Use of ritastatins in the preparation of a medicament for treating retinal artery occlusion injury

Abstract

The present invention relates to the technical field of ophthalmic drugs, and particularly relates to the use of loteprednol etabonate in the preparation of a drug for treating retinal artery occlusion injury. The present invention discovers a new use of loteprednol etabonate to solve the problem of lack of treatment means for retinal artery occlusion. Loteprednol etabonate can effectively reduce retinal artery occlusion injury and apoptosis of retinal ganglion cells. At the same time, loteprednol etabonate also has the effect of inhibiting retinal inflammatory infiltration and activation of microglia. Loteprednol etabonate also has the effect of reducing damage to retinal ganglion cells, can be used for related diseases such as neuroinflammatory injury of retinal artery occlusion, and has broad application prospects.

Description

Technical Field

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

[0002] Retinal Artery Occlusion (RAO) is clinically characterized by painless and sudden loss of unilateral vision. Due to the sudden interruption of blood flow, a large number of retinal ganglion cells (RGCs) die and a strong inflammatory response occurs, especially the activation of microglia, resulting in visual function loss. Currently, the treatment measures for RAO mainly include thrombolytic therapy, hyperbaric oxygen therapy, reduction of intraocular pressure, and ocular massage. In actual clinical work, most patients have missed the time window for thrombolytic therapy. Although other treatment measures can relieve the symptoms of some patients, it is difficult to obtain satisfactory visual improvement. The pathogenic factors of ROA are complex, and there is a lack of rapid and effective treatment drugs and regimens in clinical practice. Therefore, it is of great research significance to develop drugs with low price for treating retinal artery injury.

[0003] Lifitegrast is a new type of small molecule integrin antagonist, a compound that can be artificially synthesized, with a molecular formula of C 29 H 24 Cl2N2O7S, with a CAS number of 1025967-78-5, and the structural formula is as follows:

[0004]

[0005] Studies have confirmed that lifitegrast is a new inhibitor of intercellular adhesion factors, and it can take effect by blocking the binding between intercellular adhesion molecule-1 and integrin protein lymphocyte function-associated antigen-1. In July 2016, the US 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 to improve and treat the symptoms of dry eye. The clinical trials of this drug mainly include 1 12-week Phase II clinical trial for dry eye patients and 3 12-week Phase III clinical trials. The research results fully prove the effectiveness and safety of this drug.

[0006] Retinal artery occlusion injury belongs to the category of fundus diseases. Currently, there is no effective treatment drug, and there is no research report on lifitegrast in this disease. Therefore, it is necessary to develop a new treatment drug for treating retinal artery occlusion injury.

[0007] The present invention aims to develop a new use of ritastatins in retinal artery occlusion injury, providing treatment means and ideas for clinical research. Summary of the Invention

[0008] The object of the present invention is to provide a new use of ritastatins to solve the problem of lack of treatment means for retinal artery occlusion. Through experiments, it is found that ritastatins can effectively reduce retinal artery occlusion injury and apoptosis of retinal ganglion cells in mice. At the same time, ritastatins also have the effect of inhibiting inflammatory infiltration in the retina and activation of microglia. At the same time, ritastatins also have the effect of reducing damage to retinal ganglion cells, and can be used for related diseases such as neuroinflammatory damage of retinal artery occlusion, with broad application prospects.

[0009] The object of the present invention is achieved through the following technical solutions:

[0010] On the one hand, the object of the present invention is to provide an application of ritastatins in the preparation of a drug for treating retinal artery occlusion injury.

[0011] On the second hand, the object of the present invention is to provide an application of a composition containing ritastatins in the preparation of a drug for treating retinal artery occlusion injury.

[0012] On the third hand, the object of the present invention is to provide an application of ritastatins in the preparation of a drug for inhibiting the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6.

[0013] On the fourth hand, the object of the present invention is to provide an application of a composition containing ritastatins in the preparation of a drug for inhibiting the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6.

[0014] On the fifth hand, the application of ritastatins in the preparation of a drug for reducing apoptosis of retinal ganglion cells.

[0015] The administration method of the drug is: intravitreal injection.

[0016] The administration dose is 0.1 - 1 μg.

[0017] The action target of the drug is the microglial cell line BV2 or the retinal progenitor cell line R28.

[0018] On the sixth hand, the object of the present invention is to provide a drug for treating retinal artery occlusion injury, comprising ritastatins.

[0019] On the seventh hand, the object of the present invention is to provide a drug for inhibiting the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6, comprising ritastatins.

[0020] It also includes pharmaceutically acceptable salts and pharmaceutically acceptable carriers to form clinically acceptable drugs or preparations.

[0021] The carrier is at least one of conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, fragrances, and sweeteners in the pharmaceutical field.

[0022] The present invention has the following advantages and beneficial effects:

[0023] The present invention discovers a new use of ritastatins to solve the problem of lack of treatment means for retinal artery occlusion. Ritastatins can effectively reduce retinal artery occlusion injury and apoptosis of retinal ganglion cells. At the same time, ritastatins also have the effect of inhibiting retinal inflammatory infiltration and activation of microglia.

[0024] Ritastatins also have the effect of reducing retinal ganglion cell damage and can be used for related diseases such as neuroinflammatory damage of retinal artery occlusion, showing broad application prospects.

[0025] In the present invention, ritastatins have the effect of inhibiting the activation of microglia, and the object of this effect is the microglial cell line BV2.

[0026] In the present invention, ritastatins have the function of alleviating retinal ganglion cell damage, and the object of this function is the retinal progenitor cell line R28. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It shows the detection of electroretinogram (ERG) of mice in Example 2;

[0028] Figure 2 It shows the pathological result pictures of HE staining of the retina of mice in Example 2;

[0029] Figure 3 It shows the RGC result pictures of immunofluorescence staining of retinal whole mounts of mice in Example 2;

[0030] Figure 4 It shows the effect of ritastatins at different concentrations on the viability of BV2 cells in Example 3;

[0031] Figure 5 It shows the effect of ritastatins on the expression of iNOS in BV2 cells in Example 3;

[0032] Figure 6 It shows the effect of ritastatins on the expression of TNF-α in BV2 cells in Example 3;

[0033] Figure 7 It shows the effect of ritastatins on the expression of IL-1β in BV2 cells in Example 3;

[0034] Figure 8 Effect of rituximab in Example 3 on IL-6 expression in BV2 cells;

[0035] Figure 9 Effect of rituximab in Example 4 on oxygen-glucose deprivation injury in R28 cells. Detailed implementation manners

[0036] For a better understanding of the present invention, the following examples further illustrate the present invention, but the content of the present invention is not limited to the following examples.

[0037] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail. Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary. Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0038] The present invention provides a mouse model of retinal ischemia-reperfusion injury. Further, this mouse model simulates the pathological process of the disease by surgically occluding the pterygopalatine artery of the mouse for a period of time and then restoring blood flow. Further, the occlusion time of the pterygopalatine artery of the mouse is 2 hours, and the blood reperfusion time is 7 days. Further, the occlusion of the pterygopalatine artery is achieved by using a thread embolization method. The present invention also develops a method for treating mouse retinal artery occlusion injury with ritacalimod. Further, the administration method of this method is intravitreal injection, the administration concentration is 1 μg / μl, the volume is 1 μl, and the administration time is once every two days. The present invention also provides an effect of ritacalimod in inhibiting microglial activation. Further, the object of this effect is the mouse microglial cell line BV2. The present invention also provides a function of ritacalimod in alleviating retinal ganglion cell injury. The object of this effect is the rat retinal progenitor cell line R28.

[0039] Example 1: Establishment of an animal model of mouse retinal artery occlusion injury:

[0040] Prepare 8-week-old male C57BL / 6 mice with their body weight strictly controlled at 20 - 25 g. Then prepare a gas anesthesia device. Anesthetize the mice with a nitrous oxide / oxygen mixture of 1.5 - 2% isoflurane through a rubber tube. Maintain the body temperature at 37 ± 0.5 °C during the operation. Disinfect the surgical instruments with 75% alcohol. Place the mouse supine on a heating blanket and expose its neck. Then remove the neck hair of the mouse, disinfect the skin and cut the skin along the midline of the neck. Pull apart the cervical gland with two forceps. Directly separate the left common carotid artery, internal carotid artery and external carotid artery without squeezing the adjacent nerves and veins. Then clamp the common carotid artery and internal carotid artery with a vascular clamp. Suture with 8-0 silk thread and tie a knot at the distal end of the external carotid artery and a slip knot at the proximal end. Make a small incision between the two suture knots of the external carotid artery with an ophthalmic scissors, and then insert a special thread embolization along the incision into the external carotid artery and the common carotid artery. Next, remove the internal carotid artery clamp, cut the external carotid artery at the opening of the external carotid artery, pull the thread embolization back to the bifurcation of the common carotid artery, and reverse-insert it into the internal carotid artery, pterygopalatine artery and ophthalmic artery. Insert the thread embolization to the end of the pterygopalatine artery, about 6 mm from the bifurcation. The tail of the thread embolization is almost at the bifurcation, blocking the ophthalmic artery. Then tighten the slip knot and suture the skin. The mouse can move freely during arterial embolization. After 120 minutes of embolization, carefully remove the thread embolization from the pterygopalatine artery without major bleeding. Remove the common carotid artery clamp and restore arterial reperfusion. Then suture the skin wound and routinely feed the mouse during reperfusion.

[0041] Example 2: In vivo effect of ritacalimod on mouse retinal artery occlusion injury:

[0042] After intravitreal injection of loteprednol etabonate into mice for 24 hours, a mouse model of retinal artery occlusion was established. After 7 days, the mice were dark adapted for 12 hours, and then the electroretinogram of the mice was measured. The mouse eyeballs were carefully removed and placed in a special eyeball fixing solution for more than 2 hours, followed by paraffin embedding, sectioning and HE staining. At the same time, the retinas of the other half of the mice were carefully dissected under a surgical microscope to ensure the integrity and cell viability of the retinas, and then fixed and immunofluorescent stained with Brn3a, followed by photographing and analysis. Figure 1 For the electroretinogram (ERG) detection of the mice in Example 2, as can be seen from the figure: the amplitude of the ERG of the mice treated with loteprednol etabonate was higher, the retinal thickness increased compared with the control group, and the retinal ganglion density increased, indicating that loteprednol etabonate can effectively alleviate the damage of retinal ganglion cells in retinal artery occlusion and contribute to the recovery of visual function.

[0043] Figure 2 For the pathological result pictures of HE staining of the mouse retina in Example 2, as can be seen from the figure: the retinal thickness of the mice in the loteprednol etabonate group increased and the damage was alleviated.

[0044] Figure 3 For the RGC result pictures of immunofluorescent staining of the mouse retinal flat mount in Example 2, as can be seen from the figure: the number of ganglion cells in the retina increased after treatment with loteprednol etabonate.

[0045] Example 3: Effect of loteprednol etabonate on the activation and metabolism of microglia:

[0046] The microglial cell line BV2 of mice was cultured in vitro. During the logarithmic growth phase of cell growth, the cells were plated and grouped into a control group, a lipopolysaccharide (LPS) stimulation group and an LPS + loteprednol etabonate group. The concentration of LPS was 200 ng / ml and the concentration of loteprednol etabonate was 100 ng / ml. After stimulation for 24 hours, the cells were collected, RNA was extracted, the concentration and quality of RNA were measured, and the mRNA expression levels of Inos, Tnf-α, Il-1β, and IL-6 were detected by Real-Time PCR to analyze the changes in relative expression levels. The experimental results showed that loteprednol etabonate could effectively inhibit the metabolism and activation of microglia.

[0047] Figure 4 For the effect of loteprednol etabonate at different concentrations on the activity of BV2 cells in Example 3, as can be seen from the figure: the concentration of loteprednol etabonate from 0 to 100 ng / ml did not affect the activity of BV2 cells.

[0048] Figure 5 For the effect of loteprednol etabonate on the expression of iNOS in BV2 cells in Example 3, as can be seen from the figure: loteprednol etabonate could inhibit the gene expression level of iNOS and reduce the activation of microglia.

[0049] Figure 6 For the effect of ritastatins in Example 3 on the TNF-α expression of BV2 cells, it can be seen from the figure that ritastatins can inhibit the gene expression level of TNF-α and reduce the activation of microglia.

[0050] Figure 7 For the effect of ritastatins in Example 3 on the IL-1β expression of BV2 cells, it can be seen from the figure that ritastatins can inhibit the gene expression level of IL-1β and reduce the activation of microglia.

[0051] Figure 8 For the effect of ritastatins in Example 3 on the IL-6 expression of BV2 cells, it can be seen from the figure that ritastatins can inhibit the gene expression level of IL-6 and reduce the activation of microglia.

[0052] Example 4: The effect of ritastatins on oxygen-glucose deprivation injury of retinal ganglion cells:

[0053] The rat retinal ganglion cell precursor cell line R28 was cultured in vitro. At the logarithmic growth phase of cell growth, the cells were plated and grouped, and then subjected to oxygen-glucose deprivation treatment for 6 hours and reoxygenated for 24 hours. They were divided into a solvent control group and a ritastatins group with a ritastatins concentration of 100 ng / ml. The stimulation time was 24 hours. The cells were collected, digested into single-cell suspensions, resuspended with PBS, and then stained with Annexin V and PI, and flow cytometry was used to analyze the apoptosis of the cells. Figure 9 For the effect of ritastatins in Example 4 on oxygen-glucose deprivation injury of R28 cells, it was found that the proportion of apoptotic cells decreased significantly after treatment with ritastatins. The experimental results show that ritastatins can reduce the injury of retinal ganglion cells.

[0054] The above is the preferred embodiment of the present invention. Of course, the scope of the rights of the present invention cannot be limited by this. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and changes can be made, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. Use of lifalast in the preparation of a drug for treating retinal artery occlusion damage.

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

3. The use according to claim 1, characterized in that: The rifalast is used to reduce apoptosis of retinal ganglion cells.

4. Use of a composition containing lifalast in the preparation of a drug for treating retinal artery occlusion damage.

5. The use according to claim 4, characterized in that: The composition containing lifalast is used to inhibit the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6.

6. The use according to claim 4, characterized in that: The composition containing rifalast is used for alleviating apoptosis of retinal ganglion cells.

7. The use according to any one of claims 1 to 6, characterized in that: The administration method of the drug is: intravitreal injection.

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