containing litaoer in the preparation of a drug for inhibiting the gene expression of at least one of Inos, Tnf-alpha, Il-1beta and IL-6.

[0013] On the fifth aspect, the use of litaoer in the preparation of a drug for reducing the apoptosis of retinal ganglion cells.

[0014] The administration mode of the drug is intravitreal injection.

[0015] The administration dose is 0.1-1ug.

[0016] The drug acts on the microglial cell line BV2 or the retinal precursor cell line R28.

[0017] On the sixth aspect, the purpose of the present application is to provide a drug for treating retinal artery obstruction injury, comprising litaoer.

[0018] On the seventh aspect, the purpose of the present application is to provide a drug for inhibiting the gene expression of at least one of Inos, Tnf-alpha, Il-1beta and IL-6, comprising litaoer.

[0019] The pharmaceutically acceptable salt and the pharmaceutically acceptable carrier are further included to form a clinically acceptable medicine or preparation.

[0020] The carrier is at least one of a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, an aromatic, and a sweetener in the pharmaceutical field.

[0021] The present application has the following advantages and beneficial effects:

[0022] The present application finds a new use of Rilostane to solve the problem of lack of treatment means for retinal artery obstruction. Rilostane can effectively reduce the damage of retinal artery obstruction and the apoptosis of retinal ganglion cells. At the same time, Rilostane also has the effect of inhibiting the inflammatory infiltration and activation of microglia in the retina.

[0023] Rilostane also has the effect of reducing retinal ganglion cell damage and can be used for related diseases such as retinal artery obstruction and neuroinflammatory damage, and has a wide application prospect.

[0024] In the present application, Rilostane has the effect of inhibiting the activation of microglia, and the object of this effect is the microglia cell line BV2.

[0025] In the present application, Rilostane has the function of relieving the damage of retinal ganglion cells, and the object of this function is the retinal precursor cell line R28. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a mouse electroretinogram (ERG) detection condition of Example 2;

[0027] Figure 2 is a mouse retinal HE staining pathological result picture of Example 2;

[0028] Figure 3 is a mouse retinal smear immunofluorescence staining RGC result picture of Example 2;

[0029] Figure 4 is the effect of different concentrations of Rilostane on the activity of BV2 cells of Example 3;

[0030] Figure 5 is the effect of Rilostane on the expression of iNOS in BV2 cells of Example 3;

[0031] Figure 6 is the effect of Rilostane on the expression of TNF-α in BV2 cells of Example 3;

[0032] Figure 7 is the effect of Rilostane on the expression of IL-1β in BV2 cells of Example 3;

[0033] Figure 8 is the effect of Rilostane on the expression of IL-6 in BV2 cells of Example 3;

[0034] Figure 9 shows the effect of retaspimycin on R28 cell oxygen glucose deprivation injury of Example 4. DETAILED DESCRIPTION

[0035] In order to better understand the present application, the following examples are further illustrations of the present application, but the scope of the present application is not limited to the following examples.

[0036] The following detailed description is presented to enable any person skilled in the art to make and use the present application. For purposes of explanation, specific nomenclatures are set forth to provide a thorough understanding of the present application. Descriptions of well-known functions and constructions are omitted so as to not obscure the disclosure with details that are well known to those skilled in the art. The detailed description is presented primarily for the purpose of enabling others skilled in the art to make and use the application. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described herein. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control. Various modifications and changes can be made to the specific embodiments described herein by those skilled in the art without departing from the scope or spirit of the present application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given herein are exemplary and not intended to be limiting, of the scope or spirit of the application. In this document, the terms "compri sing," "comprises," "comprised," "comprising," "having," "including," and the like, are synonymous with the term "containing" and are used in the inclusive, rather than the exclusive, sense. The terms "comprising," "comprises" and "comprised of" as used herein are to be interpreted symmetrically with "containing" or "includes" or "including." It is intended that the application not be limited to the specific embodiments disclosed, but will include all embodiments falling within the scope of the appended claims.

[0037] The present application provides a mouse model of retinal ischemia-reperfusion injury, further, the mouse model is simulated by occluding the pterygopalatine artery of the mouse for a period of time and then reperfusing the blood to simulate the pathological process of the disease. Further, the occlusion time of the mouse pterygopalatine artery is 2 hours, and the blood reperfusion time is 7 days. Further, the occlusion of the pterygopalatine artery is achieved by the method of wire plug. The present application also develops a method for treating mouse retinal artery occlusion injury with retaspimycin, further, the administration method of the method is intravitreal injection, the administration concentration is 1 ug / ul, the volume is 1 ul, and the administration time is once every two days. The present application also provides the function of retaspimycin in inhibiting the activation of microglial cells, further, the object of the function is the mouse microglial cell line BV2. The present application also provides the function of retaspimycin in relieving retinal ganglion cell injury, and the object of the function is the rat retinal precursor cell line R28.

[0038] Example 1: Establishment of a mouse retinal artery occlusion injury animal model:

[0039] Prepare 8-week-old C57BL / 6 male mice, whose body weight is strictly controlled at 20-25 g. Then prepare the gas anesthesia device. Anesthetize the mice with a 1.5-2% isoflurane nitrous oxide / oxygen mixture through a rubber tube. The body temperature during the operation is maintained at 37±0.5℃. The surgical instruments are disinfected with 75% alcohol. The mice are supine on the heating blanket and their necks are exposed. Then the fur on the neck of the mouse is removed, the skin is disinfected and the skin is cut along the midline of the neck. The submandibular gland is pulled apart with two forceps. The left common carotid artery, internal carotid artery and external carotid artery are separated directly, but do not squeeze the adjacent nerves and veins. Then the common carotid artery and internal carotid artery are clamped with a vascular clamp. The external carotid artery is sutured with 8-0 silk thread, and the distal end is knotted and the proximal end is tied. A small incision is made between the two suture knots of the external carotid artery with an ophthalmic scissors, and then a special thread embolism is inserted into the external carotid artery and common carotid artery along the incision. Next, remove the internal carotid artery clip, cut the external carotid artery at the opening of the external carotid artery, pull the thread embolism back to the bifurcation of the common carotid artery, reverse it and insert it into the internal carotid artery, the pterygopalatine artery and the ophthalmic artery. The thread embolism is inserted into the end of the pterygopalatine artery, about 6 mm from the bifurcation. The tail of the thread embolism is almost at the bifurcation, blocking the ophthalmic artery. Then tie the slipknot and suture the skin. The mice can move freely during the arterial embolization. After 120 minutes of embolization, the thread embolism is carefully removed from the pterygopalatine artery without significant bleeding. Remove the common carotid artery clip and restore arterial reperfusion. Then suture the skin wound and feed the mice routinely during reperfusion.

[0040] Example 2: In vivo effect of Litak on mouse retinal artery occlusion injury:

[0041] After 24 hours of intravitreal injection of Litak in mice, the mouse retinal artery occlusion model was established, and after 7 days, the mice were dark adapted for 12 hours, and the mouse retinal potential was determined; Carefully remove the mouse eyeball and place it in a special eyeball fixing solution for more than 2 hours for paraffin embedding, sectioning and HE staining; At the same time, the other half of the mice were carefully separated under the operating microscope to ensure the integrity and cell activity of the retina, fixed and immunofluorescently stained for Brn3a, and photographed for analysis. Figure 1 is the detection of mouse retinal electrogram (ERG) in Example 2, from which it can be seen that the amplitude of the ERG of the Litak-treated mice is higher, the retinal thickness is increased compared with the control group, and the retinal ganglion density is increased, indicating that Litak can effectively alleviate the damage to retinal ganglion cells in retinal artery occlusion and help restore visual function.

[0042] Figure 2 is the HE staining pathological result picture of the mouse retina in Example 2, from which it can be seen that the retinal thickness of the Litak-treated mice is increased and the damage is reduced.

[0043] Figure 3 is the RGC result picture of the mouse retinal smear immunofluorescent staining in Example 2, from which it can be seen that the number of ganglion cells in the retina after Litak treatment is increased.

[0044] Example 3: Effect of Rilostane on the metabolism of microglial cells:

[0045] The mouse microglial cell line BV2 was cultured in vitro, and plated into groups in the logarithmic phase of cell growth, divided into a control group, a bacterial lipopolysaccharide (LPS) stimulation group and an LPS + Rilostane group, the LPS concentration was 200 ng / ml, and the Rilostane concentration was 100 ng / ml. After 24 hours of stimulation, the cells were collected, RNA was extracted, and the concentration and quality of the RNA were determined, Real-Time PCR was performed to detect the mRNA expression of Inos, Tnf-α, IL-1β and IL-6, and the relative expression level changes were analyzed. The experimental results showed that Rilostane can effectively inhibit the metabolism and activation of microglial cells.

[0046] Figure 4 is the effect of different concentrations of Rilostane on the activity of BV2 cells in Example 3, from which it can be seen that the concentration of Rilostane from 0 to 100 ng / ml does not affect the activity of BV2 cells.

[0047] Figure 5 is the effect of Rilostane on the expression of iNOS in BV2 cells in Example 3, from which it can be seen that Rilostane can inhibit the gene expression level of iNOS and reduce the activation of microglial cells.

[0048] Figure 6 is the effect of Rilostane on the expression of TNF-α in BV2 cells in Example 3, from which it can be seen that Rilostane can inhibit the gene expression level of TNF-α and reduce the activation of microglial cells.

[0049] Figure 7 is the effect of Rilostane on the expression of IL-1β in BV2 cells in Example 3, from which it can be seen that Rilostane can inhibit the gene expression level of IL-1β and reduce the activation of microglial cells.

[0050] Figure 8 is the effect of Rilostane on the expression of IL-6 in BV2 cells in Example 3, from which it can be seen that Rilostane can inhibit the gene expression level of IL-6 and reduce the activation of microglial cells.

[0051] Example 4: Effect of Rilostane on retinal ganglion cell damage by oxygen-glucose deprivation:

[0052] The rat retinal ganglion cell line R28 was cultured in vitro, and plated into groups in the logarithmic phase of cell growth, and subjected to oxygen-glucose deprivation treatment. Oxygen-glucose deprivation was performed for 6 hours, and re-oxygenation was performed for 24 hours. The cells were divided into a solvent control group and a litakast group, and the concentration of litakast was 100 ng / ml. The stimulation time was 24 hours. The cells were collected, and the cells were digested into a single cell suspension. After resuspension with PBS, Annexin V and PI staining was performed, and flow cytometry was used to analyze the apoptosis of the cells. Figure 9 is the effect of litakast on oxygen-glucose deprivation injury of R28 cells according to Example 4. It was found that the proportion of apoptotic cells was significantly reduced after litakast treatment. The experimental results show that litakast can reduce the injury of retinal ganglion cells.

[0053] The above is only a preferred embodiment of the present application, and of course cannot limit the scope of protection of the present application. It should be pointed out that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and changes can be made, and these improvements and changes are also considered to be within the scope of protection of the present application.

Claims

1. Use of a retaspimycin in the manufacture of a medicament for treating damage from retinal arterial occlusion.

2. Use according to claim 1, characterized in that: The retaspimycin is used to inhibit gene expression of at least one of Inos, Tnf-α, Il-1β, IL-6.

3. Use according to claim 1, characterized in that: The retaspimycin is used to reduce retinal ganglion cell apoptosis.

4. Use of a retaspimycin-containing composition in the manufacture of a medicament for treating damage from retinal arterial occlusion.

5. Use according to claim 2, characterized in that: The retaspimycin-containing composition is used to inhibit gene expression of at least one of Inos, Tnf-α, Il-1β, IL-6.

6. Use according to claim 2, characterized in that: The retaspimycin-containing composition is used to reduce retinal ganglion cell apoptosis.

7. The use according to any one of claims 1 to 6, characterized in that, The medicament is administered by intravitreal injection.

8. Use according to any one of claims 1 to 6, characterized in that: The medicament is used on a microglial cell line BV2 or a retinal precursor cell line R28.

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