Pharmaceutical Use of PLX5622

Abstract

The present invention relates to the field of medicine, and particularly to the pharmaceutical use of PLX5622. The present invention discovers that PLX5622 or its pharmaceutically acceptable salt can be used to prepare a drug for preventing and treating retinal ischemia-reperfusion injury caused by retinal artery occlusion, which results in damage to the retinal structure and / or function, and solves the problem in the prior art that it is difficult for patients with retinal artery occlusion to recover their vision after blood reperfusion.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the pharmaceutical use of PLX5622. Background Art

[0002] Retinal artery occlusion (RAO) is a severe ophthalmic emergency. Its main cause is the sudden occlusion of the central retinal artery, which leads to acute dysfunction of retinal tissue. Acute occlusion of the retinal artery causes axoplasmic stasis and intracellular edema, usually resulting in permanent retinal ischemia and irreversible death of retinal ganglion cells (RGCs) within a few hours. Currently, the clinical treatment of RAO mainly focuses on the treatment of retinal occlusion emboli. The treatment methods include anterior chamber puncture, acetazolamide treatment, eye massage, and thrombolytic treatment with drugs or interventional surgery for arteries and veins. The treatment goal is to improve and restore retinal circulation and avoid the occurrence of retinal necrosis. However, the treatment time window for RAO is short. Moreover, after the retinal artery resumes blood perfusion, not only the original retinal function fails to recover, but the structural damage becomes more severe, and even irreversible damage occurs. This phenomenon is called retinal ischemia-reperfusion injury (RIRI). It is reported that only 20% of patients can have limited vision recovery after treatment. Currently, the pathogenesis of both RAO and its ischemia-reperfusion injury has not been fully elucidated. How to maximize the rescue of ischemic but not yet infarcted retina after RAO treatment, reduce the further oxidative damage caused by RIRI, and restore the structure and function of the retina has become a difficult problem to be solved urgently. Summary of the Invention

[0003] The inventors found that using PLX5622 after the reperfusion of retinal artery occlusion can prevent the occurrence of problems of damaged retinal structure and / or function, and solve the problem that it is difficult to restore vision after reperfusion in patients with retinal artery occlusion in the prior art. Based on this, the present invention provides the use of PLX5622 or its pharmaceutically acceptable salt in the preparation of a drug for preventing and treating retinal structure damage and / or function damage caused by retinal ischemia-reperfusion injury induced by retinal artery occlusion.

[0004] The technical solution provided by the present invention is specifically as follows:

[0005] The present invention provides the use of PLX5622 or its pharmaceutically acceptable salt in the preparation of a drug for preventing and treating retinal structure damage and / or function damage caused by retinal ischemia-reperfusion injury induced by retinal artery occlusion.

[0006] In some embodiments of the present invention, the damage to the retinal structure is caused by the injury or death of retinal ganglion cells.

[0007] In some embodiments of the present invention, the drug is a therapeutic drug for retinal artery occlusion or a prophylactic drug for complications after the treatment of retinal artery occlusion.

[0008] In some embodiments of the present invention, the therapeutic drug is an arterial thrombolytic drug, and PLX5622 or a pharmaceutically acceptable salt thereof is used as an auxiliary component in the arterial thrombolytic drug, and the auxiliary component is used to prevent damage to the retinal structure and / or function.

[0009] In some embodiments of the present invention, the arterial thrombolytic drug contains a fibrinolytic component and / or a vasodilating component as the main component.

[0010] In some embodiments of the present invention, the treatment of retinal artery occlusion includes one or more of anterior chamber paracentesis, acetazolamide treatment, ocular massage, thrombolysis with arterial thrombolytic drugs, and thrombolysis by interventional surgery.

[0011] In some embodiments of the present invention, the complication is damage to the retinal structure and / or function.

[0012] In some embodiments of the present invention, PLX5622 is in the form of a pharmaceutically acceptable acid addition salt.

[0013] In some embodiments of the present invention, the dosage form of the drug is one or more of oral dosage forms, microcapsule preparations, injections, suppositories, eye drops, eye washes, ophthalmic creams, and ophthalmic gels.

[0014] In some embodiments of the present invention, the symptoms of structural damage are at least one of white lesions in the fundus oculi and retinal edema; the symptoms of functional damage are at least one of a sharp decline in visual acuity and visual field defect.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] In view of the problem of decreased visual acuity after reperfusion of retinal artery occlusion, there is a lack of effective therapeutic drugs in the prior art. The present invention provides the use of PLX5622 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating retinal ischemia-reperfusion injury caused by retinal artery occlusion, which results in damage to the retinal structure and / or function, and solves the problem that it is difficult for patients with retinal artery occlusion to recover their vision after reperfusion in the prior art. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0018] Figure 1 It is the result of immunofluorescence staining of retinal ganglion cells in mice with the oculopalatine artery and ophthalmic artery occlusion model after 7 days of intervention with low-dose (40 mg / kg / d) PLX5622; among them: Con represents the healthy group, PLX5622 Con represents the PLX5622-administered healthy group, PLX5622 UPOAO represents the PLX5622 intervention group, and UPOAO represents the control group.

[0019] Figure 2 It is the result of fundus photography of the retinal type and optical coherence tomography (OCT) in mice with the artery occlusion model after 7 days of intervention with low-dose (40 mg / kg / d) PLX5622; among them: Con represents the healthy group, PLX5622 Con represents the PLX5622-administered healthy group, PLX5622 UPOAO represents the PLX5622 intervention group, and UPOAO represents the control group.

[0020] Figure 3 is Figure 2 the statistical result of the inner retinal thickness in the OCT image of

[0021] Figure 4 It is the comparison of the waveforms of the a-wave and b-wave of electroretinogram (ERG) in mice with the artery occlusion model after 7 days of intervention with low-dose (40 mg / kg / d) PLX5622; among them: Con represents the healthy group, PLX5622 Con represents the PLX5622-administered healthy group, PLX5622 UPOAO represents the PLX5622 intervention group, and UPOAO represents the control group.

[0022] Figure 5 There is no significant difference in the counting results of retinal microglia in immunofluorescent mice with the artery occlusion model after 7 days of intervention with low-dose (40 mg / kg / d) PLX5622; among them: Con represents the healthy group, and PLX5622 Con represents the PLX5622-administered healthy group.

[0023] Figure 6 It is the result of CCK8 cell viability detection after intervention with different concentrations of PLX5622 (0.1 - 5 μM) on BV2 cells. Detailed implementation manners

[0024] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0025] PLX5622 is a small molecule inhibitor that targets colony stimulating factor 1 receptor (CSF1R). The prior art has utilized PLX5622 to study the role of microglia in retinal protection. Research has shown that PLX5622, as a CSF1R inhibitor, exhibits different effects in various retinal disease models by depleting microglia. In some cases, such as light-induced injury and retinal vein occlusion, PLX5622 can slow down retinal function degeneration and delay retinal degeneration. However, in the glaucoma model, microglia depletion exacerbates the loss of retinal ganglion cells. These research results indicate that microglia have a complex dual role in retinal protection, being both likely to protect retinal function and likely to exacerbate damage, depending specifically on the disease model and experimental conditions.

[0026] The present invention conducted low-dose (40 mg / kg / d) PLX5622 intervention on a mouse model after reperfusion of retinal artery occlusion and found that PLX5622 did not eliminate microglia, but had a protective effect on the retinas of mice after reperfusion of retinal artery occlusion, preventing the retina from being structurally and / or functionally damaged due to RIRI, which is different from the role played by high doses reported in the past for tissue microglia clearance.

[0027] The present invention provides the use of PLX5622 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating retinal structural and / or functional damage caused by retinal ischemia-reperfusion injury induced by retinal artery occlusion. In the present invention, a small amount of PLX5622 exhibits a significant protective effect on the retinas of mice after reperfusion of retinal artery occlusion. This finding not only provides a new strategy for the treatment of retinal artery occlusion, but also reveals that the protective effect of PLX5622 on the retina is not achieved by completely eliminating microglia. This finding provides a theoretical basis and experimental evidence for the development of new drugs for retinal artery occlusion and its related complications, and is expected to bring better treatment options and prognoses for patients with retinal artery occlusion.

[0028] In some embodiments of the present invention, the damage to the retinal structure is caused by the injury or death of retinal ganglion cells. The present invention verifies that PLX5622 has a protective effect on retinal ganglion cells after the treatment of retinal artery occlusion, thereby protecting the structure and function of the retina from damage. By means of technical means such as tissue section staining, the present invention detailedly observed the survival status of retinal ganglion cells in different mice. The results showed that the number of RGCs cells stained with Rbpms in the retina of the control group (UPOAO) was significantly reduced compared with that of healthy mice (Con), indicating that RIRI led to the death of RGCs cells; while the number of RGCs cells stained with Rbpms in the retina of the PLX5622 intervention group (PLX5622 UPOAO) was increased compared with that of the control group (UPOAO), indicating that PLX5622 effectively inhibited the apoptosis process of RGCs cells during RIRI. In addition, through the detection of electroretinogram, the present invention found that the retinal function of the PLX5622 intervention group was also significantly improved compared with that of the control group, specifically manifested as the amplitude and latency of the electrical signal both recovered to a level close to that of healthy mice, further confirming the dual protective effect of PLX5622 on the retinal structure and function. These findings not only deepen the understanding of the pharmacological action mechanism of PLX5622, but also provide a new perspective and potential drug treatment targets for the clinical treatment of retinal artery occlusion.

[0029] In some embodiments of the present invention, the drug is a therapeutic drug for retinal artery occlusion or a prophylactic drug for complications after the treatment of retinal artery occlusion. PLX5622 can be used during the treatment of retinal artery occlusion or after the treatment of retinal artery occlusion, and can protect the retina after the restoration of blood supply in retinal artery occlusion, preventing its structure and function from being damaged.

[0030] In some embodiments of the present invention, the therapeutic drug is an arterial thrombolytic drug, and PLX5622 or its pharmaceutically acceptable salt is used as an auxiliary component in the arterial thrombolytic drug, and the auxiliary component is used to prevent damage to the retinal structure and / or function. The time window period for the treatment of RAO is short. Using PLX5622 as an auxiliary component in the arterial thrombolytic drug can start to play a preventive role during the arterial thrombolysis process, avoiding damage to the retinal structure and / or function caused by too late administration.

[0031] In some embodiments of the present invention, the arterial thrombolytic drug contains a fibrinolytic component and / or a vasodilating component as the main component. The arterial thrombolytic drug can use the fibrinolytic component and / or the vasodilating component as the main component and PLX5622 as the auxiliary component.

[0032] In some embodiments of the present invention, the treatment of retinal artery occlusion includes one or more of anterior chamber paracentesis, acetazolamide treatment, ocular massage, thrombolysis with arterial thrombolytic drugs, and thrombolysis by interventional surgery. These treatment methods for retinal artery occlusion can all restore blood supply to the retinal artery, but they will trigger RIRI. Administering PLX5622 immediately after the treatment of retinal artery occlusion can prevent damage to the structure and / or function of the patient's retina and avoid irreversible decline in the patient's vision.

[0033] In some embodiments of the present invention, the complications are damage to the retinal structure and / or function. In the experiments of the present invention, it was verified that the waveforms of the a-wave and b-wave of electroretinogram (ERG) in mice with an arterial occlusion model did not change 7 days after intervention with low-dose (40 mg / kg / d) PLX5622, demonstrating that intervention with PLX5622 after the treatment of retinal artery occlusion can prevent damage to retinal function.

[0034] In some embodiments of the present invention, PLX5622 is in the form of a pharmaceutically acceptable acid addition salt.

[0035] In some embodiments of the present invention, the dosage form of the drug is one or more of oral preparations, microcapsule preparations, injections, suppositories, eye drops, eye washes, ophthalmic creams, and ophthalmic gels. Optionally, the oral preparation is one or more of capsules, tablets, granules, and oral liquids; the injection is preferably an intravitreal injection solution. The present invention has confirmed that the low-dose oral preparation of PLX5622 can prevent and treat retinal ischemia-reperfusion injury caused by retinal artery occlusion, resulting in damage to the retinal structure and / or function. Compared with preparations such as injections, suppositories, eye drops, eye washes, ophthalmic creams, and ophthalmic gels, the oral preparation of PLX5622 is convenient to use, has good patient compliance, and good safety.

[0036] In some embodiments of the present invention, the symptoms of structural damage are at least one of white lesions in the fundus and retinal edema; the symptoms of functional damage are at least one of a sharp decline in vision and visual field defect. In the examples of the present invention, the OCT images showed white lesions in the fundus of the control group mice after retinal ischemia, and no white lesions in the fundus were seen in the PLX5622 intervention group mice, demonstrating that PLX5622 intervention can prevent white lesions in the fundus. The fundus color photographs showed obvious ischemic edema around the optic disc in the control group mice, and no optic disc ischemic edema was seen in the PLX5622 intervention group, demonstrating that PLX5622 intervention can prevent retinal edema. The ERG results showed a significant decrease in the b-wave of the control group mice, suggesting a sharp decline in vision, and a significant increase in the b-wave of the ERG in the PLX5622 intervention group, demonstrating that PLX5622 intervention can significantly delay the decline in vision and play a role in protecting retinal function.

[0037] The technical solution provided by the present invention will be described in detail below in conjunction with the embodiments.

[0038] All the experimental animals used, C57BL / 6 mice, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (certificate number: No. 210726231100785184).

[0039] The silicone wire embolism No. 602156 (diameter 0.19 ± 0.01 mm, length 6.0 ± 0.1 mm) used was ordered from Pingdingshan Yushun Biotechnology Co., Ltd.

[0040] The drugs and reagents used: Isoflurane and oxygen / nitrous oxide mixed gas were purchased from Shenzhen Rewod Technology Co., Ltd. Other conventional antibodies and reagents are all conventional commercially available products.

[0041] The instruments and apparatuses used: The surgical microscope is Olympus SZX7 from Japan, the electroretinogram (ERG) uses RetiMINER4.0 from Chongqing Aierxi, and the fluorescence confocal microscope is TCS-SP8 from Germany. Others are all common commercially available products.

[0042] Unless otherwise specified, the PLX5622 working solution and the control solvent used in the examples were prepared by the following methods and administered in the following manner:

[0043] ① Preparation of the PLX5622 stock solution: PLX5622 was dissolved in dimethyl sulfoxide (DMSO) to obtain a PLX5622 stock solution with a concentration of 100 mg / mL.

[0044] ② Preparation of the PLX5622 working solution: Polyethylene glycol 12-hydroxystearate (Solutol HS-15, TargetMol), polyethylene glycol 400 (PEG400, TargetMol) and double-distilled water (dd water) were mixed into a dilution solvent according to a volume ratio of 2:4:13. The PLX5622 stock solution (100 mg / mL) was added to the dilution solvent according to a volume ratio of 1:9, and mixed evenly to obtain a PLX5622 working solution with a concentration of 5 mg / mL.

[0045] ③ Preparation of the control solvent: DMSO and the dilution solvent were mixed according to a volume ratio of 1:19 to obtain the control solvent.

[0046] ④ Before daily gavage administration, weigh the mice, and adjust the dosage according to the daily body weight change. Calculate the volume of the PLX5622 working solution according to a PLX5622 dosage of 40 mg per kilogram of body weight per day.

[0047] Example 1:

[0048] (1)Establishment of the unilateral pterygopalatine ophthalmic artery embolization (UPOAO) mouse model

[0049] Preparation of experimental mice:

[0050] Select 20 healthy male C57BL / 6 mice aged 8 - 12 weeks with a body weight controlled at 21g ± 1g. The left eye of each mouse is the model group, and the right eye is the control group. They are raised in an IVC system environment at a temperature of 24 - 27°C, a relative humidity of 60%, with free access to food and water for one week.

[0051] Preparation of silicone thread embolization:

[0052] Prepare the thread embolization in advance. Select a silicone with a diameter of 0.19 ± 0.01mm, a silicone length of 6mm ± 0.1mm, and a No. 0.4 fishing line. Place it in a 35mm sterile petri dish, soak it in 75% alcohol for 1h, and let it air dry naturally in a biosafety cabinet for standby.

[0053] Establishment of the unilateral pterygopalatine ophthalmic artery embolization (UPOAO) mouse model:

[0054] S10. Anesthetize, fix, and control the body temperature of C57BL / 6 mice: Anesthetize the mice by inhaling a mixture of 2.0% isoflurane and oxygen / nitrous oxide. Place the mice in a supine position and fix them. Moisten the mouse hair on the neck with 75% medical alcohol, shave it off with a blade, and wipe it clean. Insert a rectal temperature probe to keep the body temperature at 37 ± 0.5°C.

[0055] Isolate the pterygopalatine artery: Perform a neck exposure to isolate the left common carotid artery, internal carotid artery, and external carotid artery. Use an 8-0 silk thread to ligate the distal end of the external carotid artery. Thread another 8-0 silk thread through the external carotid artery and make a slipknot near the bifurcation of the common carotid artery. Make a midline incision in the neck to expose the left common carotid artery, internal carotid artery, and external carotid artery. Use an 8-0 silk thread to ligate the distal end of the external carotid artery. Thread another 8-0 silk thread through the external carotid artery and make a slipknot near the bifurcation of the common carotid artery. Use artery clamps to clamp the internal carotid artery and common carotid artery respectively. Make a small incision 1mm below the ligation line of the external carotid artery.

[0056] S20. Insert a No. 602156 silicone thread embolization into the pterygopalatine artery to construct the model: Insert a sterile No. 602156 silicone thread embolization through the 1mm incision below the ligation line of the external carotid artery, and gently push the thread embolization with forceps until all the silicone of the thread embolization enters the pterygopalatine artery.

[0057] Model completion: Release the internal carotid artery clamp, cut the external carotid artery at the small opening, withdraw the suture embolism, turn it over to make it enter the internal carotid artery, and insert it outward into the pterygopalatine artery. The insertion depth of the suture embolism is about 6±1 mm from the bifurcation of the common carotid artery. The silicone tail of the suture embolism is roughly located at the bifurcation of the common carotid artery, and the slipknot is slightly tightened. After 60 minutes of ischemia, remove the suture embolism, ligate the proximal end of the external artery, and release the artery clamp. The reperfusion of blood flow in the common carotid artery and internal carotid artery can be observed. Suture the wound to obtain UPOAO model mice.

[0058] (2)RAO treatment

[0059] Take an appropriate amount of UPOAO model mice and randomly divide them into two groups:

[0060] PLX5622 intervention group (abbreviated as PLX5622 UPOAO): While the mice are fed a regular diet every day, they are continuously gavaged with the PLX5622 working solution for 7 days (from the 1st day to the 7th day), and the dosing dose is 40 mg PLX5622 per kilogram of body weight per day.

[0061] Control group (abbreviated as UPOAO): While the mice are fed a regular diet every day, they are gavaged with a control solvent of the same volume as the UPOAO model mice in the PLX5622 intervention group.

[0062] At the same time, take several healthy male C57BL / 6 mice of the same age and divide them into two groups:

[0063] Healthy group (abbreviated as Con): While the mice are fed a regular diet every day, they are gavaged with a control solvent of the same volume as the mice in the PLX5622 intervention group.

[0064] PLX5622-administered healthy group (abbreviated as PLX5622 Con): While the mice are fed a regular diet every day, they are continuously gavaged with the PLX5622 working solution for 7 days (from the 1st day to the 7th day), and the dosing dose is 40 mg PLX5622 per kilogram of body weight per day.

[0065] (3)RAO treatment effectiveness evaluation

[0066] Take 3 mice treated with RAO from each group, intraperitoneally inject 2% pentobarbital sodium for general anesthesia at a dose of 0.3 mL per 100 g body weight, and instill compound tropicamide mydriatic eye drops for 5 minutes of mydriasis. Place the mydriatic mice on the small animal fundus imaging operation table and use the fundus and optical coherence tomography (Optical Coherence Tomography, abbreviated as OCT) module to collect fundus color photos and OCT of the retinal structure. As Figure 2As shown, white lesions appeared in the fundus of the retinas of the mice in the control group after retinal ischemia, and ischemia-induced whitening and edema occurred around the optic disc. The OCT images showed that the inner retinal structure was damaged. No white lesions were observed in the fundus of the mice in the PLX5622 intervention group, and no whitening or edema occurred around the optic disc. The OCT images showed that the inner retinal structure was relatively clear and the retinal thickness increased (as shown by the box in the figure). Compared with the control group (UPOAO), the retinal structure in the PLX5622 intervention group (PLX5622 UPOAO) was intact. Figure 3 The thickness of the inner retina in the OCT images was measured, and it was found that compared with the control group (UPOAO), the thickness of the inner retina (nerve fiber layer and ganglion cell layer, RNFL+GCL) in the PLX5622 intervention group (PLX5622 UPOAO) increased significantly (*: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001), indicating that PLX5622 had a significant protective effect on the retinal structure of the UPOAO model mice.

[0067] Eight RAO-treated mice were selected from each group. Intraperitoneal injection of 2% sodium pentobarbital was performed for general anesthesia at a dose of 0.3 mL per 100 g body weight. After the mice were stably anesthetized, compound tropicamide mydriatic eye drops were instilled for 15 minutes of mydriasis. The mydriatic mice were placed on the electroretinogram (ERG) operation table with their heads in the frontal position and both eyes at the same height, fully exposed. The tail electrode was clamped at the rear end of the mouse tail, and the subcutaneous electrode was inserted into the skin at the back of the neck. The left and right corneal contact electrodes were placed on the model eye and the normal eye, and the amplitude changes of the electroretinogram a-wave and b-wave were recorded by different light intensity stimulations of ERG. As Figure 4 The waveforms of the electroretinogram a-wave and b-wave of the mice in the PLX5622 intervention group and the control group are shown. Comparing the PLX5622 intervention group with the control group, it can be seen that at different stimulation intensities, there was no statistical difference in the a-wave, and the b-wave increased significantly (*: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001), indicating that low-dose PLX5622 had a protective effect on the visual function of the UPOAO model mice.

[0068] To further verify the protective effect of PLX5622 on retinal ganglion cells (RGCs), retinal samples from mice in the PLX5622 intervention group and the control group were taken, fixed with 4% paraformaldehyde for 45 minutes, dissected for retinal separation, and embedded to make retinal sections. The retinal sections were rinsed with PBS, immersed in 5% BSAT solution (a mixture formed by adding 0.3% Triton X-100 to immunofluorescence blocking solution (BSA)), and placed in a refrigerator at 4°C for overnight blocking. The primary antibody, Rbpms goat serum, was incubated in a refrigerator at 4°C for 48 hours. After rinsing in PBS buffer, the secondary antibody, AlexaFluor488 donkey anti-goat fluorescent antibody, was incubated overnight at 4°C. After rinsing in PBS buffer, the sections were sealed with an anti-fluorescence quenching mounting medium and photographed using a confocal microscope for RGCs counting. The results are as Figure 1 shown. Compared with the healthy group, the number of RGCs stained with Rbpms on the retinal sections of mice in the control group was significantly reduced, while the number of RGCs stained with Rbpms on the retinal sections of mice in the PLX5622 intervention group was significantly increased compared with the control group, confirming that PLX5622 has a protective effect on RGCs in the UPOAO model mice.

[0069] (4)The neuroprotective effect of PLX5622 intervention on RAO was not achieved by clearing microglia

[0070] (4.1)To verify that the protective effect of PLX5622 on retinal ganglion cells (RGCs) is not achieved by clearing microglial cells, Iba1 immunofluorescence staining was performed on the isolated retinal samples to evaluate the effect of PLX5622 on the number of microglial cells:

[0071] Six UPOAO model mice were randomly selected, with 3 mice in each group. The healthy group administered with PLX5622 was gavaged with a dose of 40 mg / kg body weight of PLX5622 per day for 7 days, and the healthy group was gavaged with an equal volume of control solvent for 7 days. After 7 days of gavage, the mice were euthanized, the eyeballs were removed, the cornea and iris were cut off with scissors, fixed in 4% paraformaldehyde solution for 1 hour, the eyeballs were rinsed with PBS, the retina was immersed in 5% BSAT solution, and placed in a refrigerator at 4°C for overnight blocking. The primary antibody, Iba1 rabbit serum, was incubated in a refrigerator at 4°C for 48 hours. After rinsing in PBS buffer, the secondary antibody, AlexaFluor594 donkey anti-rabbit fluorescent antibody, was incubated overnight at 4°C. After rinsing in PBS buffer, the retina was flattened with the optic nerve layer facing up, sealed with an anti-fluorescence quenching mounting medium and photographed using a confocal microscope for microglial cell counting. As Figure 5As shown, there was no statistical difference in microglia on the retinal optic nerve layer of mice in the healthy group after PLX5622 administration compared with the healthy group, which confirmed that PLX5622 did not eliminate microglia in the retinal tissue at a dosage of 40 mg / kg / d, and also indicated that PLX5622 did not protect the nerves of RAO by eliminating microglia.

[0072] (4.2)In in vitro experiments, the present invention used PLX5622 to intervene in the microglial cell line BV2 to further verify that PLX5622 does not act by reducing and eliminating microglia:

[0073] Using high-glucose DMEM complete medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin), the working solution of PLX5622 was diluted to a series of concentration gradients of 0.1, 0.25, 0.5, 1, and 5 μM. As a control for the experiment, the corresponding volume of the control solvent was diluted to the same dilution ratio as the 5 μM PLX5622 working solution as a negative control.

[0074] BV2 cells were cultured with the above medium for 24 hours, and the CCK8 cell viability detection kit was used to analyze the cell viability of each group to evaluate the effect of PLX5622 on the proliferation and viability of BV2 cells. The results were as Figure 6 shown. In the media with different concentrations of PLX5622, the cell viability of BV2 cells was not affected, which confirmed that low-dose PLX5622 had no effect on the cell viability of the in vitro microglial cell line BV2, and also confirmed that the protection of PLX5622 on the mouse retina was not achieved by eliminating BV2 cells.

[0075] In the description of this specification, the description referring to terms such as "one embodiment / way", "some embodiments / ways", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment / way or example are included in at least one embodiment / way or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment / way or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments / ways or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments / ways or examples described in this specification and the features of different embodiments / ways or examples.

[0076] It should be noted that in the present invention, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element. In the present invention, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise specifically defined.

[0077] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of PLX5622 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating retinal structural damage and / or functional damage caused by retinal ischemia-reperfusion injury resulting from retinal artery occlusion; the symptoms of the structural damage include at least one of white lesions in the fundus oculi, retinal edema, and damage to the inner retinal structure; the symptoms of the functional damage include a sharp decline in vision; the retinal edema is ischemic, whitened, and edematous around the optic disc.

2. The application according to claim 1, characterized in that: The drug is a therapeutic drug for retinal artery occlusion or a prophylactic drug for complications after the treatment of retinal artery occlusion.

3. The application according to claim 2, wherein: The therapeutic drug is an arterial thrombolytic drug, and PLX5622 or a pharmaceutically acceptable salt thereof is used as an auxiliary component in the arterial thrombolytic drug, and the auxiliary component is used to prevent retinal structural damage and / or functional damage.

4. The application according to claim 3, characterized in that: The arterial thrombolytic drug contains a fibrinolytic component and / or a vasodilating component as the main component.

5. The application according to claim 2, wherein: The treatment of retinal artery occlusion includes one or more of anterior chamber paracentesis, acetazolamide treatment, eye massage, thrombolysis with an arterial thrombolytic drug, and thrombolysis by interventional surgery.

6. The application according to claim 2, characterized in that: The complication is retinal structural damage and / or functional damage.

7. The application according to claim 1, characterized in that: The PLX5622 is in the form of a pharmaceutically acceptable acid addition salt.

8. The application according to claim 1, characterized in that: The dosage form of the drug is one or more of oral preparations, microcapsule preparations, injections, suppositories, eye drops, eye washes, ophthalmic creams, and ophthalmic gels.

Images